Vegetal Production

C H A P T E R

6.2 Vegetal Production

Gases in Agro-food Processes https://doi.org/10.1016/B978-0-12-812465-9.00013-X

153

# 2019 Elsevier Inc. All rights reserved.

S U B C H A P T E R

6.2.1 CO2 for Greenhouses Philippe Girardon Air Liquide, Paris, France

6.2.1.1 INTRODUCTION Boosting the CO2 level in commercial greenhouses has been in use for more than 40 years. The most common approach was to recover the CO2 from the flue gases resulting from burning the natural gas (for example, from piping distribution of methane or from liquefied propane in a vessel) that was used for heating the greenhouses. The combination of these cleaner fuels and improved burners enabled recovery of the CO2 to become one of the largest selfproductions of “industrial” CO2 made directly by the user. In the hotter summer period when no heating is required, the question was the cost of producing CO2 from combustion compared to the cost of industrial CO2 proposed by the gas industry. Further trends were to use higher levels of CO2. Initially, most systems aimed to keep the levels comparable to those found in the outside air, but growers obtained much higher yields from their crops by the use of around 2–3 times the ambient air CO2 levels. Today, greenhouse heating technology management consists of matching the CO2 production according to night and day frequency

periods with heat store use. Typically, the heating system runs during the day, producing the necessary CO2 while pumping the heat into a heat store. At night, the heating system is switched off and the heat store gives the necessary heating back to the greenhouse.

6.2.1.2 HIGH TECH GREENHOUSE MANAGEMENT Recent greenhouse designs consist of closed construction and ventilation provided by only mechanical systems, full air control, heat pumps and heat storage, temperature and humidity control, tight control of CO2, nutrient solution irrigation, and use of artificial lighting. Such interrelated systems are very efficient in terms of energy use and CO2 consumption while providing optimum growing conditions for the highest crop yields. Maximizing growth does not necessarily produce more fruit or flowers. Tomatoes fruit is one of the major products grown under plant production in greenhouses vs open field (Fig. 6.2.1.1).

6. AGRICULTURE

6.2.1.4 PHOTOSYNTHESIS BACKGROUND

FIG. 6.2.1.1

155

Tomatoes fruit is one of the major products grown under plant production in greenhouses vs open field.

6.2.1.3 INFLUENCE OF CO2 AND FUEL COSTS In the 1970s, the escalation of fuel prices and a progressive fall in the market price for CO2 resulted in a change in the economics of the application in favor of “external” CO2 use.

6.2.1.4 PHOTOSYNTHESIS BACKGROUND Plant growth is the result of photosynthesis in which light energy from the sun is used as a source of energy for the synthesis of organic

matter from atmospheric CO2 and water. Chemically, this phenomenon, which is accompanied by oxygen production by the plant, can be represented by the formula below: 6H2 O + 6CO2 + light energy ! C6 H12 O6 + 6O2 This equation shows that CO2 is one of the three main factors that combine to produce the organic elements necessary to the constitution of the structure of the plant. The reverse reaction is breathing in use among all living organisms, and which is to burn carbohydrates by releasing water, CO2, and energy. Green elements absorb CO2 normally present in the air at a concentration of 0.03% in volume on

6. AGRICULTURE

156

6.2. VEGETAL PRODUCTION

average (300 ppm) through pores (stomata) in the plant cells. This value is less than the optimum of that required for the growth of plants. If the concentration falls to 0.012%–0.015% (120–150 ppm), this will even stop photosynthesis. There are many other carbohydrates and organic compounds that are formed together with mineral nutrients and water to build the plant tissues and organs.

6.2.1.5 ADVANTAGES OF CO2 ENRICHMENT Without the outside input of CO2, this critical level can be reached in a few hours in a greenhouse full of plants. CO2 enrichment allows restoring the minimum content required. The results are variable according to the cultivated species and varieties. Researchers and growers rapidly found that much higher rates of growth were possible with elevated CO2 levels. Enrichment of CO2 from 300 up to about 800 ppm can ensure an increase of yield both in weight and number of fruits, faster development giving earlier harvesting, better bloom color quality, more vigorous stems, healthy growth with improved disease resistance, and longer lasting flowers, as indicated in Tables 6.2.1.1–6.2.1.4. In these tables the advantages of CO2 enrichment are recorded for the following specific crops: tomatoes, cucumbers, lettuce, peppers, and roses for the main. The role of illumination is not negligible as shown in the following curves, knowing that due to dissipation of sun energy caused by the water in air, there is a big gap between theoretical availability and reality. This is mostly in northern countries, for example, 1000 watt m2 on the Earth’s surface at the equator versus 150 in Benelux (Fig. 6.2.1.2).

TABLE 6.2.1.1 Optimum CO2 Content for Some Products Plants Grown

CO2 Concentration (ppm)

VEGETABLES Tomatoes

600–1000

Cucumbers

800–1000

Lettuce

1000

Melons

1000

PLANTS IN POTS Begonias

800

Hydrangeas

1500–2000

St-Paulia

800

GREEN PLANTS Ficus

800

Croton

800

CUT FLOWERS Roses

800

Mums

800–1000

Tulips

1200

Eyelets

800

TABLE 6.2.1.2 Additional CO2 Average Registered, and Estimated Value for a 400 ppm CO2 Concentration kg CO2 (m22 year21)

kg CO2 (m22 day21)

4

11  103

TABLE 6.2.1.3 Impact of CO2 on the Production of Cucumbers From Mid-March to Mid-August CO2 concentration (ppm)

400

1000

Number of fruits (m2 week1)

2

2.3

6. AGRICULTURE

157

6.2.1.6 TYPICAL INSTALLATION

TABLE 6.2.1.4

Total Consumption of CO2 (Fumes + Additional) According to the CO2 Concentration CO2 Concentration 600 ppm 22

kg CO2 (m

21

year )

1000 ppm 22

kg CO2 (m

21

day )

22

kg CO2 (m

21

year )

kg CO2 (m22 day21)

Peppers

6.5

17.8  102

12.7

35  103

Tomatoes

6.1

16.8  103

12.7

35  103

Roses

6.7

18.3  103

12.7

35  103

Photsynthesis rate (mg CO2/m3/s)

2

300

1.8

250

1.6 1.4

200

1.2

150 100

1 0.8 0.6

50

0.4 0.2

Watt/m2

CO2 concentration (ppm) FIG. 6.2.1.2

Photosynthesis rate at different levels of illumination and CO2 concentration.

6.2.1.6 TYPICAL INSTALLATION CO2 is usually supplied into the circulating airstream from a liquid CO2 tank (18 bar pressure). The pipes used for CO2 distribution inside the greenhouse are those used for the CO2 stream coming from the fume recovery, whatever the heating or nonheating period. The CO2 passes

through a hand valve prior to injection directly into the ventilation air stream. In such a configuration, the injected liquid CO2 will produce a fine spray of solid CO2 snow and gas into the airstream. The warm air easily provides the heat necessary to ensure rapid sublimation of the snow particles. When the heater is switched off, a specific CO2 heater must be used to avoid any

6. AGRICULTURE

158

6.2. VEGETAL PRODUCTION

liquid CO2 encroachment in the airstream pipe causing possible material embrittlement. The most common method of CO2 distribution throughout the greenhouse is the use of a very lightweight tube made from a plastic film (for example, polyethylene) perforated at regular intervals to provide the necessary flow rates. These plastic film-forming tubes are lying on the floor at the bottom of the vegetable plants and rooting medium.

6.2.1.7 SAFETY The concentration of CO2 in the greenhouse atmosphere controlled from the normal atmospheric level of around 340 ppm up to around 2000 ppm is well below the 5000 ppm permissible exposure limit. The basic safety interlock is that CO2 cannot be injected unless the ventilation system is operating.

6.2.1.9 CONCLUSION AND PERSPECTIVES 6.2.1.9.1 Vertical Vegetal Greenhouses In the recent past, a few companies have been involved in closed growing equipment that integrate state-of-the art technologies: fluorescent lighting with an output wavelength optimized for vegetable growth, air-conditioning systems that maintain a constant temperature and moisture level, tracking control of growth, and sterilization systems for packing material. Strategic interests for these companies are probably based on LED lighting technology development appearing in that sector versus traditional lighting. Refer to Toshiba and Philips communication: https://www.toshiba.co.jp/about/press/ 2014_09/pr3001.htm http://www.lighting.philips.com/main/ products/horticulture On another side, a vertical greenhouse concept has been developed in the trend of urbanization of agriculture.

6.2.1.8 ECONOMICS In order to optimize the product yields, growers worked with research institutes and consultants to find the optimum economic conditions according to the costs of fuels and CO2 that can fluctuate depending of the periods versus the market selling price of the vegetable that is also subject to fluctuation at different periods of the year. The professionals did this atmosphere optimization like they did for the whole growing process, for example, the rooting medium, the hydroponic watering composition, the lighting, the variety selection, etc. There is no room for independent parameter evaluation, everything being linked. Practically, a breakeven assumption was estimated a few years ago with the following figures: fuel barrel equivalent price and CO2 price per ton both around 100€, meaning accurate management of combustion versus pure CO2 uses by the farmer.

6.2.1.9.2 Hygiene The plant factories described above in close to sterile conditions minimizing germ contamination and the consequences on food safety can contribute to extend the freshness and shelf life of vegetables, which is a major concern for retailers, convenience stores, and supermarkets. Could we observe in the future a more integrated production step for fresh-cut salad plants? The hydroponic nutrient flow brought to the culture is a source of contamination when recycled. Generally, it is steamed for sterilization before controlling the three macronutrients used by plants. These macronutrients are nitrogen (N), phosphorus (P), and potassium (K), NPK for short. Ozonation has been tested as an alternative solution with an onsite generator. See the related chapter on ozone sanitation.

6. AGRICULTURE

6.2.1.9 CONCLUSION AND PERSPECTIVES

159

FIG. 6.2.1.3 Homogeneous blooming can be obtained with ethylene boosting during the ripening step of tomato growth.

6.2.1.9.3 Ripening of Tomatoes Recent European legislation banned the use of ethephon (C2H6ClO3P), a plant growth regulator promoting fruit ripening, abscission, and flower induction as well as generating ethylene used at the last step in the ripening of tomatoes before picking. Tests regarding replacement by gaseous ethylene are being validated and legal approval is being sought through some technical

centers, growers, and universities, mostly in Belgium (Fig. 6.2.1.3).

6.2.1.9.4 Influence of Other Gases Complete gas control management in a perspective of growing optimization points out a last parameter concerning oxygen content in hydroponic solutions with some encouraging results on some varieties.

6. AGRICULTURE

S U B C H A P T E R

6.2.2 Controlled Atmospheres for Fruit and Vegetable Storage and Ripening Tongchai Puttongsiri, Anthony Keith Thompsona King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand

6.2.2.1 INTRODUCTION The postharvest effects of gases on fresh fruits and vegetables was first referred to as “gas storage” (Kidd and West, 1927), but in the 1930s, that was changed to “controlled atmosphere (CA) storage”. The scientific basis for the effects of gases during the storage of fresh fruit and vegetables probably began at the University of Montpellier in France in 1819 (Berard, 1821). They found that fruit absorbed O2 and gave out CO2 and showed that climacteric fruit stored in atmospheres containing no O2 did not ripen, but if they were held for only a short period and then placed in air they continued to ripen. In the 1860s, Benjamin Nyce experimented in the United States with modifying the atmosphere in an apple store by making it airtight. Franklin Kidd and Cyril West carried out experiments in the early 20th century on CA storage and by 1920 were able to set up trials at a farm in the United Kingdom to test their laboratory findings in small-scale commercial practice. In 1929, a commercial CA store for apples was built by a grower in the United Kingdom. By 1938, there were more than 200 commercial CA stores for apples in the a

United Kingdom (Thompson, 2010). Currently, CA storage is increasingly used on an increasing number of fresh fruits and vegetables in an increasing number of countries. An enormous number of papers have been published in the scientific literature on postharvest science and technology; some journals are even exclusively dedicated to the subject. This chapter attempts to review the role of gases in the postharvest science and technology of fresh fruits and vegetables.

6.2.2.2 PRESTORAGE EFFECTS The condition of fruits and vegetables before being put into store can have a profound effect on their postharvest life. Harvest maturity, especially with climacteric fruit such as apples, needs to be specified before harvesting them for longterm storage. Some cultivars are known to have a longer storage life than others and the responses of different crops to levels of O2 and CO2 may vary considerably. Also, some chemical treatments can be applied to them to control diseases and physiological disorders while rendering them less sensitive to ethylene.

Formally: Cranfield University, Cranfield, United Kingdom.

6. AGRICULTURE

6.2.2.3 CONTROL OF GASES IN CA STORES

6.2.2.3 CONTROL OF GASES IN CA STORES The optimum levels O2, CO2, and other gases, especially ethylene and water vapor, vary between different types of fruits and vegetables and even between cultivars and areas of production. These have been determined to some degree and data is presented for some in many publications, for example Thompson (2010). Many recommendations may not reflect the optimum conditions but rather the most suitable conditions in relation to the technology available at the time. Development of technology that enables O2 levels to be maintained safely at very low levels has led to improved conditions in storage, especially ultralow oxygen (ULO) storage, that is, O2 levels of 1% or less. The first experiments in the early part of the 20th century on the effects of gases on fresh fruits and vegetables used metal cylinders of compressed gas that had been made up with the various proportions of the gases to be studied. Subsequently, gas levels in commercial CA stores were measured by frequently extracting a sample of the store air, analyzing it in an Orsat gas analyzer or other equipment, and adjusting the levels manually. This method was clearly laborious and did not permit sufficient accuracy in controlling the gas levels in the store. Currently, the atmosphere in many modern CA stores is regularly analyzed automatically for CO2 levels, using an infrared gas analyzer and O2 levels with analyzers using electrochemical cells or sometimes a paramagnetic analyzer. The analyzers are monitored and controlled by a computer, which can then automatically adjust their levels. This method is based on prior knowledge of the optimum gas combination that suits the type and cultivar of fruit or vegetable to be stored. Subsequently, systems and equipment have been developed that use the physiological responses of the fruit or vegetable to determine the optimum gas level and also to control it. Yearsley et al. (1996) concluded that determining lower

161

O2 limits on the steady-state internal atmosphere of the apples estimated the true lower O2 limit more accurately than those estimated from the store atmosphere. Equipment to determine the chlorophyll fluorescence (CF) and respiratory quotient (RQ) have been developed to measure physiological responses for this purpose. Temperature interacts with gas levels on the effects on fruits and vegetables during storage. Kidd and West (1927) reported that there is evidence that CA storage is only successful when applied at low temperatures. Standard refrigeration units are therefore integral components of CA stores. In commercial practice for long-term CA storage, the store temperature is initially reduced to 0°C for a week or so, whatever the subsequent storage temperature will be. This would clearly not be applicable to fruits and vegetables that can suffer from chilling injury at relatively high temperatures (10–13°C). Also, CA stores are normally designed to a capacity that can be filled in 1 day, so fruits are loaded directly into the store and cooled the same day. In the United Kingdom, the average CA store size was given as about 100 tons with variations between 50 and 200 tons, in continental Europe about 200 tons, and in North America about 600 tons (Bishop, 1996). In the United Kingdom, the smaller rooms are preferred because they facilitate the speed of loading and unloading.

6.2.2.3.1 Water Vapor Most fruits and vegetables require high humidity when kept in storage. Generally, the closer the humidity is to saturation the better, so long as moisture does not condense on the fruit or vegetables as this might result in disease infection. A major reason for comparatively low humidity developing in a store is that the cooling coils on the refrigeration unit are set at too high a temperature differential from the required store air. This results in condensation on the cooling coils that can cause desiccation

6. AGRICULTURE

162

6.2. VEGETAL PRODUCTION

from the fruit or vegetables. Evaporator coil refrigerant temperature, store humidity, cooldown period, and mass loss rates in a commercial 1200 bin apple store were studied by Hellickson et al. (1995). They reported that stores in which the evaporator coil refrigerant temperatures were dictated by cooling demand resulted in a significantly longer time required to achieve the desired humidity than stores in which the evaporator coil temperatures were controlled by a computer. A whole range of humidifying devices can also be used to increase the humidity in the store, including spinning disc and sonic humidifiers. In a laboratory experiment, Dijkink et al. (2004) described a system that could maintain humidity very precisely in 500-L containers. They were able to maintain 90.5  0.1% RH using a hollow fiber membrane contactor and a liquid desiccant.

contact equally with all the fruit in the room. At the same time, the CO2 given off by the fruit, which can impede ripening initiation, is not allowed to concentrate around the fruit. The application of ethylene can be as a liquid, for example, Ethrel, which is sprayed on fruit and hydrolyzed to produce ethylene. Gas cylinders containing ethylene under pressure are also used. Typical mixtures are 95% N2 and 5% ethylene. The method of application was to meter the gas into the ripening room containing the fruit through a pipe. Currently, ethylene generators are most commonly used (Fig. 6.2.2.1). These are devices that give a slow release of ethylene over a protracted period, commonly 16 h. The generators are used by placing them in the ripening room, pouring a liquid provided by the manufacturer into a container within the generator, and plugging the generator into an

6.2.2.3.2 Ethylene Ethylene is synthesized by plant cells. In climacteric fruit, the biosynthesis of ethylene initiates ripening. Ripening rooms have been used for climacteric fruit (especially bananas) that are harvested in a preclimacteric state and subsequently placed in a ripening room and exposed to ethylene gas under controlled conditions. They are also used for degreening citrus fruits. The primary requirements for ripening rooms are that they should have a good temperature control system, have good and effective air circulation, be gas tight, and have a good system for introducing fresh air and ethylene gas. Over the past few decades, in some countries there has been an increasing demand for all the fruit being offered for sale in a supermarket to be of exactly the same stage of ripeness so that it has an acceptable and predictable shelf life. This has led to the development of a system called “pressure ripening.” The system involves the circulating air in the ripening room being channeled through boxes of fruit so that exogenous ethylene gas, which initiates ripening, is in

FIG. 6.2.2.1 ripening.

6. AGRICULTURE

Catalytic ethylene generator used in fruit

6.2.2.3 CONTROL OF GASES IN CA STORES

electrical supply. The liquid is probably ethanol because if ethanol is heated in the presence of a copper catalyst, ethylene gas is produced. Ethylene can also have negative effects on fresh fruits and vegetables, especially when accumulated in stores. For example, Wills et al. (1999) showed that the postharvest life of some nonclimacteric fruit and vegetables could be extended by up to 60% when stored in an atmosphere containing less than 5 parts per billion ethylene compared with those stored in 100 parts per billion. Solomos and Biale (1975) showed that exposure to ethylene during storage results in an increased respiration rate (Table 6.2.2.1). 1-Methylcyclopropene (1-MCP) is used commercially to slow the ripening of fruits in store. Its mode of action involves 1-MCP tightly binding to the ethylene receptor sites in plant cells, thereby blocking the effects of ethylene by inhibiting the activities of ACC (1-aminocyclopropane-1carboxylic acid). This also delays the peaks in the ACC synthase activity and ACC concentration TABLE 6.2.2.1 Effects of Ethylene on Respiration Rate of Selected Crops Respiration Rate (μL O2 g21 h21) Crop

Control

Ethylene

Apple

6

16

Avocado

35

150

Beet

11

22

Carrot

12

20

Cherimoya

35

160

Grapefruit

11

30

Lemon

7

16

Potato

3

14

Rutabaga

9

18

Sweet potato

18

22

163

as well as gene expression of enzymes and of ethylene receptors at the transcript level (Ma et al., 2009). N-dimethylaminosuccinamic acid (DPA, Daminozide, Alar, B9, or B995) is a plant growth regulator. Treated apples were shown to be less sensitive to ethylene than nontreated apples during storage, but this response varied between cultivars (Knee and Looney, 1990). However, it has been withdrawn from the market in several countries because of suggestions that it might be carcinogenic. Aminoethoxyvinylglycine (AVG) is used in apple orchard sprays to inhibit their ethylene biosynthesis. Its mode of action is to inhibit the activity of ACC-synthase. McIntosh apples sprayed with AVG were shown to have a delayed onset of the climacteric during subsequent storage (Robinson et al., 2006). There are various ways in which ethylene can be removed from stores, including absorption, reaction, ozone scrubbers, and catalytic converters. Filters are available commercially in several forms that can be placed in the air circulation system. These contain an active alumina carrier impregnated with potassium permanganate that oxidizes the ethylene (Fig. 6.2.2.2). Catalytic converters remove ethylene by chemical reaction. Air from the store is passed through a device (Fig. 6.2.2.3) where it is heated to more than 200°C in the presence of an appropriate catalyst, usually platinum (Wojciechowski, 1989).

6.2.2.3.3 Carbon Dioxide

Adapted from Solomos, T., Biale, J.B., 1975. Respiration in fruit ripening. Colloq. Int. C. N. R. S. 238, 221–228.

When CO2 levels in store are too high, fruit or vegetables can be damaged. The optimum CO2 level in store varies for different crops and situations (Thompson, 2010). There are many different types of scrubbers that can remove CO2 from CA stores, but they can basically be divided into two types. One uses a chemical that reacts with CO2 and thus removes it from the store; this is sometimes called “passive scrubbing.” The other is renewable, sometimes called “active scrubbing.” Passive scrubbing is where bags or pallets, usually calcium hydroxide, are placed

6. AGRICULTURE

164

6.2. VEGETAL PRODUCTION

FIG. 6.2.2.2 Ethylene-absorbing potassium permanganate granules, (A) a filter blanket containing granules (B). Reproduced with permission of Molecular Products Limited.

FIG. 6.2.2.3 Portable Tubamet Swingcat ethylene scrubber in use in a wholesale packhouse in the United Kingdom in the 1990s.

inside the store, where they can keep CO2 levels low (usually about 1%). For greater control, the bags or pallets of lime may be placed in a separate airtight small room. When the CO2 level in the store is above that which is required, a fan draws the store atmosphere through the room containing the bags of lime until the required level is reached. In passive scrubbers, the time taken for the levels of these two gases to reach the optimum (especially for the O2 to fall from 21 kPa in fresh air) can reduce the maximum storage life of the crop. It is common therefore to fill the store with the crop, seal it, inject N2 gas until the O2 has reached the required level, and then maintain it in the way described above. The N2 may be obtained from large liquid N2 cylinders or from N2 generators (Fig. 6.2.2.4). Active scrubbers use molecular sieves and activated carbon that can hold CO2 and organic molecules such as ethylene. When fresh air is passed through these substances, the molecules are released. This means that they can be used in a two-stage system where the store air is being passed through the substance to absorb the CO2 and ethylene while the other stage is being cleared by the passage of fresh air. After an appropriate period, the two stages are reversed. Hydrated aluminum silicate or aluminum calcium silicate is used. The regeneration of the

6. AGRICULTURE

6.2.2.3 CONTROL OF GASES IN CA STORES

FIG. 6.2.2.4

165

Nitrogen generator used to reduce the O2 level in CA stores.

molecular sieve beds can be achieved when they are warmed to 100°C to drive off the CO2 and ethylene. This system of regeneration is referred to as “temperature swing” where the gases are absorbed at low temperature and released at higher temperature. Regeneration can also be achieved by reducing the pressure around the molecular sieve, which is called “pressure swing.” During the regeneration cycle, the trapped gases are usually ventilated to the outside. Burdon et al. (2005) reported that the method of CO2 control may affect the volatile composition of the room atmosphere, which in turn may affect the volatile content of fruit. They compared activated carbon scrubbing, hydrated lime scrubbing, N2 purging, and storage in air on kiwi stored at 0°C in 2 kPa O2 + 5 kPa CO2. After storage, the fruits were allowed to ripen at 20°C and the volatile profiles differed between CA stored and air stored fruits, and also among fruits from the different CO2 scrubbing systems. However, the different CO2 scrubbing systems did result in measurable differences in ripe fruit volatile profiles.

6.2.2.3.4 Oxygen The way that traditional CA storage systems were operated was that the rooms were sealed and when the O2 reached the level required for the particular crop through respiration, it was maintained at that level by frequently introducing fresh air from outside. Sharples and Stow (1986) reported that tolerance limits were set at 0.15% for O2 levels below 2%, and 0.3% for O2 levels of 2% and above. With continuing equipment development, the precision with which the set levels of CO2 and O2 can be maintained is increasing. With recent developments in the control systems used in CA stores, it is possible to control O2 levels close to the theoretical minimum. This is because modern systems can achieve a much lower fluctuation in gas levels, and ULO storage (levels around 1%) is now common. If the O2 level in CA stores is too low, the type of respiration of the fruit can change from aerobic respiration to fermentation, producing various volatiles including acetaldehyde, ethyl acetate,

6. AGRICULTURE

166

6.2. VEGETAL PRODUCTION

and ethanol. Considerable advantages have been shown in storing fruit as close as possible to that low O2 point, called the anaerobic compensation point (ACP) (Bishop, 1996; Prange et al., 2014). North and Cockburn (1975) describe detection for ethanol in CA stores using indicator tubes that could be connected to an alarm system so that when fermentation was just beginning, the O2 level could be slightly increased to prevent the fruit from being damaged. This technology was subsequently developed and Schouten et al. (1997) described a system that he called “dynamic control of ultra low O2 storage” based on headspace analysis of ethanol levels that were maintained at less than 1 μL L1 where O2 levels were maintained at 0.3–0.7 kPa in the store. This method of monitoring and control is called “dynamic controlled atmosphere” (DCA) storage (Prange et al., 2014). Therefore DCA uses the responses of actual fruits or vegetables being stored as monitors of the store atmosphere instead of an arbitrary setting of the O2 and CO2 levels in the store that had been based on previous experience and experimentation. The ACP is the critical internal O2 concentration where the metabolism of the fruit or vegetable reaches a level that results in fermentation. Two methods of detecting the ACP were described by Gasser et al. (2008) based on RQ and chlorophyll fluorescence (CF) signal monitoring, which were used to detect the critical O2 concentration during DCA storage. DCA storage under a stress atmosphere maintained a flat fluorescence (Fα) baseline while those with lower O2 produced an Fα spike. O2-induced Fα shifts occurred quickly at the lower O2 limit of the fruit and were measurable but returned quickly to the prestressed level when the O2 level was raised above the lower O2 limit (Prange et al., 2002, 2003). A commercial technology called HarvestWatch based on CF measurement of stress, which occurs when there is insufficient O2 for aerobic metabolism, has been developed, commercialized, and patented. Prange et al. (2014)

TABLE 6.2.2.2 The Effects of Time in Storage on the Lower Oxygen Limit Detected by Dynamic Controlled Atmosphere-Chlorophyll Fluorescence (DCA-CF) on Four Apple Cultivars Lower O2 Limit (kPa) Apple Cultivar

October 10–19

December 1–4

Delicious

0.85

0.47

Golden Delicious

0.92

0.45

Honeycrisp

0.90

0.50

Empire

0.90

0.88

Adapted from Prange, R.K., Wright, A.H., DeLong, J.M., Zanella, A., 2014. History, current situation and future prospects for dynamic controlled atmosphere (DCA) storage of fruits and vegetables, using chlorophyll fluorescence. Acta Hortic. 1012, 905–915.

used DCA-CF to calculate the lower O2 limit for apples and showed that this varied between cultivars and was also reduced considerably during storage (Table 6.2.2.2). Another method developed for ULO storage is Safepod, which has also been patented and commercialized. Yearsley et al. (1996) investigated the internal levels of concentrations of acetaldehyde, ethyl acetate, and ethanol in stored apples as a means of determining their ACP and fermentation threshold in order to ascertain their optimum CA conditions. They concluded that determining lower O2 limits on the steady-state internal atmosphere of the apples estimated the true lower O2 limit more accurately than those estimated from the store atmosphere. They considered, therefore, that the fermentation threshold RQ represented the safest estimate of the true lower O2 limit for optimizing storage atmospheres.

6.2.2.3.5 HarvestWatch HarvestWatch is commercial CA technology based on CF measurement of stress that has been patented and commercialized. Isostore is software that incorporates the HarvestWatch signals, CO2, O2, and temperature data into a

6. AGRICULTURE

6.2.2.3 CONTROL OF GASES IN CA STORES

167

FIG. 6.2.2.5 Chlorophyll fluorescence (HarvestWatch) showing the system (A) and individual kennels with avocados readily prepared to be placed in the store (B). Reproduced with permission of Robert Prange.

single real-time display. Samples of fruit are placed in plastic boxes called “kennels” that are placed among the fruit to be stored (Fig. 6.2.2.5). Each kennel has appropriate monitoring equipment that is linked to a computer for monitoring and control of O2 levels in the store. CF is light reemitted by chlorophyll molecules during return from excited to nonexcited states (DeEll et al., 1999). Prange et al. (2003) reported that “a new chlorophyll fluorescence (F) sensor system called FIRM (fluorescence interactive response monitor) was developed that measures F at low irradiance. This system can produce a theoretical estimate of Fo at zero irradiance for which they have coined a new fluorescence term, Fα. The ability of Fα to detect fruit and vegetable low-O2 stress was tested in short-term (4-day) studies on chlorophyllcontaining fruit. In all of these fruit and vegetables, Fα was able to indicate the presence of low-O2 stress. As the O2 concentration dropped below threshold values of 0 to 1.4 kPa, depending on the product, the Fα value immediately and dramatically increased. At the end of the short-term study, O2 was increased above the threshold level, whereupon Fα returned to approximately pre-stressed values.” Prange et al. (2003) reported a study with apples stored at 0.9, 0.3, or 1.5 kPa O2 for 9 months using

HarvestWatch. The apples stored at 0.9 kPa had the highest firmness, lowest concentration of fermentation volatiles, and lowest total disorders. Sensory ratings for off-flavor, flavor, and preference indicated no discernible differences among the three O2 storage conditions. Burdon et al. (2008) reported that CF in avocados remained constant at 0.8 at 6°C in O2 levels down to 1 kPa. However, below that O2 level, the CF rapidly dropped to 0.68 within 24 h. When the fruit was kept for 6 days, then returned to a nonstressed atmosphere, the CF rapidly returned to 0.8. They also found that after DCA storage, using CF, the avocados ripened in 4.6 days at 20°C compared with 7.2 days for “static” CA stored fruit and 4.8 days for fruit stored throughout in air.

6.2.2.3.6 Safepod Storage Control Systems Ltd. developed Safepod (US Patent 8739694, Canadian Patent CA2746152). Each Safepod holds around 60– 70 kg of fruit. It is exposed to the same storage conditions as the rest of the store but is isolated from the store’s atmosphere at regular intervals. The control is based on stress detection on the 60–70 kg representative sample of fruit. The Safepod sits in the CA store and thus has the same temperature, humidity, pressure, and

6. AGRICULTURE

168

6.2. VEGETAL PRODUCTION

FIG. 6.2.2.6 Safepod unit (A) and Safepod ready for loading into a ULO store (B). Reproduced with permission of David Bishop, Storage Control Systems Ltd.

atmosphere as the store (Fig. 6.2.2.6). Periodically, the valves are closed and the fruit inside is then tested for CO2 produced and O2 consumed and its RQ is calculated using Safepod software. The RQ is then used as a basis to make the necessary small adjustments to the overall conditions of the store (Thompson and Bishop, 2016). Wollin et al. (1985) had previously discussed the possibility that RQ may be used to calculate the lowest O2 level that can be tolerated in fruit storage to be incorporated in an automated CA system. RQ is the measure of moles CO2 evolved to moles O2 absorbed in plant cells. It is 1 when the substrate in carbohydrate but lower for lipids and proteins. Burton (1952) measured RQ in potatoes stored at 10°C in 5–7 kPa CO2 for up to 14 weeks. The increased CO2 reduced both O2 uptake and CO2 output by about 25%–30%, but the RQ was unaffected and remained close to 1. Bessemans et al. (2016) described DCA storage to control O2 and CO2 in storage containers for apples, based on measurements of RQ. Ethanol concentrations in the fruit were found to be very low (<0.028 g L1), indicating the RQ-DCA system managed to store the fruit close to the ACP. Also, the fruit metabolism did not shift from aerobic respiration to fermentation. However, during RQ-DCA storage, the RQ breakpoint was

found to vary between 0.25 and 0.4 kPa O2, and there were difficulties in obtaining a reliable RQ measurement at very low O2 levels. This was due to reduced fruit metabolism or when the pressure of the storage container increased due to increased atmosphere pressure as a result of leakage in the storage container.

6.2.2.3.7 Other Methods Hypobaric and hyperbaric conditions have also been evaluated in relation to the control of gases in fruit and vegetable stores; their effect is primarily on O2 content. They are not included in this chapter but were reviewed by Burg (2004) and Thompson (2015).

6.2.2.4 INTERRUPTED CA STORAGE Where CA storage has been shown to have detrimental side effects on fruits and vegetables, the possibility of alternating CA storage with air storage has been studied. Results have been mixed with positive, negative, and in some cases no affect. Anderson (1982) described experiments where peaches and nectarine were stored at 0°C in 5 kPa CO2 + 1 kPa O2, which was

6. AGRICULTURE

6.2.2.5 DELAYED CA STORAGE

interrupted every 2 days by removing the fruit to 18–20°C in air. When subsequently ripened, fruits from this treatment had little of the internal breakdown found in fruits stored in air at 0°C. Intermittent exposure of Haas avocados to 20 kPa CO2 increased their storage life at 12°C and reduced chilling injury during storage at 4°C compared to those stored in air at the same temperatures (Marcellin and Chaves, 1983). Parsons et al. (1974) stored tomatoes at 13°C in 3 kPa O2 + 0, 3 or 5 kPa CO2, which was interrupted each week by exposing them to air for 16 h. This interrupted storage had no measurable effect on the storage life of the fruit but increased the level of decay that developed on fruit when it was removed from storage to higher temperatures to simulate shelf life.

6.2.2.5 DELAYED CA STORAGE In some cases, a delay in establishing CA conditions can be advantageous. Watkins et al. (1997) showed that the sensitivity of Braeburn apples to CO2-induced injury declined and skin disorders in Empire apples could be reduced when fruits were held for a period in air before CA was established. Streif et al. (2003) found that flesh browning in Elstar apples was reduced when the establishment of the CA conditions was delayed for up to 40 days compared to establishment immediately after harvest. However, there is a balance that needs to be made because the delays can have negative effects. For example, Argenta et al. (2000) found that delaying establishment of CA conditions for up to 12 weeks significantly reduced the severity of brown heart in Fuji apples, but resulted in softer, less acidic fruit compared to the establishment of 1.5 kPa O2 + 3 kPa CO2 directly after harvest. Wang et al. (2000) also found that CO2 linked disorders were reduced in apples by storage at 3°C in air before CA storage, but excessive ripening and associated loss of flesh firmness occurred. Drake and Eisele (1994) found that

169

immediate establishment of CA conditions of apples after harvest resulted in good quality fruits after 9 months storage. Reduced quality was evident when CA establishment was delayed by as little as 5 days, even though the interim period was in air at 1°C. However, softening was quicker than in fruits where CA conditions were established directly after harvest. Streif et al. (2003) found that the firmness loss was much higher in Elstar apples when CA conditions were established directly after harvest. Colgan et al. (1999) also showed that apple scald was controlled less effectively when establishment of CA conditions was delayed. Landfald (1988) recommended prompt CA storage to reduce the incidence of lenticel rot in Aroma apples. Watkins and Nock (2012) investigated whether treatment with 1-MCP could counter the effects of delayed application of CA storage to apples. 1-MCP suppressed the internal ethylene concentrations of several apple cultivars during the14-day period before CA conditions were established, but the extent of the suppression was lower in fruit with high internal ethylene concentrations at harvest. 1-MCP treatment resulted in firm fruit after delayed CA up to 14 days, but the most consistent effects were found in the cultivar Empire, which had lower initial ethylene concentrations than McIntosh. Delaying CA storage has been shown to affect crops other than apples. With nectarines (Prunus persica), Zhou et al. (2000) found that both delayed storage for 48 h at 20°C before storage and CA storage at 10 kPa CO2 + 3 kPa O2 alleviated or prevented chilling injury, which can result in woolliness, during storage for 4 or 6 weeks at 0°C. Control fruits showed 80% woolliness after 4 weeks at 0°C and 100% after 6 weeks. Delayed storage and CA were similar in their beneficial effect after 4 weeks, but CA was better after 6 weeks storage. Tonini and Tura (1997) showed that storage of kiwi in 4.8 kPa CO2 + 1.8 kPa O2 reduced rot caused by infections with Botrytis cinerea, but the reduction

6. AGRICULTURE

170

6.2. VEGETAL PRODUCTION

was greatest the quicker the CA storage conditions were established. With a delay of 30 days, the CA storage conditions were ineffective in controlling the rot. Gadalla (1997) found that CA storage of onion bulbs was most effective when applied directly after curing, but a delay of up to one month was almost as good.

6.2.2.6 RESIDUAL EFFECTS OF CA STORAGE Storing fruits and vegetables in CA conditions can affect their subsequent marketable life. Day (1996) indicated that minimally processed fruit and vegetables stored in 70 kPa and higher O2 levels deteriorated more slowly on removal, even more so than those that had been freshly prepared. Bell peppers exposed to 1.5 kPa O2 for 1 day exhibited suppressed respiration rates for at least 24 h after transfer to air (Rahman et al., 1993a). This residual effect after CA storage has been shown for minimally processed fruit and vegetables (Day, 1996), cabbages (Berard, 1985), avocados (Burdon et al., 2008), and raspberries (Goulart et al., 1990). Mencarelli et al. (1983) showed that storage of courgettes in low O2 reduced chilling injury, but on removal to air storage at the same chilling temperature, the protection disappeared within 2 days. This effect has also been shown for climacteric fruit. For example, Palma et al. (1993) stored cherimoya fruit (Annona cherimola Mill.) at 10°C in 5, 10, or 20 kPa O2 for 30 days. On removal to 20°C in air, those that had been stored in 5 kPa O2 took 11 days to ripen, those that had been stored in 10 kPa O2 took 6 days, and those that had been stored in 20 kPa O2 ripened in 3 days. Hardenburg et al. (1977) showed that apples stored in CA for 6 months and then for 2 weeks at 21°C in air were firmer, more acidic, and had a lower respiration rate than those that had previously been stored in air. Khanbari and Thompson (1996) stored potatoes in various CA combinations at 5°C for 25 weeks

and found that there was almost complete sprout inhibition, low weight loss, and maintenance of a healthy skin for all cultivars stored in 9.4 kPa CO2 + 3.6 kPa O2. When tubers from this treatment were subsequently stored in air at 5°C for a further 20 weeks, the skin remained healthy and they did not sprout while the tubers that had been previously stored in air or other CA combinations sprouted quickly. Bananas, which had been initiated to ripen by exposure to exogenous ethylene and then immediately stored in 1 kPa O2 at 14°C, remained firm and green for 28 days but then ripened quickly when transferred to air at 21°C (Liu, 1976). However, Wills et al. (1982) showed that preclimacteric bananas exposed to low O2 took longer to ripen when subsequently exposed to air than fruits kept in air for the whole period. Imahori et al. (2013) also showed similar effects when mature green bananas were stored in 0.5, 2, or 21 kPa O2 for 7 days at 20°C before ripening was initiated by ethylene, also at 20°C. Fruit that had been in low O2 showed delayed onset of the climacteric peak but had a lower production of ester volatiles. The activity of alcohol dehydrogenase significantly increased in 0.5 kPa O2, but in 2 and 21 kPa O2, it remained very low throughout the storage period. Production of ethyl acetate, isoamyl acetate, and isobutyl acetate were suppressed by low O2 storage. In storage of onion bulbs at 0°C in 1, 3, or 5 kPa O2 + 0 or 5 kPa CO2, sprouting was reduced, but those combinations that included 5 kPa CO2 were the most effective. Also the residual effects of CA storage on sprouting were still effective after 2 weeks at 20°C. Most of the cultivars stored in 1 kPa O2 + 5 kPa CO2 and some cultivars stored in 3 kPa O2 + 5 kPa CO2 were considered still marketable after 9 months storage (Gadalla, 1997). Storage of capsicums for 5 days at 20°C in 1.5 kPa O2 resulted in poststorage suppression of their respiration rate for about 55 h after transfer to air and a marked reduction in the oxidative capacity of isolated mitochondria. Mitochondrial activity in jackfruit (Artocarpus heterophyllus L.)

6. AGRICULTURE

6.2.2.7 EFFECTS OF CA STORAGE

was suppressed for 10 h after transfer to air, but within 24 h had recovered to levels comparable to those of mitochondria from fruits stored continuously in air (Rahman et al., 1993b, 1995).

6.2.2.7 EFFECTS OF CA STORAGE 6.2.2.7.1 Phytochemicals The effects of CA storage on the phytochemical content of fresh fruits and vegetables have shown variable results. Awad and de Jager (2003) studied the effects of storage on the flavonoid content of two apple cultivars and found no losses during long-term storage in air or CA or during subsequent shelf life. Matityahu et al. (2016) compared storage of pomegranates at 7°C in air with those in 2 kPa O2 + 5 kPa CO2. CA-stored fruit were in better quality than those stored in air, but storage in air was better at maintaining the anthocyanin level and in preventing the occurrence of off-flavors. Bekele et al. (2016) stored Jonagold apples in 1 kPa O2 + 3 kPa CO2, 3 kPa O2 + 3 kPa CO2, 1 kPa O2 + 10kPa CO2 or in air. Aspartate and 1-aminocyclopropane-1-carboxylic acid were positively correlated with O2 concentration during the first 2 days and after one week of storage while glucose-6-phosphate and some amino acids such as proline, alanine, and threonine were negatively correlated with O2 concentration. Glutamate and succinate were correlated with CO2 concentration. Galactinol substantially increased with storage time. Harbaum-Piayda et al. (2010) stored Pak choi (Brassica rapa var. chinensis L.) in 1.5–2.5 kPa O2 + 5–6 kPa CO2 or air at 2°C and 99% RH. The level of flavonoids increased more in CA storage than in air, but hydroxycinnamic acids and chlorophyll content were unaffected or changed only marginally. Storage of broccoli in air showed that the keeping quality for the four most important glucose inolates was between 4 and 7 days while they kept for at least 14 days in broccoli stored in

171

1.5 kPa O2 + 15kPa CO2 (Schouten et al., 2009). Glucosinolates and their isothiocyanate hydrolysis products can protect against the development of cancer. Selcuk and Erkan (2015) stored Medlars (Mespilus germanica L.) in air, modified atmosphere packing, and CA of 2 kPa O2 + 5 kPa CO2 or 3 kPa O2 + 10 kPa CO2 at 0°C for 60 days. Total phenolic concentrations, total flavonoid concentrations, antioxidant activity, ascorbic acid retention, and total condensed tannin retention were the highest in 2 kPa O2 + 5 kPa CO2. Bartlett pears were stored at +1°C or 1°C under ULO of 0.8 kPa O2 + < 0.5 kPa CO2. ULO at 1°C suppressed the synthesis of esters, methyl and ethyl (2E,4Z)deca-2,4-dienoate, and hexyl acetate while ethyl acetate synthesis was suppressed only by ULO at 1°C. The levels of most aroma volatiles recovered after 10 days subsequent shelf life, although with significantly lower recovery for methyl and ethyl (2E,4Z)-deca-2,4-dienoate in fruit from the ULO storage. Although synthesis of aroma volatiles was most suppressed under ULO at 1°C, butyl and hexyl acetate levels recovered better in fruit under ULO storage at 1°C than at +1°C (Zlati
c et al., 2016).

6.2.2.7.2 Sensory Characteristics Martins and de Resende (2015) stored papaya at 13°C for 20 days in air or atmospheres containing 1 or 3 kPa O2 with 6 or 12 kPa CO2, with ethylene scrubbing, followed by 6 days at 23°C in air for ripening. Storage in 12 kPa CO2 negatively affected the sensory attributes of the fruit after ripening. The optimum condition that best preserved the sensory attributes was in an atmosphere of 3 kPa O2 + 6 kPa CO2. Delate and Brecht (1989) showed that storage of sweet potatoes to 2 kPa O2 + 60 kPa CO2 resulted in less sweet potato flavor and more off-flavor than those stored in air. Latocha et al. (2014) stored kiwi at 1°C and 85% RH in air or 1.5 kPa O2 + 1.5 kPa CO2 for 4 and 8 weeks. They found that storage in air was adequate for up to 4 weeks but those in CA could be stored for 8 weeks. The

6. AGRICULTURE

172

6.2. VEGETAL PRODUCTION

sensory characteristics of fruit stored in CA and then ripened during simulated shelf life were similar to those of fruit ripened on the plant. The most significant negative change in the sensory characteristics of fruit after long-term cold storage was the increase in the intensity of the bitter taste. CA storage of onion bulbs in either 2 kPa CO2 + 2 kPa O2 or 8 kPa CO2 + 2 kPa O2 reduced pungency and flavor by reducing both flavor precursors and enzyme activity compared to storage in air. The storage conditions were 0.5  0.5°C and 80  3% RH for 9 weeks (Uddin and MacTavish, 2003).

6.2.2.7.3 Disorders The Braeburn browning disorder of apples appeared to develop during the first 2 weeks of storage, and storage in air at 0°C prior to CA storage decreased both its incidence and severity (Elgar et al., 1998). Lum et al. (2016) reported that CA storage can promote the development of injury, which can be exacerbated by 1-MCP. Honeycrisp apples were treated with or without 1 μL L1 1-MCP for 24 h and then transferred to 2.5 kPa O2 + 2.5 kPa CO2 or 2.5 kPa O2 + 0.03 kPa CO2 at 3°C with no conditioning or conditioned at 10°C for 5 days prior to CA for up to 35 weeks. CA-related injury occurred with storage in both CA regimes, regardless of 1-MCP and conditioning, whereas 1-MCP exacerbated the negative impact of elevated CO2. Prestorage conditioning reduced the negative impact of 1-MCP on the incidence of CA-related injury in 2.5 kPa CO2. CA-related injury was strongly associated with changes in γ-aminobutyrate and moderately linked to total glutathione and glutathione redox status. Forney et al. (2015) stored raspberries at 1°C and 95% RH in air or 12.5 kPa CO2 + 7.5 kPa O2. CA storage strongly suppressed fruit decay but loss of fruit firmness was similar during storage in air and CA. Guavas of the cultivar Pedro Sato were stored at 12.2°C for up to 28 days in 5 kPa O2 combined with 1, 5, 10, 15, or 20 kPa CO2. CO2 injury occurred in fruit stored in 15kPa CO2 and 20kPa CO2 after 28 days with

increasing softening, pH values, and soluble pectin content. From their results they recommended that they should be stored in atmospheres with 5 kPa O2 and no more than 5 kPa CO2 (Teixeira et al., 2016).

6.2.2.7.4 Pests CA storage has been used to control insects and may be applicable to quarantine control of fruit flies and other insects in international trade in fruit and vegetables. Yahia and Kushwaha (1995) reported that Hass avocados, Sunrise papayas, and Keitt mangoes tolerated low O2 (0.5 kPa) and/or very high CO2 (50 kPa) for 1, 2, and 5 days, respectively, at 20°C. They purported that these treatments could have some potential use as insecticidal atmospheres for quarantine insect control on the basis of fruit tolerance, insect mortality, and costs. On stored vegetables, Cantwell (1995) also showed that various combinations of O2, CO2, and temperature were effective in achieving complete insect kill before the development of phytotoxic symptoms. Sweet potatoes were exposed by Delate and Brecht (1989) to low O2 and high CO2 for 1 week during curing or subsequent storage to evaluate the effect on the weevil Cylas formicarius elegantulus. Exposure to levels required for insect control was found not to be feasible during curing, but cured sweet potatoes could tolerate CA that has a potential as a quarantine procedure. Exposure of cured sweet potatoes to 2 or 4 kPa O2 + 40 kPa CO2, or 4 kPa O2 + 60kPa CO2 for 1 week at 25°C had little effect on postharvest quality but exposure to 2 kPa O2 + 60kPa CO2 resulted in increased decay and a reduction in flavor and more off-flavor. In a study by Liu (2013) of various CA treatments, complete mortality of nymphs, adults, and eggs of grape mealybug (Pseudococcus maritimus Ehrhorn) was achieved in 10 days in 50 kPa CO2 + < 0.01μL L1 O2 (q.v.) at 2°C after 2 weeks of posttreatment storage at 2°C. The CA treatments did not have a significant negative effect on grape quality and were considered safe.

6. AGRICULTURE

173

6.2.2.8 MATHEMATICAL MODELING

6.2.2.7.5 Diseases Reports on the effects of CA storage on the development of diseases of fruits and vegetables have shown mixed results. In vitro studies of Chinese cabbage inoculated with Phytophthora brassicae and stored at 1.5°C in 0.5 kPa CO2 + 1.5 kPa O2 or 3.0 kPa CO2 + 3.0 kPa CO2 showed low levels of disease development compared to those stored in air. In contrast, in vivo studies showed that the infection caused by P. brassicae was significantly higher in the CA conditions than in air after 94–97 days storage (Hermansen and Hoftun, 2005). In celery stored at 8°C, disease suppression was greatest in atmospheres containing 7.5–30 kPa CO2 + 1.5 kPa O2, but there was only a slight suppression in 4– 16 kPa CO2 + 1.5 kPa O2 or in 1.5–6 kPa O2 + 0 kPa CO2 (Reyes, 1988). Kiwi was exposed to 60 kPa CO2 + 20 kPa O2 at 30 or 40°C for 1, 3, or 5 days by Cheah et al. (1994) for the control of rot caused by Botrytis cinerea. Spore germination and growth of B. cinerea were completely inhibited in vitro by 60 kPa CO2 at 40°C and partially suppressed at 30°C. Kiwi was inoculated with B. cinerea spores, exposed to 60 kPa CO2 at 30 or 40°C, and then stored in air at 0°C for up to 12 weeks after treatment. 60kPa CO2 at 40°C reduced disease incidence from 85% in air at 20°C to about 50%, but exposure to CO2 at 40°C for longer than 1 day adversely affected fruit ripening. Several fungal species were found to be infecting blueberries (Vaccinium corymbosum L.) during storage but Botrytis spp. and Alternaria spp. were the dominant fungal pathogens causing decay. Overall, sulfur dioxide fumigation followed by CA storage (3 kPa O2 + 6 or 12kPa CO2) reduced decay. However, CO2 levels of 24 kPa resulted in quicker fruit softening and off-flavors (Cantı´n et al., 2012). Teles et al. (2014) exposed grapes to 40 kPa CO2 for 48 h prestorage followed by CA storage in 12 kPa O2 + 12kPa CO2 at 0°C. The gray mold incidence (B. cinerea) was reduced from 22% to 0.6% and from 100% to 7.4% after 4 and 7 weeks storage, respectively. Parsons et al. (1974) showed considerable reduction in disease levels on tomatoes in

TABLE 6.2.2.3 Effects of Controlled Atmosphere Storage Conditions on the Decay Levels of Tomatoes Harvested at the Green Mature Stage

Storage Atmosphere

After Removal From 6 Weeks at 13°C (%)

Plus 1 Week at 15–21°C (%)

Plus 2 Weeks at 15–21°C (%)

Air (control)

65.6

93.3

98.6

0% CO2 + 3% O2

2.2

4.4

16.7

3% CO2 + 3% O2

3.3

5.6

12.2

5% CO2 + 3% O2

5.0

9.4

13.9

Adapted from Parsons, C.S., Anderson, R.E., Penney, R.W., 1974. Storage of mature-green tomatoes in controlled atmospheres. J. Am. Soc. Hortic. Sci. 95, 791–794.

CA storage compared to storage in air with most of the effect coming from the low O2 levels with little additional effect from increased CO2 levels (Table 6.2.2.3). Some in vitro and in vivo studies have indicated that CA may have a negative effect on bacteria. Parsons and Spalding (1972) inoculated tomato fruit with soft rot bacteria and held them for 6 days at 12.8°C in 3 kPa O2 + 5 kPa CO2 or in air. Lesions were smaller on fruits stored in CA than on those stored in air, but CA storage did not entirely control decay. Amodio et al. (2003) stored slices of mushrooms at 0°C either in air or in 3 kPa O2 + 20 kPa CO2 for 24 days and found that there was a slight increase in bacteria in air but no increase or a slight decrease for those in CA. However, Daniels et al. (1985) reported that Clostridium botulinum may survive even at high CO2 levels.

6.2.2.8 MATHEMATICAL MODELING Mathematical models have been developed in relation to many aspects of CA storage, particularly for the prediction of the optimum gas combinations for various crops in various situations. Nahor et al. (2003) described a model for simulation of the dynamics of heat, moisture, and gas exchange in CA stores consisting of

6. AGRICULTURE

174

6.2. VEGETAL PRODUCTION

interacting systems between cooled space, the refrigeration unit, and the gas handling unit. They included respiration rate and product quality and reported that model predictions were usually found to be in good agreement with the experimental results. They later (Nahor et al., 2005) reported that the Michaelis-Menten type gas exchange models were appropriate for CA storage of pome fruit, as opposed to the empirical ones, due to their generic behavior. They recommended that the kinetic parameters of the model should be expressed as a function of the physiological age of the fruit to include the influence of age when the models are to be used to simulate gas exchange of bulk CA-stored fruit for longer storage periods. Gwanpua et al. (2012) proposed a model to describe the loss of firmness in apples stored under different conditions. They tested the model on Braeburn apples harvested at different maturities and stored at 1°C in various CA conditions, taking into account ethylene production, pectin degradation, and synthesis of pectindegrading enzymes. Appropriate kinetic equations for these reactions were proposed for the development of a model. Their model was found to explain up to 83.64% of the total variance of the measured data.

6.2.2.9 CONCLUSIONS Fresh fruits and vegetables being available at the time when the consumer requires them is increasingly important. Not long ago, consumers adapted to certain fruits and vegetables being available only at certain times of the year. Also, awareness is more widespread of their nutritional importance. In the 19th and 20th centuries, refrigeration became increasing used for their preservation and transport. This was supplemented by controlling the atmosphere, which enabled them to be stored for protracted periods with better maintenance of their nutritional attributes. These technologies are becoming used more widely and their control and

application more sophisticated. It is foreseeable that there will be continuous development and innovation of controlling the atmosphere during transport and storage and its cost will be reduced as it is applied in new situations and on new commodities. Its importance is reflected in the number of scientific publications, conferences, and journals devoted to the subject.

References Amodio, M.L., Colelli, G., de Cillis, F.M., Lovino, R., Massignan, L., 2003. Controlled-atmosphere storage of fresh-cut ‘Cardoncello’ mushrooms (Pleurotus eryngii). Acta Hortic. 599, 731–735. Anderson, R.E., 1982. Long term storage of peaches and nectarine intermittently warmed during controlled atmosphere storage. J. Am. Soc. Hortic. Sci. 107, 214–216. Argenta, L.C., Xuetong, F., Mattheis, J., 2000. Delaying establishment of controlled atmosphere or CO2 exposure reduces ‘Fuji’ apple CO2 injury without excessive fruit quality loss. Postharvest Biol. Technol. 20, 221–229. Awad, M.A., de Jager, A., 2003. Influences of air & controlled atmosphere storage on the concentration of potentially healthful phenolics in apples & other fruits. Postharvest Biol. Technol. 27, 53–58. Bekele, E.A., Ampofo-Asiama, J., Alis, R.R., Hertog, M.L.A.T.M., Nicolai, B.M., Geeraerd, A.H., 2016. Dynamics of metabolic adaptation during initiation of controlled atmosphere storage of ‘Jonagold’ apple: effects of storage gas concentrations & conditioning. Postharvest Biol. Technol. 117, 9–20. Berard, J.E., 1821. Memoire sur la maturation des fruits. Ann. Chim. Phys. 16, 152–183. Berard, L.S., 1985. Effects of CA on several storage disorders of winter cabbage. Controlled atmospheres for storage and transport of perishable agricultural commodities. In: 4th National Controlled Atmosphere Research Conference, July 1985, pp. 150–159. Bessemans, N., Verboven, P., Verlinden, B.E., Nicolaı¨, B.M., 2016. A novel type of dynamic controlled atmosphere storage based on the respiratory quotient (RQ-DCA). Postharvest Biol. Technol. 115, 91–102. Bishop, D.J., 1996. Controlled atmosphere storage. In: Dellino, C.J.V. (Ed.), Cold and Chilled Storage Technology. Blackie, London. Burdon, J., Lallu, N., Billing, D., Burmeister, D., Yearsley, C., Wang, M., Gunson, A., Young, H., 2005. Carbon dioxide scrubbing systems alter the ripe fruit volatile profiles in controlled-atmosphere stored ‘Hayward’ kiwifruit. Postharvest Biol. Technol. 35, 133–141.

6. AGRICULTURE

REFERENCES

Burdon, J., Lallu, N., Haynes, G., McDermott, K., Billing, D., 2008. The effect of delays in establishment of a static or dynamic controlled atmosphere on the quality of ‘Hass’ avocado fruit. Postharvest Biol. Technol. 49, 61–68. Burg, S.P., 2004. Postharvest Physiology and Hypobaric Storage of Fresh Produce. CAB International, Wallingford, Oxford, UK. Burton, W.G., 1952. Studies on the dormancy and sprouting of potatoes. III. The effect upon sprouting of volatile metabolic products other than carbon dioxide. New Phytol. 51, 154–161. Cantı´n, C.M., Minas, I.S., Goulas, V., Jim
enez, M., Manganaris, G.A., Michailides, T.J., Crisosto, C.H., 2012. Sulfur dioxide fumigation alone or in combination with CO2-enriched atmosphere extends the market life of highbush blueberry fruit. Postharvest Biol. Technol. 67, 84–91. Cantwell, M.I., 1995. Post-harvest management of fruits and vegetables. In: Barbara, G., Inglese, P., PimientaBarrios, E. (Eds.), Agro-ecology, cultivation and uses of cactus pear. In: FAO Plant Production and Protection Paper 132, pp. 120–136. Cheah, L.H., Irving, D.E., Hunt, A.W., Popay, A.J., 1994. Effect of high CO2 and temperature on Botrytis storage rot and quality of kiwifruit. In: Proceedings of the Forty Seventh New Zealand Plant Protection Conference, Waitangi Hotel, New Zealand, 9–11 August, 1994, pp. 299–303. Colgan, R.J., Dover, C.J., Johnson, D.S., Pearson, K., 1999. Delayed CA and oxygen at 1% or less control superficial scald without CO2% injury on Bramley’s Seedling apples. Postharvest Biol. Technol. 16, 223–231. Daniels, J.A., Krishnamurthi, R., Rizvi, S.S., 1985. A review of effects of CO2 on microbial growth and food quality. J. Food Prot. 48, 532–537. Day, B.P.F., 1996. High O2 modified atmosphere packaging for fresh prepared produce. Postharvest News Inf. 7, 31N–34N. DeEll, J.R., van Kooten, O., Prange, R.K., Murr, D.P., 1999. Applications of chlorophyll fluorescence techniques in postharvest physiology. Hortic. Rev. 23, 69–107. Delate, K.M., Brecht, J.K., 1989. Quality of tropical sweetpotatoes exposed to controlled-atmosphere treatments for postharvest insect control. J. Am. Soc. Hortic. Sci. 114, 963–968. Dijkink, B.H., Tomassen, M.M., Willemsen, J.H.A., van Doorn, W.G., 2004. Humidity control during bell pepper storage, using a hollow fiber membrane contactor system. Postharvest Biol. Technol. 32, 311–320. Drake, S.R., Eisele, T.A., 1994. Influence of harvest date and controlled atmosphere storage delay on the color and quality of ‘Delicious’ apples stored in a purge-type controlledatmosphere environment. HortTechnology 4, 260–263.

175

Elgar, H.J., Burmeister, D.M., Watkins, C.B., 1998. Storage and handling effects on a CO2-related internal browning disorder of ‘Braeburn’ apples. HortScience 33, 719–722. Forney, C.F., Jamieson, A.R., Munro Pennell, K.D., Jordan, M.A., Fillmore, S.A.E., 2015. Relationships between fruit composition and storage life in air or controlled atmosphere of red raspberry. Postharvest Biol. Technol. 110, 121–130. Gadalla, S.O., 1997. Inhibition of Sprouting of Onions During Storage and Marketing. (Ph.D. Thesis). Cranfield University, UK. Gasser, F., Eppler, T., Naunheim, W., Gabioud, S., Hoehn, E., 2008. Control of the critical oxygen level during dynamic ca storage of apples by monitoring respiration as well as chlorophyll fluorescence. Acta Hortic. 796, 69–76. Goulart, B.L., Evensen, K.B., Hammer, P., Braun, H.L., 1990. Maintaining raspberry shelf-life: part 1. The influence of controlled atmospheric gases on raspberry postharvest longevity. Pa. Fruit News 70, 12–15. Gwanpua, S.G., Verlinden, B.E., Hertog, M.L.A.T.M., Bulens, I., Van de Poel, B., Van Impe, J., Nicolaı¨, B.M., Geeraerd, A.H., 2012. Kinetic modeling of firmness breakdown in ‘Braeburn’ apples stored under different controlled atmosphere conditions. Postharvest Biol. Technol. 67, 68–74. Harbaum-Piayda, B., Walter, B., Bengtsson, G.B., Hubbermann, E.M., Bilger, W., Schwarz, K., 2010. Influence of pre-harvest UV-B irradiation and normal or controlled atmosphere storage on flavonoid and hydroxycinnamic acid contents of pak choi (Brassica campestris L. ssp. chinensis var. communis). Postharvest Biol. Technol. 56, 202–208. Hardenburg, R.E., Anderson, R.E., Finney Jr., E.E., 1977. Quality and condition of ‘Delicious’ apples after storage at 0 deg C and display at warmer temperatures. J. Am. Soc. Hortic. Sci. 102, 210–214. Hellickson, M.L., Adre, N., Staples, J., Butte, J., 1995. Technologias de cosecha y postcosecha de frutas y hortalizas. In: Proceedings of a Conference Held in Guanajuato, Mexico, 20–24 February. Harvest and Postharvest Technologies for Fresh Fruits and Vegetables, pp. 546–553. Hermansen, A., Hoftun, H., 2005. Effect of storage in controlled atmosphere on post-harvest infections of Phytophthora brassicae, and chilling injury in Chinese cabbage (Brassica rapa L pekinensis (Lour) Hanelt). J. Sci. Food Agric. 85, 1365–1370. Imahori, Y., Yamamoto, K., Tanaka, H., Bai, J., 2013. Residual effects of low oxygen storage of mature green fruit on ripening processes and ester biosynthesis during ripening in bananas. Postharvest Biol. Technol. 77, 19–27. Khanbari, O.S., Thompson, A.K., 1996. Effects of controlled atmosphere, temperature, and cultivar on sprouting and processing quality of stored potatoes. Potato Res. 39, 523–531.

6. AGRICULTURE

176

6.2. VEGETAL PRODUCTION

Kidd, F., West, C., 1927. A Relation Between the Concentration of O2 and CO2 in the Atmosphere, Rate of Respiration, and the Length of Storage of Apples. (Report of the Food Investigation Board London for 1925, 1926)., pp. 41–42. Knee, M., Looney, N.E., 1990. Effect of orchard and postharvest application of daminozide on ethylene synthesis by apple fruit. J. Plant Growth Regul. 9, 175–179. Landfald, R., 1988. Controlled-atmosphere storage of the apple cultivar Aroma. Norsk Landbruksforsk. 2, 5–13. Latocha, P., Krupa, T., Jankowski, P., Radzanowska, J., 2014. Changes in postharvest physicochemical and sensory characteristics of hardy kiwifruit (Actinidia arguta and its hybrid) after cold storage under normal versus controlled atmosphere. Postharvest Biol. Technol. 88, 21–33. Liu, F.W., 1976. Storing ethylene pretreated bananas in controlled atmosphere and hypobaric air. J. Am. Soc. Hortic. Sci. 101, 198–201. Liu, Y.-B., 2013. Controlled atmosphere treatment for control of grape mealybug, Pseudococcus maritimus (Ehrhorn) (Hemiptera: Pseudococcidae), on harvested table grapes. Postharvest Biol. Technol. 86, 113–117. Lum, G.B., Brikis, C.J., Deyman, K.L., Subedi, S., DeEll, J.R., Shelp, B.J., Bozzo, G.G., 2016. Pre-storage conditioning ameliorates the negative impact of 1-methylcyclopropene on physiological injury and modifies the response of antioxidants and γ-aminobutyrate in ‘Honeycrisp’ apples exposed to controlled-atmosphere conditions. Postharvest Biol. Technol. 116, 115–128. Marcellin, P., Chaves, A., 1983. Effects of intermittent high CO2 treatment on storage life of avocado fruits in relation to respiration and ethylene production. Acta Hortic. 138, 155–163. Martins, D.R., de Resende, E.D., 2015. External quality and sensory attributes of papaya cv. Golden stored under different controlled atmospheres. Postharvest Biol. Technol. 110, 40–42. Matityahu, I., Marciano, P., Holland, D., Ben-Arie, R., Amir, R., 2016. Differential effects of regular and controlled atmosphere storage on the quality of three cultivars of pomegranate (Punica granatum L.). Postharvest Biol. Technol. 115, 132–141. Mencarelli, F., Lipton, W.J., Peterson, S.J., 1983. Responses of ‘zucchini’ squash to storage in low O2 atmospheres at chilling and nonchilling temperatures. J. Am. Soc. Hortic. Sci. 108, 884–890. Nahor, H.B., Scheerlinck, N., Verboven, P., Impe, J.van & Nicolai, B., 2003. Combined discrete and continuous simulation of controlled atmosphere (CA) storage systems. Commun. Agric. Appl. Biol. Sci. 68, 17–21. Nahor, H.B., Schotsmans, W., Scheerlinck, N., Nicolaı¨, B.M., 2005. Applicability of existing gas exchange models for bulk storage of pome fruit: assessment and testing. Postharvest Biol. Technol. 35, 15–24.

North, C.J., Cockburn, J.T., 1975. Ethyl alcohol levels in apples after deprivation of oxygen and the detection of alcohol vapour in controlled atmosphere storage using indicator tubes. J. Sci. Food Agric. 26, 1151–1161. Palma, T., Stanley, D.W., Aguilera, J.M., Zoffoli, J.P., 1993. Respiratory behavior of cherimoya Annona cherimola Mill. under controlled atmospheres. HortScience 28, 647–649. Parsons, C.S., Spalding, D.H., 1972. Influence of a controlled atmosphere, temperature, and ripeness on bacterial soft rot of tomatoes. J. Am. Soc. Hortic. Sci. 97, 297–299. Parsons, C.S., Anderson, R.E., Penney, R.W., 1974. Storage of mature-green tomatoes in controlled atmospheres. J. Am. Soc. Hortic. Sci. 95, 791–794. Prange, R.K., Delong, J.M., Leyte, J.C., Harrison, P.A., 2002. Oxygen concentration affects chlorophyll fluorescence in chlorophyll-containing fruit. Postharvest Biol. Technol. 24, 201–205. Prange, R.K., Delong, J.M., Harrison, P.A., Leyte, J.C., McLean, S.D., 2003. Oxygen concentration affects chlorophyll fluorescence in chlorophyll-containing fruit and vegetables. J. Am. Soc. Hortic. Sci. 128, 603–607. Prange, R.K., Wright, A.H., DeLong, J.M., Zanella, A., 2014. History, current situation and future prospects for dynamic controlled atmosphere (DCA) storage of fruits and vegetables, using chlorophyll fluorescence. Acta Hortic. 1012, 905–915. Rahman, A.S.A., Huber, D., Brecht, J.K., 1993a. Respiratory activity and mitochondrial oxidative capacity of bell pepper fruit following storage under low O2 atmosphere. J. Am. Soc. Hortic. Sci. 118, 470–475. Rahman, A.S.A., Huber, D., Brecht, J.K., 1993b. Physiological basis of low O2 induced residual respiratory effect in bell pepper fruit. Acta Hortic. 343, 112–116. Rahman, A.K.M.M., Huq, E., Mian, A.J., Chesson, A., 1995. Microscopic and chemical changes occurring during the ripening of two forms of jackfruit (Artocarpus heterophyllus L.). Food Chem. 52, 405–410. Reyes, A.A., 1988. Suppression of Sclerotinia sclerotiorum and watery soft rot of celery by controlled atmosphere storage. Plant Dis. 72, 790–792. Robinson, T.L., Watkins, C.B., Hoying, S.A., Nock, J.F., Iungermann, K.I., 2006. Aminoethoxyvinylglycine and 1-methylcyclopropene effects on ‘McIntosh’ preharvest drop, fruit maturation and fruit quality after storage. Acta Hortic. 727, 473–480. Schouten, S.P., Prange, R.K., Verschoor, J., Lammers, T.R., Oosterhaven, J., 1997. Improvement of quality of Elstar apples by dynamic control of ULO conditions. In: Mitcham, E.J. (Ed.),. CA’97. Proceedings of the 7th International Controlled Atmosphere Research Conference. In: vol. 2. University of California, Davis, CA, pp. 71–78. Schouten, R.E., Zhang, X., Verkerk, R., Verschoor, J.A., Otma, E.C., Tijskens, L.M.M., van Kooten, O., 2009.

6. AGRICULTURE

REFERENCES

Modelling the level of the major glucosinolates in broccoli as affected by controlled atmosphere and temperature. Postharvest Biol. Technol. 53, 1–10. Selcuk, N., Erkan, M., 2015. The effects of modified and palliflex controlled atmosphere storage on postharvest quality and composition of ‘Istanbul’ medlar fruit. Postharvest Biol. Technol. 99, 9–19. Sharples, R.O., Stow, J.R., 1986. Recommended Conditions for the Storage of Apples and Pears. (Report of the East Malling Research Station for 1985)., pp. 165–170. Solomos, T., Biale, J.B., 1975. Respiration in fruit ripening. Colloq. Int. C. N. R. S. 238, 221–228. Streif, J., Saquet, A.A., Xuan, H., 2003. CA-related disorders of apples and pears. Acta Hortic. 600, 223–230. Teixeira, G.H.A., Cunha Ju´nior, L.C., Ferraudo, A.S., Durigan, J.F., 2016. Quality of guava (Psidium guajava L. cv. Pedro Sato) fruit stored in low-O2 controlled atmospheres is negatively affected by increasing levels of CO2. Postharvest Biol. Technol. 111, 62–68. Teles, C.S., Benedetti, B.C., Gubler, W.G., Crisosto, C.H., 2014. Prestorage application of high carbon dioxide combined with controlled atmosphere storage as a dual approach to control Botrytis cinerea in organic ‘Flame Seedless’ and ‘Crimson Seedless’ table grapes. Postharvest Biol. Technol. 89, 32–39. Thompson, A.K., 2010. Controlled Atmosphere Storage of Fruits and Vegetables, second ed. CAB International, Oxford. Thompson, A.K., 2015. Fruit and Vegetable Storage: Hypobaric, Hyperbaric and Controlled Atmosphere. Springer, New York. Thompson, A.K., Bishop, D., 2016. Controlled atmosphere technology. In: Reference Module in Food Sciences. Elsevier, pp. 1–8. https://doi.org/10.1016/B978-0-08-1005965.21136-0. Tonini, G., Tura, E., 1997. New CA storage strategies for reducing rots (Botrytis cinerea and Phialophora spp.) and softening in kiwifruit. In: Seventh International Controlled Atmosphere Research Conference, July 13–18, 1997. University of Caifornia, Davis, CA, p. 104 (abstract). Uddin, M.M., MacTavish, H.S., 2003. Controlled atmosphere and regular storage-induced changes in S-alk(en)yl-lcysteine sulfoxides and alliinase activity in onion bulbs (Allium cepa L. cv. Hysam). Postharvest Biol. Technol. 28, 239–245.

177

Wang, Z.Y., Kosittrakun, M., Dilley, D.R., 2000. Temperature and atmosphere regimens to control a CO2-linked disorder of ’Empire’ apples. Postharvest Biol. Technol. 18, 183–189. Watkins, C.B., Nock, J.F., 2012. Rapid 1-methylcyclopropene (1-MCP) treatment and delayed controlled atmosphere storage of apples. Postharvest Biol. Technol. 69, 24–31. Watkins, C.B., Burmeister, D.M., Elgar, H.J., Fu, W.L., 1997. A comparison of two carbon dioxide-related injuries of apple fruit. In: Postharvest Horticulture Series, vol. 16. Department of Pomology University of California, pp. 119–124. Wills, R.B.H., Pitakserikul, S., Scott, K.J., 1982. Effects of prestorage in low O2 or high CO2 concentrations on delaying the ripening of bananas. Aust. J. Agr. Res. 33, 1029–1036. Wills, R.B.H., Ku, V.W., Shohet, D., Kim, G.H., 1999. Importance of low ethylene levels to delay senescence of nonclimacteric fruit and vegetables. Aust. J. Exp. Agric. 39, 221–224. Wojciechowski, J., 1989. Ethylene removal from gases by means of catalytic combustion. Acta Hortic. 258, 131–139. Wollin, A.S., Little, C.R., Packer, J.S., 1985. Dynamic control of storage atmospheres. In: Blankenship, S. (Ed.), Controlled Atmospheres for Storage and Transport of Perishable Agricultural Commodities. Proceedings of the 4th National Controlled Atmosphere Research Conference, Raleigh, NC, USA, pp. 308–315. Yahia, E.M., Kushwaha, L., 1995. Technologias de cosecha y postcosecha de frutas y hortalizas. In: Kushwaha, L., Serwatowski, R., Brook, R. (Eds.), Proceedings of a Conference Held in Guanajuato, Mexico, 20–24 February, 1995. Harvest and Postharvest Technologies for Fresh Fruits and Vegetables, pp. 282–286. Yearsley, C.W., Banks, N.H., Ganesh, S., Cleland, D.J., 1996. Determination of lower oxygen limits for apple fruit. Postharvest Biol. Technol. 8, 95–109. Zhou, H.-W., Lurie, S., Lers, A., Khatchitski, A., Sonego, L., Ben Arie, R., 2000. Delayed storage and controlled atmosphere storage of nectarines: two strategies to prevent woolliness. Postharvest Biol. Technol. 18, 133–141. Zlati
c, E., Zadnik, V., Fellman, J., Demsˇar, L., Hribar, J.,  c, Zˇ., Vidrih, R., 2016. Comparative analysis of aroma Ceji
compounds in ‘Bartlett’ pear in relation to harvest date, storage conditions, and shelf-life. Postharvest Biol. Technol. 117, 71–80.

6. AGRICULTURE

S U B C H A P T E R

6.2.3 Pest Control Peter Meeus QHSE, Training & Expertise, EWS Group Antwerp, Antwerp, Belgium Entomologie fonctionnelle et
evolutive, Gembloux Agro-Bio Tech—Universit
e de Lie`ge, Namur, Belgium

FIRST KNOW YOUR ENEMY, THEN CHOOSE YOUR WEAPONS

6.2.3.1 CARBON DIOXIDE AS A CARRIER GAS IN SILO FUMIGATION Fumigation of cereals in vertical silos is a very common practice. Phosphine gas is used as the fumigant. As this gas is very flammable and will ignite spontaneously at higher concentrations, it cannot be used as a pure gas directly from the bottle. Here, we produce the gas out of tablets with aluminum or magnesium hydroxide. Together with the moisture in the cereals and the ambient air, we get a slow chemical reaction with phosphine gas (PH3) as the end product. The gas will be evenly distributed in the cereal mass in the silo by dosing the tablets in the grain flow during charging. During discharge of the silo, the remaining powdery residue will be removed by the dust aspiration in the transport system. In most cases, however, the need for fumigation is only defined after the silo has been filled. In such a case, the tablets can’t be evenly

distributed. The only solution is putting the fumigant on top of the cargo in the headspace. The gas has to penetrate from itself into the cereals, but this is a very slow process, and the penetration depth will normally never exceed 10 m. This will result in failed fumigation in higher silos. This problem can be solved by either the use of a recirculation system with a fan and hoses or the use of a heavier-than-air carrier gas. In the case of recirculation, we have a fan that sucks out the gas-air mixture from the headspace and injects this mixture through perforated tubing at the bottom of the silo. This creates an even distribution of the fumigant in the cereals after a while. The recirculation system can only be applied if the silo has been adapted for it before charging. See Fig. 6.2.3.1. This leaves us, for nonprepared silos, only the use of carbon dioxide as a carrier gas for the phosphine. Here we take notice of the fact that carbon dioxide is heavier than air (density ¼ 1.5). When injected slowly in the headspace of the silo, the carbon dioxide will “grab” the phosphine gas, and they both will sink together to the bottom of the silo. The speed will depend on the absorption characteristics of the cereals, but in many cases even silos up to 30 m high will already have a measurable concentration of PH3

6. AGRICULTURE

6.2.3.1 CARBON DIOXIDE AS A CARRIER GAS IN SILO FUMIGATION

FIG. 6.2.3.1

179

Phosphine recirculation system in silos for walnuts (California).

after 24 h. It’s important to note that the carbon dioxide used here has absolutely no insecticidal effect itself. It only acts in a physical way as a “carrier.” The introduction speed of the carbon dioxide depends on the kind of phosphine formulation that is used. Between 100 and 200 g of CO2 per ton of product will be needed, and the total amount of gas has to be injected during the time period when the highest amount of gas is generated out of the phosphide formulation. This will generally be about 24 h, but it’s also temperature-dependent. The moment the phosphine concentration at the bottom of the silo can

be considered as enough for insect extermination, the valves of the bottle(s) on the silo deck will have to be closed. Carrier gas fumigation will normally be executed with carbon dioxide from gas bottles without a riser tube inside, so we will only have carbon dioxide gas coming out of the bottle. See Fig. 6.2.3.2. In some cases (for example, no elevator in the silo building), dry ice will be a better choice as the source for the carbon dioxide gas. Then, the weight of the steel packaging does not have to be transported upstairs by foot, but on the other hand the injection time cannot be

6. AGRICULTURE

180

FIG. 6.2.3.2

6.2. VEGETAL PRODUCTION

Carrier gas fumigation with CO2 bottles.

controlled after the introduction of the product. The sublimation of the solid product into gas is not controllable. Another advantage of carrier gas fumigation is that no powder residue will remain in the cargo. The bags, plates, or sleeves can be removed completely before the start of the ventilation.

6.2.3.2 RODENT EXTERMINATION WITH CARBON DIOXIDE This chapter doesn’t deal with veterinary actions for purposes of public health. Mass exterminations of warm-blooded animals by means of carbon dioxide for reasons of epidemiology or public health are not considered as a part of pest control. But sometimes rodents—and more specifically, mice—get into warehouses with raw or finished food or feed materials. When the pallets are not properly stored at least 0.5 m from the walls, those animals will be attracted to jump into the palletized goods. The reason for this is very “clear” for us, but absolutely not so “clear” for the rodents because they are half-blind.

Suppose a female mouse walks into a warehouse. Such a mouse can be considered quite certainly to be pregnant, as that’s pure rodent biology. She smells the odor of tasty things, and the moment a pallet is stored too close to the wall, she sees that pallet and jumps in. Plenty of shelter, nesting material, and food. Do mice need to drink? NO! They can get enough water out of their food. As an example, the average and normal moisture content of stored cereals is about 12%. This also means that more than 10% of the grain is water! As such, 10 kg of grain contains more than 1 L of water. This female mouse will become the ancestor of a whole family of descendants, and they will not come out of the cargo until there are too many of them. That’s also why a treatment with toxic baits or “nontox” traps at the outside will not work with 100% efficiency. Especially in warehouses with finished food products, even the presence of only one (female) mouse can be catastrophic the moment those products are delivered to the end consumer. But also, mice-infested raw food materials can pose serious problems when brought into the production areas, where the

6. AGRICULTURE

6.2.3.2 RODENT EXTERMINATION WITH CARBON DIOXIDE

escaping mice can migrate toward stores with finished products. In many countries, toxic gases are not registered for use against rodents in food or feed storage. Here, we can use inert gases, not as a toxic agent but as an element that will push the oxygen away and suffocate the animal concerned, due to the absence of that necessary oxygen. Rodents as well as humans are warmblooded mammals. We need a continuous flow of oxygen to maintain the energy in our body, and especially our brain. That’s why a lack of oxygen for 5 min can be deadly for warmblooded species (Fig. 6.2.3.3). In such cases, we can choose the suffocation weapon. Large amounts of carbon dioxide will be injected into the premises in the shortest time possible, with the sole purpose of lowering the oxygen content deep enough to suffocate all target organisms. Carbon dioxide is used instead of nitrogen because of the difference in technical properties of both gases in terms of being injected out of a tank into a space using the liquid phase. In practice, the target concentration level will be 14 vol.% of oxygen. This gives a sufficient C  T (concentration-time product) dosage to kill all mammals. Meetings and preparations—and especially those concerning safety—are extremely important. Many people still consider a “nontox” pest control treatment as less dangerous than a classic fumigation. This is in general true for the environmental aspects, but absolutely not for the aspect of worker safety. We are, just like our targets, warm-blooded mammals, so we

FIG. 6.2.3.3

181

are as vulnerable to the product as they are. We may even be a bit more susceptible because we can’t hide inside a pallet during the treatment. A lack of oxygen is deadly within minutes, and there is no antidote. Special precautions have to be taken in case there are neighbors in the same building. Carbon dioxide not only “flows like water,” but it also very easily penetrates brick walls that are commonly used as separation between sections in warehouses. Always take into account the worst-case scenario: the gas concentration in the neighboring spaces will also be above safety limits, and we have to ventilate that also. After all coordination activities are finished and the spaces to treat are made gastight, adapted hoses and spray nozzles are installed. See Figs. 6.2.3.4 and 6.2.3.5. We say “adapted” because the injection will be at high pressure and very low temperatures. The last also means that special attention has to be paid to systems containing water inside the building (for example, sprinkler installations). They can freeze, with possible serious damage as a result. First, the whole CO2 injection system (hoses and nozzles) will be put on pressure by gaseous carbon dioxide (top of the tank). See Figs. 6.2.3.6 and 6.2.3.7. After that, and as soon as possible, there will be a switch to the liquid phase (bottom of the tank) to prevent possible freezing of the cargo. For this same reason, the operation, once started, cannot be stopped. Closing an injection valve before the end of the injection will result in freezing the liquid CO2 in the hoses and nozzles, which aborts the operation.

Lethality during brief exposure to low oxygen concentration.

6. AGRICULTURE

FIG. 6.2.3.4 Start of the introduction. Putting the manifold system on pressure.

FIG. 6.2.3.5 Injection hoses, nozzles, and tubes for gas concentration and temperature measurements, just before the introduction of the gas.

FIG. 6.2.3.6 gaseous phase.

Start of the introduction,

6.2.3.3 INSECT CONTROL WITH MODIFIED ATMOSPHERES AT NORMAL ATMOSPHERIC PRESSURE

FIG. 6.2.3.7

183

Last view inside the warehouse before the white dense cloud covers everything.

The operation starts mostly in the evening, after working hours, and the ventilation will take place the next morning. So the gas will have had plenty of time to penetrate inside the goods sufficiently. Especially in large structures, care has to be taken to ensure a large safety perimeter at the moment the gates are opened for ventilation. One can see the gas coming out (the “fata morgana” look), and as it’s heavier than air, it will flow over the ground. A high concentration about 100 m downwind for the first half hour is not uncommon. Also, all basement structures in the treated premises and the immediate surroundings must be checked carefully before a gas-free declaration.

6.2.3.3 INSECT CONTROL WITH MODIFIED ATMOSPHERES AT NORMAL ATMOSPHERIC PRESSURE Insects can be controlled by means of carbon dioxide or nitrogen, using the same technique as above: asphyxiation. But there is one very big difference: insects are not mammals. Insects are cold-blooded arthropods that have a completely different biology than we have.

They are much more adapted to survive hostile circumstances than we are. In general, we can conclude that when the mammals have disappeared from the Earth, insects will still be ruling this same Earth. The insects we have to deal with, the so-called “stored product insects,” all have four stages in their lives. An adult female deposits eggs, mostly in locations where she knows by instinct the chances for survival are positive for the offspring. Out of those eggs, we see emerging larvae. They will grow and molt several times until they pupate. Those pupae can survive unfavorable times (such as winter). After a certain time, when circumstances are becoming positive, we see adults coming from the pupae, and the circle is closed. Larvae only think about one thing: eating and growing. Adults only think about another thing: mating and reproduction. Insects have no lungs and no active breathing apparatus. They have more or less a dense “tubing system” (trachea) inside their body through which the air can flow freely. The capacity is controlled by opening or closing their hatches (spiracles) at the end of those trachea. That’s why insects can be compared more or less with

6. AGRICULTURE

184

6.2. VEGETAL PRODUCTION

our submarines from the former century. When there was a hostile environment, they closed all hatches and sunk themselves to the bottom of the sea. They shut down all nonvital systems, and waited until the situation was safe enough to come back to the surface and open the air supply again. It took quite a lot of time until a submarine had to surface due to lack of oxygen. In the insect development stages, we see that the eggs are very tolerant toward a lack of oxygen. They simply wait. Larvae, however, are very active. They eat a lot and their metabolism is high, so they need a lot of oxygen. Pupae can survive a certain time without oxygen. Adults do need a relatively huge amount of oxygen because they consume a lot of energy during crawling and/or flying in search of a partner. Being cold-blooded automatically means that organisms are very dependent on temperature. The hotter it is, the quicker the development. In ambient temperatures, treatments according this principle can take a relatively long time. A generalized rule says that 60% CO2, 21°C, 21 days for insects with “hidden” stages. This

FIG. 6.2.3.8

means with one or more stages developing inside the grain, like the rice weevil (Sitophilus oryzae L.). In tropical circumstances with temperatures above 30°C, the exposure time can go down until 1 week. An example of a treatment for rice against the rice weevil can be seen in Figs. 6.2.3.8 and 6.2.3.9. Here, we have a lot of 30 ft bulk containers loaded with organic rice. There was a clear infestation with weevils. Luckily, the rice was packed in a big plastic bag (the “inner liner”) in the container, so we had a quite gastight situation inside the container. The aim was to have at least 60 vol. % CO2 during 21 days. Each container was connected to a rack of bottles, and the carbon dioxide flow was adjusted in such a way that there would be a very slight overpressure during those 21 days. This was to compensate for small leaks (there are always small leaks during such fumigations). After 21 days, the containers were ventilated and rice samples were taken. Those samples were kept for 3 weeks in a warm location (>21°C) and deniable inspected on the presence of live insects.

Bulk containers with biorice, infested with rice weevil. 60% CO2, 21 days exposure at 21°C.

6. AGRICULTURE

6.2.3.4 HIGH-PRESSURE CARBON DIOXIDE TREATMENTS

FIG. 6.2.3.9

185

Sealing the loading hatches and mounting tubes for gas injection, measurement, and temperature control.

Air samples were taken on a daily basis. The O2 and CO2 concentrations of a container treated that way can be seen in Fig. 6.2.3.10. These techniques are very well suited to treat organic products. The weak point is that temperature control is extremely important to keep the exposure time commercially acceptable. Nitrogen can also be used as a gas for such operations, but at a much higher dosage. With N2, we will have to go to at least 99% gas concentration, leaving a maximum of 1% oxygen. Especially in museums, where artifacts are very valuable and vulnerable, mostly nitrogen is used because it’s absolutely harmless to the artifacts. Longer exposure times are for those purposes less important. Nitrogen is mostly generated by a nitrogen separator. Oxygen is taken out of the air by this machine and released back into the ambient air. The pure nitrogen is used to flush the fumigation chambers and to maintain the low oxygen

level. Fig. 6.2.3.11 shows an example of a nitrogen fumigation under sheets. In Fig. 6.2.3.12, we see the preparation of a treatment in a gastight fixed chamber. In such installations, the temperature can also be raised in order to reduce the exposure time.

6.2.3.4 HIGH-PRESSURE CARBON DIOXIDE TREATMENTS One of the main disadvantages of treatments with inert gases is the long exposure time needed to kill all insect stages. The solution here is the use of carbon dioxide under high pressure in an autoclave (see Fig. 6.2.3.13). Such autoclaves can be constructed to take palletized goods (horizontal construction) or bulk products (vertical construction). Carbon dioxide is injected at pressures mostly between 20 and 30 bar. Exposure periods

6. AGRICULTURE

186

6.2. VEGETAL PRODUCTION

FIG. 6.2.3.10

Carbon dioxide and oxygen concentration during treatment of a biorice container. CO2 > 60%; O2 < 10%.

FIG. 6.2.3.11

Low oxygen treatment of highly valuable organic coffee with a nitrogen separator. 6. AGRICULTURE

6.2.3.4 HIGH-PRESSURE CARBON DIOXIDE TREATMENTS

FIG. 6.2.3.12

Treatment of exotic herbs in a chamber for low oxygen treatment.

FIG. 6.2.3.13

High-pressure CO2 treatment in an autoclave.

range between 2 and 3 h, depending on the pressure used. Due to the high pressure, the carbon dioxide is “pumped” into the body of the insects (all stages). Mortality is caused by a kind of

187

acidification of the body fluids. At the end of the treatment, the pressure is released relatively rapidly. The dissolved CO2 in the body fluids will try to escape the same way as when shaking

6. AGRICULTURE

188

6.2. VEGETAL PRODUCTION

and opening a soda bottle. Sometimes insects, and also insect eggs, literally explode during this phase of the process. This is a quick treatment method, but the capacity is low. Of course the investment cost of the autoclave, pumps, and tanks will have a serious impact on the exploitation costs. Organic products or products with high added value will be the targets for this technique. For example, the company Martin Bauer (Germany) has two horizontal autoclaves connected to each other. At the end of the fumigation cycle in one of them, the carbon dioxide is pumped in the other one as the start of that other cycle. This results in the use of less gas and thus cost savings.

6.2.3.5 LOW-PRESSURE PRESERVATION AND FUMIGATION TREATMENTS The opposite of high pressure is low pressure. Treatments at very low pressures (20 millibar) showed very promising results for stored product protection. Even insect eggs were killed in

FIG. 6.2.3.14

relatively short times. However, such low pressures cannot easily be obtained and maintained in commercial circumstances. Also, there are very few to no commercially available containers or packaging that can withstand such low pressures for a longer time. On the other hand, we now have strong barrier films with extremely low oxygen permeability. With the appropriate sealing devices, we can make from these foils bigbag-size bags. The aim is very clear: lower the partial oxygen pressure by a partial vacuum in such a way that the insects cannot find enough oxygen to breathe normally. As we already know, insects have no lungs, so they can’t actively take in the air they need to get their oxygen. All their stigmata will now be opened completely, desperately searching for enough air. At that moment, we add another “weapon” into the battle: we flush with either carbon dioxide or nitrogen, and then we go back to low pressure. With every cycle of “flushing and vacuumizing,” the oxygen content goes down. See Fig. 6.2.3.14. After that, the whole suffocation process can also be enhanced by raising the temperature. The higher the temperature, the higher the

Long-term insect control using VacQPack technology.

6. AGRICULTURE

189

6.2.3.6 CONCLUSIONS

FIG. 6.2.3.15

Sealing the special valve after vacuumizing.

activity and metabolism of the insects and the higher their oxygen demand. With their spiracles wide open, they will also lose a lot of water because the vapor pressure of the gas mixture is very low. This effect plays an evenly important role in insect mortality. Low oxygen in vacuumizing big bags not only has an effect on insect control, but by reducing the oxygen content, there will be less oxidation of the free fatty acids in the product, so deterioration processes can be slowed down or even inhibited. This means a significant increase in the shelf life of the product. Also, mold forming will be inhibited because the moisture content inside the packaging will be significantly diminished by replacing the ambient air with completely dry gases during each flushing cycle. Of course, the semivacuum has to be kept during the whole storage period. Four factors will determine the quality of the system:

• Oxygen permeability of the foil used. • Quality of the seals in the foil. • Presence and/or quality of a valve for extraction/injection. • Kind of cargo. All those operations can be carried out using either nitrogen or carbon dioxide as the flushing agent. They both have advantages and disadvantages. In such specialized markets, it’s advisable to ask for an expert opinion (Fig. 6.2.3.15).

6.2.3.6 CONCLUSIONS Inert gases for pest control are already well established. However, creativity is very important in these subjects. There are still so many subjects that are not investigated yet… Quite a challenge!!!

6. AGRICULTURE

S U B C H A P T E R

6.2.4 Algae Culture Philippe Granvillain, Rayen Filali, Francis A. Kurz R&D, ALGAE NATURAL FOOD SAS, Illkirch-Graffenstaden, France

6.2.4.1 CO2, GLOBAL WARMING, AND INDUSTRIAL EMISSIONS One major cause of global warming is now widely known as the large and steady increase of the CO2 level in the atmosphere since the advent of the industrial revolution in the mid1800s. The CO2 level, one of the prime sources of the greenhouse gas emission effect, rose over the past 40 years due to the exponential emissions of flue gases from industrial plants and fossil fuel modes of transportation. A typical industrial flue gas contains up to 20% CO2. Even though many nations, citizens, and NGOs worldwide have tried to limit the production of excessive levels of CO2, the exponential rise of the worldwide population has boosted the global demand for more products, impairing the efforts of global CO2 reduction. Carbon biological sequestration is one of the promising ways to reduce the excess CO2 from the atmosphere and industrial plants. Complete physical techniques are already well known and implemented: absorption, adsorption, cryogeny, and membranes (Velea and Dragos, 2009). Today, the cost to capture the CO2 produced by a standard thermal power facility is still too high, $128–967 per tons of carbon (IEA, n.d.), and therefore not economically sustainable. The only way to bring down the cost of capture is to both capture and fix efficiently the CO2 by a

system that may be a chemical, geological, or biological one, and turn CO2 into useful commercial molecules. Thus, CO2 from industrial processes might be valorized as one component for a new process, considered as belonging to the context of the circular economy. Hence, extensive studies have been carried out since then for CO2 sequestration. CO2 gas may be generated in agrofood facilities such as breweries, malthouse plants, or any plant where some fermentation processes are involved. The sequestration of the CO2 emissions by the food-processing industry is a promising step because no toxic byproduct gases such as SOx and NOx are produced, unlike those produced by power plants or boilers. One of the most promising ways for CO2 biofixation concerns the application of microalgae cells. Indeed, microalgae are photosynthetic microorganisms that are able to convert carbon dioxide into biomass, oxygen, and high-value molecules. Thus, many studies are focused on CO2 mitigation by cultures of microalgae strains. This biological process doesn’t require any separation and/or purification step of CO2 from flue gases: those can be directly used in the culture, and save around 70% of the total cost of CO2 fixation. The industrial CO2 emissions may be a valuable and potentially cheap source of carbon for growing plants and algae as a whole. Microalgae culture is considered a

6. AGRICULTURE

6.2.4.2 CARBON SOURCE FOR A BIOLOGICAL SYSTEM: CARBONATE SYSTEM

promising way for CO2 biofixation, also called CO2 capture, mitigation, sequestration, or removal. However, many flue gases contain other toxic gases such as SOx and NOx, which are toxic with a 50 ppm concentration for microalgae growth (Lee and Lee, 2003). Then, some upstream treatment might be necessary before using such gases in cultivation. Moreover, several flue gases usually contain particulate matter, heavy metals, and halogen acids that could end up in the biomass harvested that could not be used as valuable end products.

6.2.4.2 CARBON SOURCE FOR A BIOLOGICAL SYSTEM: CARBONATE SYSTEM Carbon dioxide is naturally dissolved into aqueous liquid, where it is slowly hydrated to form the diacide form, or carbonic acid: H2CO3 (pKa 2.84). It should be noted that CO2(aq) and H2CO3 are hard to distinguish. Then, H2CO3 dissociates quickly to form another mineral aqueous form: HCO3  , called bicarbonate ion, featuring a pKa of 6.35 (Dreybrodt et al., 1997). Bicarbonate quickly loses its last H+ at a pH above 10.33 representing its last pKa. This growth of living microorganisms depends on the carbon source, in the form of carbon dioxide, bicarbonate, and carbonate. The equilibrium of these forms on the aqueous solution is related to the carbonate system, considering the pH of the medium. To summarize, in an aqueous liquid environment where pH ranges from 6.35 to 10.33, which is suitable and experimentally observed for microalgae cultivation, the predominant form of the inorganic carbon is HCO3  , as summarized in Fig. 6.2.4.1. The carbon dioxide transfer from the gas to the liquid defines the quantity of the CO2 absorbed, which depends on the diffusion rate constant (kL), the effective mass transfer area (α), and the driving force concentration difference (ΔCO2). Thus, the CO2 mass transfer rate

191

FIG. 6.2.4.1 CO2 dissociation equations in aqueous solution (Aslam and Mughal, 2016).

NCO2 (mg L1 h1) is related to the volumetric gas-liquid mass transfer coefficient and CO2 concentration upon the following formula:   NCO2 ¼ kL α C∗CO2 L  CCO2 L where kL is the liquid-phase mass transfer coefficient (m h1), α is the specific area available for the mass transfer (m1), CCO2L∗ (mg L1) is the carbon dioxide concentration in CO2 in equilibrium with the outlet gas phase, given by Henry’s law, and CCO2L (mg L1) is the carbon dioxide concentration in the liquid (Markl, 1977). Current studies try to increase kL and α in order to raise the CO2 mass transfer rate by various sparging strategies and PBR configurations. Because of its acidic feature, and as displayed in Fig. 6.2.4.1, CO2 solubility also depends on the pH of the solution. Naturally, as gas solubility increases as the temperature decreases, a lower temperature promotes CO2 solubility; the outcome is the same for applying a higher inlet (sparging) gas pressure. But what is less known is that the more salt presence in the culture media, the less CO2 solubility (Liu et al., 2013). It should be noted that CO2 gas solubility is much higher than for O2 gas by a magnitude of 100: 1496 g CO2 L1 under pressure of 1 atmosphere at 25°C in water, explaining why the CO2 mass transfer from the gas form to liquid is rather fast (Aslam and Mughal, 2016). To conclude, the use of that kind of carbon source, either CO2 or HCO3  , by microalgae is therefore one of the envisaged credible ways to CO2 biofixation. This chemical process enables a better assimilation of the carbon by

6. AGRICULTURE

192

6.2. VEGETAL PRODUCTION

microalgae, and therefore illustrates perfectly the CO2 sequestration. This mineral carbon therefore turns into the organic form thanks to the microalgae metabolic way.

6.2.4.3 BIOLOGICAL MECHANISM FOR CO2 FIXATION BY MICROALGAE Microalgae are microorganisms that convert light energy and carbon sources into biomass and other high-value molecules. The primary metabolism is the photosynthetic activity. Photosynthesis by microalgae represents a bioenergetics process, unlike that developed by terrestrial plants. The mechanism of CO2 fixation is rather simple: the mineral form of carbon goes through the plasmatic membrane thanks to an active transport; the same phenomenon is carried out with the chloroplast membrane. The key enzyme involved for CO2 utilization into the well-known Calvin cycle is the Rubisco, which stands for ribulose-1,5-bisphosphate carboxylase/oxygenase (only the carboxylase aspect for fixing CO2 is of interest in this chapter) and is mainly responsible of the CO2 fixation efficiency (Badger and Spalding, 2000). The CO2 concentrating mechanism, or CCM, was a concept created to help understand the many ways all aerobic photosynthetic cells use for carbon fixation, which aim to set suitable conditions for the Rubisco carboxylation performances (Broda, 1975). Several components constitute the CCM: the active capture of CO2, the energy supply during photosynthesis (energy biological system ATP/NADPH), the intermediary species of CO2 (essentially HCO3  ), the CO2 release mechanism outside the cell by flowing (for cyanobacteria, that CO2 could be absorbed again into the cell by a recycling process), the CO2 concentrating section around Rubisco, the loss of CO2 diminution in CO2 generation sit by carbonic anhydrase, and the kinetic properties alteration of Rubisco (Badger

and Spalding, 2000). Interestingly, the intermediary species of the CO2 CCM component is HCO3  , which is localized into the chloroplast stroma for the microalgae eukaryotic cell and into the cytoplasm for the cyanobacteria cell. It should be noted that both the CO2 and HCO3  present in the aqueous media would potentially be absorbed and fixed by the microalgae cell. Another major enzyme involved in that process, the carbon anhydrase, turns HCO3  into CO2 into the cell. The local rise of the CO2 level enables the Rubisco to function well for producing the very first organic components. Fig. 6.2.4.2 shows an overview of the mechanisms involved for cyanobacteria (Ducat et al., 2011).

6.2.4.4 MICROALGAE CULTURE Cultivating microalgae is both a science and an art. As a science, since the 1960s, the literature has plenty of technical articles describing various cultivation system designs developed for specific microalgae strains, cultures conditions, and applications. As an art, it is common to think an effective microalgae cultivation as the right combination of three components: the right choice and selection of microalgae species, the identification and optimization of the microalgae environmental conditions, and the suitable culture system. About the selection of the microalgae and depending on the application, the most considered criteria concern the specific growth rate and high tolerance of the microalgae strains to an industrial flue gas containing high levels of CO2, NOx, and SOx, and potentially the tolerance to high temperature that usually comes with supplying flue gas. To select the right strain to be studied or produced, it is interesting to recoup both a bibliographical work with primary cultivation trials that might be performed into a laboratory photobioreactor, or microplates as displayed in the Fig. 6.2.4.3.

6. AGRICULTURE

6.2.4.4 MICROALGAE CULTURE

193

FIG. 6.2.4.2 Overview of cyanobacterial organization: (A) cross-section of a cyanobacteria: Synechococcus elongatus, (B) diagram of the complexes involves in the light-dependent ETC (Ducat et al., 2011).

FIG. 6.2.4.3

Microplate used for screening microalgae cultivation at Algae Natural Food, Riquewihr, France.

6. AGRICULTURE

194

6.2. VEGETAL PRODUCTION

In regard to the right strains to use, selected strains are either isolated, identified, and purified from natural and various habitats, or should be bought from a public or private microalgae bank. A strategy for improving cultivation systems is to perform physiological stress on the strains, such as severe culture conditions like pH, temperature, and light, to isolate the robust algae cells that deserve to be inoculated for further cultivation. However, genetic manipulation on microalgae cells to improve performance cultivation is not a current and global way of research, nor is it recommended for that purpose. Three microalgae cultivation modes are used so far: microalgae cells may grow (1) autotrophically, where mineral compounds and light are used, (2) heterotrophically, where organic compounds such as glucose and a dark mode is used, and (3) mixotrophycally, which is a blend of the first two modes, like using organic compounds and light to grow microalgae. Modes (1) and (3) are related to the purpose of that chapter being using flue gases for cultivation. Regarding the microalgae environmental condition, one of the most important steps concerns the identification of the optimal growth parameters and the composition of the medium. Water salinity is linked to the type of water, which may be freshwater, marine water, or hypersaline water. It also may display various minerals in different concentrations that would eventually affect cultivation and the nutritive medium that should be complemented. Moreover, for massive microalgae cultivation, the current stream of R&D is to valorize agrofood wastewaters with high mineral and biocomponent composition in order to avoid using artificial nutritive media to be bought and added to the cultivation. Formulation of wastewater for microalgae culture is more challenging as it represents a complex liquid system combining both minerals and organic parts, from small molecules to huge ones, and in equilibrium in a particular pH and temperature. Light is the second

major component to be addressed. Usually, cultivation uses natural sum beam and respects a light-dark cycle, called the photoperiod. Geographical aspects such as the latitude of the cultivation area, the season, and the time of the day determine the daily access of light for suitable growth. Artificial light may be used either for extra illumination supply, especially during winter or for Northern regions, or when an absolute industrial cultivation control is required by the commercial client, which is the case for highvalued molecules or microalgae extracts for the cosmetics industry. Then sparging rate, gas distribution, mixing quality, air quality, and composition are considered. This is not only to provide a suitable mass transfer of CO2 as a primary source of carbon, but also to avoid microalgae cell sedimentation, especially for those not equipped with flagella, that would impair growth because of the limitation of light access and bacteria development. Concerning the cultivation systems, two major systems are usually utilized: ponds or photobioreactors. Ponds, also called open ponds or open systems, may take many forms such as a natural pond where microalgae develop more or less monospecifically, that is, for one specific strain, as is the case for some African lakes where Spirulina develops naturally and is taken out and eaten by local inhabitants. Artificial ponds are also well known with a so-called raceway, typically designed in a U-shape. Other names exist such as round ponds or racetracktype ponds. Many sizes, lengths, deepnesses, and designs exist, from the lab scale to hundreds of cubic meters. The latter design represents the least expensive cultivation system for massive microalgae cultivation. Indeed, for artificial ponds, a mere large hole dug in the soil with a liner, all of which is equipped with a mechanical system to circulate the culture to avoid cell sedimentation, is the minimum required to produce microalgae for a low Capex. Working on, harvesting, and cleaning such a system displays a low Opex as well. However, a large area is

6. AGRICULTURE

6.2.4.4 MICROALGAE CULTURE

needed for setting up ponds, and this may represent a real issue where access to land is either difficult because of the topography, or expensive. Also, the growth productivity is usually low as the ratio of illuminated surface on useful culture volume is unfavorable. Moreover, because of their design, ponds are open to whatever comes from the air such as pollution, aerosols, etc., which would eventually contaminate the microalgae cultivation, including by other microalgae species. This explains why it is so difficult to cultivate a single microalgae strain in such a system usually required by the client. Finally, ponds display a poor ability to control culture parameters, such as controlling the light beam or water quality. For all the reasons

195

mentioned above, the microalgae biomass usually obtained in raceway ponds is not that much, around 0.5–1 g L1. This is unlike photobioreactors, which can display up to 4 g L1 (Davis et al., 2011), even though this high cell concentration induces serious shading phenomena that greatly limit the growth. From an area perspective, the productivity may attain 96 g m2 d1, giving a theoretical value of 350 ton ha1 year1 (Zamalloa et al., 2001). From an area perspective, the productivity may attain 96g m2 d1, giving a theoretical value of 350 ton ha1 year1 (Zamalloa et al., 2001). From the photobioreactor perspective, also called a closed system, many strategic designs have been developed for decades, such as the ones displayed in Fig. 6.2.4.4, and further

FIG. 6.2.4.4 Some photobioreactor designs: (A) flat PBR, (B) biocoil 1000 L tubular PBR, (C) plastic bag, and (D) air-lifted tubular PBR (Andersen, 2005).

6. AGRICULTURE

196

6.2. VEGETAL PRODUCTION

creation and development of designs and equipment are the current research. The simplest one is the column photobioreactor. It may be made of soft plastic forming a plastic bag, which is the cheapest photobioreactor to be made because these are industrially manufactured as rolls from which the operator can cut the proper length to get the volume wanted for microalgae cultivation. It may also be made of plain plastic.

6.2.4.5 CO2 BIOFIXATION BY MICROALGAE: MICROALGAE SPECIES AND PERFORMANCES CO2 biofixation by microalgae depends on the right selection of microalgae strain to be cultivated into its proper cultivation system (specific customized pond or PBR) and optimized under the local environment (sunlight access, fresh or seawater, wastewater) and specific parameters (sparging rate and quality, pH control, nutritive medium). The scientific literature is full of references to microalgae tolerance for high levels of CO2 (Tang et al., 2011) and those used for CO2 mitigation such as Botryococcus braunii, Chlorella vulgaris, Chlorella kessleri, Chlorocuccum littorale, Scenedesmus sp., Chlamydomonas reinhardtii, and Spirulina sp. (Aslam and Mughal, 2016), even though strain selection is not easy because of their morphology, growth features, or various environmental factors (Velea and Dragos, 2009). Specifically, it regularly describes such a performance with the microalgae Chlorella. Indeed, the strain C. vulgaris features an outstanding capacity to fix CO2 at a pace of more than 6 g L1 d1 (Cheng et al., 2006). It has also been demonstrated than the strain Chlorella sp. can also remove nitrogen oxides and sulfur dioxides from gas and CO2 at a pace of 0.8–1 g L1 d1 (Keffer and Kleinheinz, 2002), and it has been confirmed that Chlorella is a great CO2 removal algae (Chiu et al., 2008).

Microalgae uses CO2 for two main reasons: creating and developing its own cell organites for biomass productivity or for assuring that its own metabolism (Chiu et al., 2009) can survive and develop, and where carbon mitigation may be lowered, as demonstrated for some species where optimal CO2 removal occurred at 1%, even though higher biomass productivity is observed beyond that CO2 rate (Ramanan et al., 2010). Moreover, the more CO2 is used for microalgae growth, the more lipids and fatty acids the latter may produce, representing an obvious environmental and economical field of interest for mankind. Indeed, several microalgae show an increase production of polyunsaturated fatty acids (PUFAs) and total lipids when sparged with 30%–50% of CO2 (Chiu et al., 2008; Tang et al., 2011), which could be caused by a diminution of the O2 level affecting enzymatic desaturation (Vargas et al., 1998). For instance, Chlorella concentrated fatty acids starting from 0.04% up to 4%–5% of CO2 (Tsuzuki et al., 1990). Generally, it is now accepted by the scientific community that a level of CO2 that ranges from 0.2% to 5% in the gas flow is recommended to achieve sufficient biomass productivity for most microalgae species. For instance, Nannochloropsis occulata grows well at 2% of CO2 but its growth is stopped at 5% (Hsueh et al., 2007). Some species develop well with sparging gas containing between 5% and 20% of CO2, which is considered a significant level. However, a level above 20% which is considered high, may impair the cultivation and reduce biomass productivity (Silva and Pirt, 1984; Lee and Tay, 1991), even though it depends on the microalgal cell concentration and pH (Chiu et al., 2008, 2009; Olaizola, 2003). Finally, few specific strains grow quickly under a CO2 level up to 20%. It has even been shown that some microalgae displayed a high rate of photosynthesis when grown under 40% of CO2 concentration, and may show a good CO2 tolerance up to 100% of CO2 (Matsumoto et al., 1997; Olaizola, 2003).

6. AGRICULTURE

6.2.4.5 CO2 BIOFIXATION BY MICROALGAE: MICROALGAE SPECIES AND PERFORMANCES

rate of more than 1 g CO2 L1 d1 for Chlorella sp. (freshwater species) and Chlorococcum littorale (marine species) (Murakami and Ikenouchi, 1997). The main criterion to quantify the CO2 mitigation from a biological system is to determine the CO2 efficiency, given by the following formula (Chiu et al., 2009):

That CO2 tolerance limitation could be explained either by the stress the CO2 high level represents for microalgae photosystem II, which is inhibited (Xu et al., 2003), or by the rise of the osmotic pressure on the microalgal cell (Soletto et al., 2008). It should be noted that CO2 tolerance surprisingly depends on nutrients and light as well (Soletto et al., 2008). Identification of a strain that grows under a higher CO2 level is one of the current research methods. Using a lab photobioreactor, researchers demonstrated that some microalgae absorb CO2 gas generated by coal power plants. One of the parameter used to demonstrate sequestration performances is the CO2 conversion efficiency into algal biomass. For instance, 1.5 kg of CO2 may be transformed into 1 kg dry weight of Chlorella (Velea and Dragos, 2009); a similar observation gives 2 tons of CO2 generating 1 ton of microalgae biomass (Stepan et al., 2002). Another scientific source reported a CO2 fixation TABLE 6.2.4.1

197

CO2 efficiency ð%Þ ¼

ðCO2 influent  CO2 effluentÞ∗ 100%=CO2 influent

Table 6.2.4.1 illustrates the CO2 efficiency for some microalgae species. According to Table 6.2.4.1, the CO2 removal rate is similar to the CO2 fixation rate, RCO2, measured in (g CO2 m3 h1), and can be related to the carbon content CC, in g/g of cell dry weight, of the microalgae biomass by the following formula (Yun et al., 1997): RCO2 ¼ CC μL ∗ ðMCO2 =Mc Þ

CO2 Removal Efficiency From Some Microalgae Species (Aslam and Mughal, 2016)

Microalgae Species

Cultivation System

Temperature (°C)

Lipid Content (% Dry Weight)

Chlorella sp.

Sequential bioreactor

27

18–48

15

1

85.6

Andersen (2005) and Velea and Dragos (2009)

Chlorococcum littorale

Fate PBR

25

19.3

20

0.4

16.7

Ono and Cuello (2007), Ramanan et al. (2010), and Andersen (2005)

Monoruphidium minutum

Flask

25

–

13.6

1

90

Silva and Pirt (1984) and Soletto et al. (2008)

Nannochloropsis sp. Cylindrical glass PBR

25–27

35.7

2–15

0.17

11–47

Lee et al. (2000) and Sierra et al. (2008)

Spirulina sp.

30

4–16.6

6

0.22

53.29

Andersen (2005), Rodolfi et al. (2009), Roncallo et al. (2012), and Sanchez Miron et al. (2000)

Tubular PBR

CO2 Concentration (%)

CO2 Growth Rate Removal (g L21 d21) (%)

6. AGRICULTURE

Reference

198

6.2. VEGETAL PRODUCTION

where μL is the volumetric growth rate (g dry weight m3 h1) in the linear growth phase, and MCO2 and Mc are, respectively, the molecular weight of CO2 and C. However, it has been observed that the fixation rate of CO2 by microalgae is low. Usually, the flue gas generated by a plant is high in temperature: the strains to be chosen should therefore be thermotolerant, like the ones that naturally grow in hot spring water or that grow well around or above 30°C. Hence, a microalgae cultivation area should be set up next to such an industrial gas-emitting plant. For instance, hot spring water Chlorella in Japan has been cultivated under a CO2 level of at least 40% and 42°C (Wang et al., 2008). Regarding the cyanobacteria Spirulina sp., the productivity attained under 12% of CO2 was 0.22 g L1 d1 with a yield of 3.50 g L1 DW (Murakami and Ikenouchi, 1997). Another example is for preselected Scenedesmus and Chlorella that have been grown under 50% of CO2 with a good growth rate (Hanagata and Takeuchi, 1992).

6.2.4.6 THE USE OF NOX AND SOX BY MICROALGAE For untreated flue gases displaying upper levels of CO2, SOx, and NOx, the common scientific approach is to select strains that are capable of biologically resisting such gases and that present a high growth rate along with a sufficient cell density (IEA, n.d.). Otherwise, the culture may be at least inhibited and eventually die.

Also, the more tolerant the strain to those untreated gases, the less the potential cost of gas pretreatment is considered. A good example concerns the new Chlorella called KR-1, which has not been inhibited under 100 ppm of NO, 50 ppm of SO2, and 20% of CO2 (Lee et al., 2002; Sung and Lee, 1998). Table 6.2.4.2 gives insight into such a tolerance with microalgae growth performance.

6.2.4.7 TOLERANCE AND REMOVAL OF NOX FROM MICROALGAE The biofixation process of NO and NO2 is quite efficient during the exponential phase of the microalgal culture, but might inhibit the growth if added too early at the beginning of the growth. Several parameters are considered when studying the NOx tolerance. Among them, the microalgae strain selection is one of the most considered parameters; the growth phase and NOx concentration at a given flow rate are also important parameters (Aslam and Mughal, 2016). Nitrogen oxide NO represents the most of NOx from flue gases, more than 90%, and is interesting to be eliminated by a microalgal system because of its poor aqueous solubility ( Jin et al., 2005). To avoid this limitation, researchers added chelatant agents to turn the NO in gas form into an aqueous and stable form, like a metal-nitrosyl complexes. For instance, adding ethylenediaminetetraacetic acid chelated with

TABLE 6.2.4.2 Growth Features of Microalgae Suitable for CO2 Absorption Under Toxic Gases (Aslam and Mughal, 2016; Lee and Lee, 2003) Microalgae

CO2 (%)

NOx (ppm)

SOx (ppm)

Growth Rate in Linear Phase (g L21 day21)

C. littorale

70

50

30

0.47

Chlorella HA-1

20

100

50

0.51

Chlorella KR-1

30

100

100

0.78

6. AGRICULTURE

6.2.4.8 TOLERANCE AND REMOVAL OF SOX FROM MICROALGAE

Iron (II): Fe(II)EDTA to NO(g) rapidly generates the aqueous form of NO: Fe(II)EDTA-NO(aq) ( Jin et al., 2008). Other chelating agents may be used: nitrilotriacetic acid (NTA), methyliminodiacetic acid (MIDA), or dimercaptopropanesulfonic acid (DMPS). However, adding such a chelating agent might be considered a micropollutant and might impair the subsequent use of the microalgal biomass. Moreover, it has been demonstrated that the NOx removal rate is quite low in a microalgal system, for example, 0:74gNOx mreactor 3 d1 (Van Den Hende et al., 2011), and does not represent a major way to get rid of it. Some complexes are also unstable under long sunlight exposure, which is the case for iron EDTA (Lockhart and Blakeley, 1975). Nannochloropsis sp. cultivation removed half the NOx present at 300 ppm from flue gases (Yoshihara et al., 1996). It should be noted that NO partially oxidized into the aqueous molecule of NO2 by reacting with dioxygen generated from the algae culture during photosynthesis, and therefore may inhibit the cultivation of microalgae (Matsumoto et al., 1997).

6.2.4.8 TOLERANCE AND REMOVAL OF SOX FROM MICROALGAE Microalgae are known to be more sensitive to SOx uptake and can easily be inhibited. Also, the SO2 may be literally toxic for some species (Lee et al., 2000). The admitted threshold for SO2 is 50 ppm for many strains (Yanagi et al., 1995). This is partly due to the decrease of the cultivation media pH following SOx sparging; for instance, 400 ppm of sulfur dioxide SO2 decreases the pH to 4, sufficiently acidic to stop microalgae growth (Matsumoto et al., 1997; Stepan et al., 2002). But the process is not irreversible: adding some basic chemical agents such as NaOH increases pH and the culture grows normally, even with SOx (Matsumoto

199

et al., 1997). Another reason for that potential detrimental effect is the anion bisulfite HSO3ðaqÞ  concentrations, as this plays the role of oxidizing or reducing agent. That could eventually generate highly oxidative molecules that can damage microalgae organites and biomolecules and therefore can stop microalgae growth. HSO3ðaqÞ  is formed by easily dissolving SO2 in gas form with carbonate CO3ðaqÞ 2 . An illustration about the toxicity concerns concentrations below 104 mg L1 of NaHSO3, the aqueous form of sulfur HSO3ðaqÞ  is a good S-source for B. braunii once oxidized into sulfate SO4 2 , the preferred form for microalgae assimilation; but a level above 104 mg L1 of NaHSO3 is shown toxic for that microalgae (Yang et al., 2004). This can be explained by the oxidative entities, such as hydroxyl radicals, superoxide anions, or hydrogen peroxides, that are formed during the oxidation from HSO3  to SO4 2 and cause the peroxidation of the lipids building membranes and bleaching of chlorophyll (Giordano et al., 2005). So, an artificial rise of pH during cultivation, to at least pH 6, should limit the formation of HSO3  and its potential detrimental consequences on cultivation (Lee et al., 2000). Not much scientific data has been released so far about the removal rate of SO2 by microalgae. Besides, it is a common practice in the microalgae world to use the vessel volumes per minute (vvm) as a measure of the flow rate; this is merely the standard gas that is a gas volume running per minute, the gas flow rate, but also per bioreactor volume. A study displayed a rate of only 3.2 mg SO2 L1 d1, conveyed into a very low gas flow rate of 0.0050 vvm containing 572 mg N m3 SO2 from flue gas. As expected, the SO4 2 increased at the same time from 50.7 to 54.9 mg L1 in the liquid cultivation media (Van Den Hende et al., 2011). From an industrial perspective, once the biomass is harvested from culture, the remaining cultivation media would be discharged for subsequent water treatment. However, a higher sulfate level would not let that wastewater be eligible for treatment

6. AGRICULTURE

200

6.2. VEGETAL PRODUCTION

because of regulations, which is for instance from 90 to 150mgSO4 2 L1 . This constitutes a roadblock for flue gas SO2 removal, much more so than microalgae SO2 tolerance (Aslam and Mughal, 2016).

6.2.4.9 EFFECTS OF EXTERNAL PARAMETERS 6.2.4.9.1 Mass Transfer, Flow, and Mixing For a microalgal cultivation system, mass transfers involves the absorption by the microalgae (solid phase) of the nutritive components dissolved in the cultivation media (liquid phase) and including CO2 both in gas form (gas phase) and aqueous form (HCO3  ) (liquid phase), as summarized in Fig. 6.2.4.5. Optimization comes both from new theoretical equations (Sanchez Miron et al., 2000) and experiments by varying CO2 concentration, bubble size, gas flow rate, gas holdup, and superficial aeration velocity. CO2 removal is enhanced when the adequate algae species is used under optimized operating conditions, as illustrated in Table 6.2.4.3 for Chlorella sp. (Chiu et al., 2008). It has also been demonstrated that a gradual supply of CO2 for microalgae cultivation to a significant level could increase the growth and CO2 removal rate more than a constant rate. This

FIG. 6.2.4.5 Mass transfer involved in a microalgae system (Velea and Dragos, 2009).

TABLE 6.2.4.3 CO2 Removal for Chlorella sp. (Chiu et al., 2008) CO2 (v/v)

CO2 Removal Efficiency (%)

2

58

5

27

10

20

15

16

can be explained by a better adaptation of the microalgae for CO2 tolerance (Yun et al., 1996). The optimal aeration rate recommended, embodied by the gas volumetric flow rate per unit volumetric culture medium (vvm), ranges from 0.025 to 1, and depends on the species and PBR configuration. In that configuration, 5%–10% (v/v) of CO2 seems to be effective for a satisfying CO2 removal by microalgae (Sierra et al., 2008). Above that range, the aeration rate will eventually cause shear stress due to bubble generation, coalescence, and breakup.

6.2.4.9.2 Artificial and Natural Light Two common strategies exist about the light supply for microalgae cultivation: (1) natural light and (2) artificial light. The main interest of the latter is that the light is controlled by operators both in intensity (measured in W m2 or μmol m2 s1, even in lux, which provides an idea about light intensity but is not recognized as a thorough light measure), and light beam direction (meaning the right incident angle into the microalgae culture). This approach may be used whenever strict cultivation control is required, typically for producing high-value compound purposes, that is, in the biotech, cosmetics, or health industry, or to supply extra light at natural light when the season, weather, or production site location is unfavorable. However, the main roadblock is the cost of artificial light because it is usually high in Capex and

6. AGRICULTURE

6.2.4.10 VALUABLE COMPOUNDS FROM MICROALGAE

Opex, where a high power supply consumption is needed to operate that kind of lighting. Current development about artificial light is the utilization of LEDs, optical fibers, and thorough work on the photoperiod, which is the right balance between dark and light duration for a period of time and for a given strain. When thinking in terms of a large-scale cultivation area, the most economical way to supply light is obviously the use of sunlight, which is the cheapest way to supply light for a massive cultivation facility. For instance, the highest light intensity is 1100 W m2 at midday (Miyake et al., 1999). Stated otherwise, a typically sunny day in France gives around 1350 μmol m2 s1 (80,000 lux). Interestingly, only dozens of μmol m2 s1 are enough to let the microalgae cell do its photosynthesis overcoming its respiration mode, and then starting to produce biomass and growth; for example, only 34μmol m2 s1 are enough to yield a maximum cell density of 37.5 106 cell mL1 for the strain Nannochloropsis sp. in a 25-cm diameter vertical photobioreactor (Roncallo et al., 2012). Unfortunately, sunlight is not constant during the course of the days, seasons, or latitudes. Moreover, as that way for lightening is used for outdoor open pond cultivation, sunlight comes with UV-radiations those might damage microalgae cells (Chen et al., 2008), and also implies a day-night cultivation mode where half of the period of time of a culture is not illuminated. All those factors largely impair the cultivation and explain why the growth rate is low in such a cultivation mode. That general lack of light energy impairs the light conversion efficiency, impacting the metabolism of microalgae cells where the biocomposition of algae is modified; that is, the carbohydrate content decreases for Chlorella pyrenoidosa at night (Ogbonna and Tanaka, 1998).

6.2.4.9.3 Temperature Temperature greatly influences the microalgae cultivation performance because it is directly linked to enzyme optimum functioning

201

and therefore, metabolism. Hence, it is common to cultivate Spirulina strains under 35°C in situ for achieving maximum productivity; that explains why such a strain is massively produced in sunny regions and inside a greenhouse to promote high temperature for cultivation. Another example is for the Nannochloropsis strain, which grows better when the temperature is less than 25°C. However, the flue gas used as a CO2 inlet usually comes with very high temperatures, usually around 120°C (that is, in power plants). For such a cultivation mode, the strategy is to select strains tolerant to high temperature to both assure a workable biomass production and to save on the cost of cooling flue gases (Ono and Cuello, 2007). Therefore, either mesophilic microalgae, where optimal growth occurs between 13°C and 45°C, or thermophilic ones that range from 42°C to 75°C, with both displaying a high tolerance to CO2, are the best candidates for achieving significant biomass production when using flue gases. The main issue of working at those high temperatures is eventually water evaporation of the cultivation medium that concentrate dissolved nutritional salts, increase the shadowing effect by rising the cell density, and might impact the pH, perturbating the smooth run of the microalgae cultivation.

6.2.4.10 VALUABLE COMPOUNDS FROM MICROALGAE By choosing specific microalgae with high CO2 fixation capability and growth rate, the valorization of the microalgae biomass can be considered for the production of valuable and sellable biocompounds, CO2 level increase may at least be offset, no saying decreasing on the long run. Clearly, there are two ways to value biocompounds produced by microalgae when grown for CO2 biofixation. The first consists of recovering compounds that may be edible, either for the

6. AGRICULTURE

202

6.2. VEGETAL PRODUCTION

feed or the food industry. This may be possible only when CO2 gas from an agroplant is used for microalgae culture, which does not usually occur unless treated flue gas is used. Thus, many feed additives such as antioxidants, amino acids, pigments, crude proteins, PUFAs, and pharmaceuticals may be obtained from such cultivation. Selling prices per kg are usually higher than those of nonedible compounds, potentially reaching $100,000 per kg for very high value products. The other way is to extract compounds for nonedible utilization, and this is the preferred way when using CO2 from flue gas containing toxic SOx and NOx. For the latter, hydrocarbons may be extracted by solvent in order to make some biofuels by thermochemical liquefaction. Another way is to mix a synthetic polymer with microalgae to produce new building material. This was done by mixing C. vulgaris with polyvinylchloride into molds to create new structures (Velea and Dragos, 2009), but again, the economic yield has to be thoroughly checked.

6.2.4.11 CONCLUSION So much work has been done so far to understand the mechanism of absorption and use of various gases by microalgae, and especially the flue gas ones. By that way, microalgae are fully entering into a new business model, the circular economy, where algae should be grown industrially in a very large scale in a profitable way while using a massive quantity of flue gas with a beneficial environmental impact. Hence, the issue about gas transportation for making that algae cultivation concept true. The new business model should be based on a solid, win-win, and durable industrial partnership between a large-scale algae producer and an industrial plant wanting to get rid of its gas for various reasons. Worldwide, very rare microalgae producers have already installed their algae culture plant aside to an industrial flue gas producer; Algae Natural Food from

Riquewihr, France, has done it since 2014 by setting up its Spirulina cultivation facilities next to a malting plant in Strasbourg, France, that produces malt for the beer industry from barley. In that business model, Algae Natural Food uses not only the rinsing water from barley as the liquid base to cultivate Spirulina inside, saving hundreds of cubic meters of fresh water, but also it uses calorific energy that the malting plant produces to keep the right cultivation temperature, especially during winter, and of course the CO2 gas coming from the respiration of barley during the germination into malt process. The short distance between the algae cultivation platform and the malting plant enables getting a low CAPEX and OPEX for production microalgae, and consequently offers an attractive selling price for the food and feed industries. Reusing byproducts of plants such as flue gases in order to restart new primary biological productions as microalgae not only permits industrially acting in an economically friendly way, but also significantly decreases production costs and opens new markets. The circular economy fully applies here and is now a reality for producing microalgae that way, and will develop for the next generations to come.

References Andersen, R.A., 2005. Algal Culturing Techniques, first ed. Academic Press. Aslam, A., Mughal, T.A., 2016. A review on microalgae to achieve maximal carbon dioxide (CO2) mitigation from industrial flue gases. Int. J. Res. Advent Technol. 4, 12–29. Badger, M.R., Spalding, M.H., 2000. In: Leegood, R.C., Sharkey, T.D., von Caemmerer, S. (Eds.), Photosynthesis: Physiology and Metabolism. Kluwer, pp. 369–397. Broda, E., 1975. The Evolution of Bioenergetic Processes. Pergamon Press, Oxford, pp. 135–137. Chen, C.Y., Saratale, G.D., Lee, C.M., Chen, P.C., Chang, J.S., 2008. Phototrophic hydrogen production in photobioreactors coupled with solar-energy-excited optical fibers. Int. J. Hydrogen Energy 33 (23), 6886–6895. Cheng, L., Zhang, L., Chen, H., Gao, C., 2006. Carbon dioxide removal from air by microalgae cultured in a membranephotobioreactor. Sep. Purif. Technol. 50 (3), 324–329.

6. AGRICULTURE

REFERENCES

Chiu, S.Y., Kao, C.Y., Chen, C.H., Kuan, T.C., Ong, S.C., Lin, C.S., 2008. Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Bioresour. Technol. 99 (9), 3389–3396. Chiu, S.Y., Kao, C.Y., Tsai, M.T., Ong, S.C., Chen, C.H., Lin, C.S., 2009. Lipid accumulation and CO2 utilization of Nannochloropsis occulata in response to CO2 aeration. Bioresour. Technol. 100 (2), 833–838. Davis, R., Aden, A., Pienkos, P.T., 2011. Techno-economic analysis of autotrophic microalgae for fuel production. Appl. Energy 88 (10), 3524–3531. Dreybrodt, W., Eisenlohr, L., Madry, B., Ringer, S., 1997. Precipitation kinetics of calcite in the system CaCO3 H2O CO2: the conversion to CO2 by the slow process H+ + HCO3 ! CO2 + H2O as a rate limiting step. Geochim. Cosmochim. Acta 61 (18), 3897–3904. Ducat, D.C., Way, J.C., Silver, P.A., 2011. Engineering cyanobacteria to generate high-value products. Trends Biotechnol. 29 (2), 103–195. Giordano, M., Beardall, J., Raven, J.A., 2005. CO2 concentrating mechanisms in algae: mechanisms, environmental modulation, and evolution. Annu. Rev. Plant Biol. 56, 99–131. Hanagata, N., Takeuchi, T., 1992. Tolerance of microalgae to high CO2 and high temperature. Phytochemistry 31, 3345–3348. Hsueh, H.T., Chu, H., Yu, S.T., 2007. A batch study on the bio-fixation of carbon dioxide in the absorbed solution from a chemical wet scrubber by hot spring and marine algae. Chemosphere 66 (5), 878–886. IEA, n.d. Carbon Dioxide Capture From Power Stations. www.ieagreen.org.uk. Jin, Y., Veiga, M.C., Kennes, C., 2005. Bioprocesses for the removal of nitrogen oxides from polluted air. J. Chem. Technol. Biotechnol. 80 (5), 483–494. Jin, H.F., Santiago, D.E., Park, J., Lee, K., 2008. Enhancement of nitric oxide solubility using Fe(II) EDTA and its removal by green algae Scenedesmus sp. Biotechnol. Bioprocess Eng. 13 (1), 48–52. Keffer, J.E., Kleinheinz, G.T., 2002. Use of Chlorella vulgaris for CO2 mitigation in a photobioreactor. J. Ind. Microbiol. Biotechnol. 29 (5), 275–280. Lee, J.-S., Lee, J.-P., 2003. Review of advances in biological CO2 mitigation technology. Biotechnol. Bioprocess Eng. 8, 354–359. Lee, Y., Tay, H.S., 1991. High CO2 partial pressure depresses productivity and bioenergenetic growth yield of Chlorella pyrenoidosa culture. J. Appl. Phycol. 3, 95–101. Lee, J.H., Lee, J.S., Shin, C.S., Park, S.C., Kim, S.W., 2000. Effects of NO and SO2 on growth of highly-CO2-tolerant microalgae. J. Microbiol. Biotechnol. 10 (3), 338–343. Lee, J.S., Kim, D.K., Lee, J.P., Park, S.C., Koh, J.H., Cho, H.S., Kim, S.W., 2002. Effects of SO2 and NO on growth of Chlorella sp. KR-1. Bioresour. Technol. 82 (1), 1–4.

203

Liu, R., Liu, Y., Z, C., 2013. Development of an efficient CFDsimulation method to optimize the structure parameters of an airfilt sonobioreactor. Chem. Eng. Res. Des. 91 (2), 220–221. Lockhart, H.B., Blakeley, R.V., 1975. Aerobic photodegradation of iron (III)-(ethylenedinitrilo) tetraacetate (ferric EDTA). Implications for natural waters. Environ. Sci. Technol. 9 (12), 1035–1038. Markl, H., 1977. CO2 transport and photosynthetic productivity of a continuous culture of algae. Biotechnol. Bioeng. 19 (12), 1851–1862. Matsumoto, H., Hamasaki, A., Sioji, N., Ikuta, Y., 1997. Influence of CO2, SO2 and NO in flue gas on microalgae productivity. J. Chem. Eng. Jpn. 30 (4), 620–624. Miyake, J., Wakayama, T., Schnackenberg, J., Arai, T., Asada, Y., 1999. Simulation of the daily sunlight illumination pattern for bacterial photo-hydrogen production. J. Biosci. Bioeng. 88 (6), 659–663. Murakami, M., Ikenouchi, M., 1997. The biological CO2 fixation and utilization project by rite—screening and breeding of microalgae with high capability in fixing CO2. Energ. Convers. Manage. 38, S493–S497. Ogbonna, J.C., Tanaka, H., 1998. Cyclic autotrophic/heterotrophic cultivation of photosynthetic cells: a method of achieving continuous cell growth under light/dark cycles. Bioresour. Technol. 65 (1), 65–72. Olaizola, M., 2003. Microalgal removal of CO2 from flue gases: changes in medium pH and flue gas composition do not appear to affect the photochemical yield of microalgal cultures. Biotechnol. Bioprocess Eng. 8 (6), 360–367. Ono, E., Cuello, J.L., 2007. Carbon dioxide mitigation using thermophilic cyanobacteria. Biosystem Eng. 96 (1), 129–134. Ramanan, R., Kannan, K., Deshkar, A., Yadav, R., Chakrabarti, T., 2010. Enhanced algal CO2 sequestration through calcite deposition by Chlorella sp. and Spirulina platensis in mini-raceway pond. Bioresour. Technol. 101 (8), 2616–2622. Rodolfi, L., Chini Zittelli, G., Bassi, N., Padovani, G., Biondi, N., Bonini, G., Tredici, M.R., 2009. Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102 (1), 100–112. Roncallo, O.P., Garcia Freites, S., Paternina Castillo, E., Bula Silvera, A., Cortina, A., Acuna, F., 2012. Comparison of two different vertical column photobioreactors for the cultivation of Nannochloropsis sp. J. Energy Res. Technol. 135, 1–7. Sanchez Miron, A., Garcia Camacho, F., Contreras Gomez, A., Grima, E.M., Chisti, Y., 2000. Bubble-column and airlift photobioreactors for algal culture. AIChE J. 46 (9), 1872–1887. Sierra, E., Acien, F.G., Fernandez, J.M., Garcia, J.L., Gonzales, C., Molina, E., 2008. Characterization of a flat

6. AGRICULTURE

204

6.2. VEGETAL PRODUCTION

plate photobioreactor for the production of microalgae. Chem. Eng. J. 138 (1), 136–147. Silva, H., Pirt, S.J., 1984. Carbon dioxide inhibition of photosynthetic growth of Chlorella. J. Gen. Microbiol. 130, 2833–2838. Soletto, D., Binaghi, L., Ferrari, L., Lodi, A., Carvalho, J.C.M., Zilli, M., Converti, A., 2008. Effects of carbon dioxides feeding rate and light intensity on the fed-batch pulsefeeding cultivation of Spirulina platensis in helical photobioreactor. Biochem. Eng. J. 39 (2), 369–375. Stepan, D.J., Shockey, R.E., Moe, T.A., Dorn, R., 2002. Carbon Dioxide Sequestering Using Microalgal System. University of North Dakota. Sung, K.D., Lee, J.S., 1998. Isolation of a new highly CO2 tolerant fresh-water microalga Chlorella KR-1. Korean J. Chem. Eng. 15, 449–450. Tang, D., Han, W., Li, P., Miao, X., Zhong, J., 2011. CO2 biofixation and fatty acid composition of Scenedesmus obliquus and Chlorella pyrenoidosa in response to different CO2 levels. Bioresour. Technol. 102 (3), 3071–3076. Tsuzuki, M., Ohnuma, E., Sato, N., Takaku, T., Kawaguchi, A., 1990. Effects of CO2 concentration during growth on fatty acid composition in microalgae. Plant Physiol. 93 (3), 851–856. Van Den Hende, S., Vervaeren, H., Desmet, S., Boon, N., 2011. Bioflocculation of microalgae and bacteria combined with flue gas to improve sewage treatment. New Biotechnol. 29 (1), 23–31. Vargas, M.A., Rodriguez, H., Moreno, J., Olivares, H., Del Campo, J.A., Rivas, J., Guerrero, M.G., 1998. Biochemical composition and fatty acid content of filamentous nitrogen-fixing cyanobacteria. J. Phycol. 34 (5), 812–817. Velea, S., Dragos, N., 2009. Biological sequestration of carbon dioxide from thermal power plant emissions, by aborbtion in microalgal culture media. Rom. Biotechnol. Lett. 14 (4), 4485–4500. Wang, B., Li, Y., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79 (5), 707–718. Xu, M., Chen, S., Liu, G., Hu, Z., 2003. Pilot study of physiological and morphological acclimation of Scenedesmus armatus under extreme high CO2 stress. Wuhan Bot. Res. 22 (5), 439–444. Yanagi, M., Watanabe, Y., Saiki, H., 1995. CO2 fixation by Chlorella sp. HA-1 and its utilization. Energ. Convers. Manage. 36 (6), 713–716. Yang, S., Wang, J., Cong, W., Cai, Z., Ouyang, F., 2004. Effects of bisulfite and sulfite on the microalga Botryococcus braunii. Enzyme Microb. Technol. 35 (1), 46–50. Yoshihara, K.I., Nagase, H., Eguchi, K., Hirata, K., Miyamoto, K., 1996. Biological elimination of nitric oxide

and carbon dioxide from flue gas by marine microalga NOA-113 cultivated in a long tubular photobioreactor. J. Ferment. Bioeng. 82 (4), 351–354. Yun, Y.S., Park, J.M., Yang, J.W., 1996. Enhancement of CO2 tolerance of Chlorella vulgaris by gradual increase of CO2 concentration. Biotechnol. Tech. 10 (9), 713–716. Yun, Y.S., Lee, S.B., Park, J.M., Lee, C.I., Yang, J.W., 1997. Carbon dioxide fixation by algal cultivation using wastewater nutrients. J. Chem. Technol. Biotechnol. 69 (4), 451–455. Zamalloa, C., Vulsteke, E., Albrecht, J., Verstraete, W., 2001. The techno-economic potential of renewable energy through the anaerobic disgestion of microalgae. Bioresour. Technol. 102 (2), 1149–1158.

Further Reading Benemann, J.R., 1997. CO2 mitigation with microalgae systems. Energ. Convers. Manage. 38, S475–S479. Brown, L.M., 1996. Uptake of carbon dioxide from flue gas by microalgae. Energ. Convers. Manage. 37 (6), 1363–1367. De Morais, M.G., Costa, J.A.V., 2007a. Biofixation of carbon dioxide by Spirulina sp. and Scenedesmus obliquus cultivated in a three-stage serial tubular photobioreactor. J. Biotechnol. 129 (3), 439–445. De Morais, M.G., Costa, J.A.V., 2007b. Carbon dioxide fixation by Chlorella kessleri, C. vulgaris, Scenedesmus obliquus and Spirulina sp. cultivated in flasks and vertical tubular photobioreactors. Biotechnol. Lett. 29 (9), 1349–1352. De Morais, M.G., Costa, J.A.V., 2007c. Isolation and selection of microalgae from coal fired thermoelectric power plant for biofixation of carbon dioxide. Energ. Convers. Manage. 48 (7), 2169–2173. Giavarini, C., Maccioni, F., Santarelli, M.L., 2010. CO2 sequestration from coal fired power plants. Fuel 89 (3), 623–628. Hu, Q., Kurano, N., Kawachi, M., Iwasaki, I., Miyachi, S., 1998. Ultrahigh-cell-density culture of a marine green alga Chlorococcum littorale in a flat-plate photobioreactor. Appl. Microbiol. Biotechnol. 49 (6), 655–662. Iwazaki, I., Hu, Q., Kurano, N., Miyachi, S., 1998. Effect of extremely high-CO2 stress on energy distribution between photosystem I and photosystem II in a “highCO2” tolerant green algae, Chlorococcum littorale and intolerant green alga Stichococcus bacillaris. J. Photochem. Photobiol. B: Biol. 44 (3), 184–190. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel production and other applications: a review. Renew. Sustain. Energy Rev. 14 (1), 217–232.

6. AGRICULTURE

S U B C H A P T E R

6.2.5 Liquid Nitrogen: A Sustainable Solution to Cryopreserve Life ` s Camus, Olivier Couture, Anne Linda Van Kappel, Guy Delhomme, Cl
emence Lesimple, Agne Andr
es Gonzalez, Richard LeBoucher, Christian Beau, Eric Schmitt IMV Technologies, L’Aigle, France

6.2.5.1 WHY CRYOPRESERVING? It would not be a surprise for a lab operator not to be amazed when he looks at cryopreserved cells coming back to life after years in liquid nitrogen. As a step back in time, the heat provided gradually melts ice crystals, wakes up complex biochemical machinery, and finally brings back life. In 1949, when Polge et al. successfully exploited the cryoprotective effect of glycerol to freeze the first mammalian cell (a spermatozoa, supposedly programmed to die fast), they definitely reached a milestone toward time control. Thanks to the following discoveries in this field, cryopreservation has led to many applications in animal research, farming, and health. Year after year, research has allowed the freezing of embryos, ovarian tissue, oocytes, and testicular tissue while providing precious tools to manage species and breeds. Today, this method is widely used in artificial insemination of cattle, small ruminants, equine, swine, fish, pets (that is, dogs), and endangered species (to help maintain biodiversity). Cryopreservation applied to artificial insemination plays a decisive role in the management of animal genetic resources, which is “the need to ensure that livestock can continue fulfilling

the roles that make them so important to the lives and livelihoods of so many people around the world, and that the value embodied in livestock biodiversity is not lost.” (FAO, 2012). It makes it possible to recreate a breed from conserved cells, even if only a small proportion of the breeds in the world can be considered as properly preserved: cattle (16% of breeds), goats (9%), sheep (9%), pigs (9%), and chickens (3%). Thawed cells (spermatozoa, oocyte, and embryos) give back genetics to first create new ancestors and later to optimize genetic diversity, minimizing inbreeding and genetic drift. In commercial breeding programs of animal species, cryopreservation is used to store the genetics of some individuals, to store alleles of interest, and more generally to collect images of past genetics. It is therefore possible to grow the family of a male, test their performance (milk production, meat yield, and disease resistance), and come back to the gene bank to collect straws full of marketable genetics. In a genetic environment of rapid adaptation to domestication, evolution, and selection, frozen straws are a freeze-frame of husbandry. When you control time, you can control distance. Cryopreservation deeply changed the artificial insemination industry, allowing male gametes to travel the world. It was the beginning

6. AGRICULTURE

206

6.2. VEGETAL PRODUCTION

of the globalization of genetics and the dispersion of some bull genes. One of the first famous bulls, Hanoverhill Starbucks, who was born in Port Perry on April 26, 1979, provided 685,000 straws sold in 45 countries. The record has now been set by Toy Story (May 1, 2001), who provided 2.4 million doses of frozen semen. Today, breeders use cryopreservation to manage the genetics of their population as well as to optimize fertility, health control, and hygiene. The holy trinity is a cell medium, a freezing curve, and an adapted container. Liquid nitrogen, Cassou straws (Cassou et al., 1994), and cryoprotectants are all choices that shaped the industry of different species and their history.

6.2.5.2 ARTIFICIAL INSEMINATION AND SEMEN PACKAGING 6.2.5.2.1 A Brief History of AI Artificial insemination (AI) in viviparous species dates back quite a long time, perhaps as far back as 1320 AD where an Arab chieftain purportedly mated mares with natural sponges using the sperm collected in the vagina of mares belonging to rival groups. The first reported successful insemination was performed on a bitch by Lazzaro Spallanzani of Italy in 1780 (Heape, 1897; Spallanzani, 1785; Walters et al., 2009). Spallanzani has since been known as the inventor of AI. In 1897, the British zoologist Walter Heape successfully inseminated rabbits, dogs, and horses. After pioneering semen work in the late 1890s on birds, cattle, horses, and sheep, Russia’s Ilya Ivanovich Ivanov in 1922 developed the first semen extender (a liquid medium where sperm cells are better preserved in time) and methods of artificial insemination similar to those we know today (Ombelet and Robays, 2015). Most of Ivanoff’s work was taken over by Viktor Milovanov, another Russian scientist who, in 1931, mass

bred almost 20,000 cows in Russia using AI. In 1938, Milovanov established major cattle breeding projects in Russia, a country known today as the first country having performed AI on a large scale (Ombelet and Robays, 2015). The later part of the 1930s was therefore the true start of AI. In 1936, Denmark’s Eduard Sorensen established the world’s first bovine AI cooperative with some 1100 cows (Bartlett, 1946; Foote, 2002). Sorensen invented the first “straw” (made of oat) in order to package semen inside (Foote, 2002), and F.J. Perry established the first AI cooperative in North America in 1938. Shortly after, the planet saw a massive increase of AI cooperatives being set up worldwide. Indeed, the dairy cattle industry proved to be the largest beneficiary of AI as AI brought four major advantages to the industry: (1) Fast genetic improvement of livestock using genetically superior sires (Walters et al., 2009), (2) Control of diseases (Critser and Russel, 2000; Knight and Abbott, 2002), (3) Increased fertility rates (Kuczynski et al., 2001; Nalesnik et al., 2004), and (4) Maintenance of genetic diversity (Critser and Russel, 2000; Walters et al., 2009).

6.2.5.2.2 Semen Packaging: Past and Present Up to the mid-1960s, glass ampules (Fig. 6.2.5.1) were the container of choice, whether frozen on dry ice or in LN2. However, besides the issues mentioned above, glass ampules had several problems, such as breaking easily or having lower fertility results due to their large volume/surface area (sperm cells freeze/thaw better when the volume surface ratio of the container decreases). In 1964, Robert Cassou of France, then founder and director of one of the first AI cooperatives in that country (L’Aigle 1947), revolutionized the AI industry by manufacturing a small 1.2-mL tube made of extruded PVC and a plug with a “tripartite” plug

6. AGRICULTURE

6.2.5.2 ARTIFICIAL INSEMINATION AND SEMEN PACKAGING

FIG. 6.2.5.1

207

First semen conditioning structure: glass ampules.

(Fig. 6.2.5.2). The straw was sealed at the opposite end first with polyvinylic alcohol, then mechanically by ultrasound. With the straw, the production of one center went from a few thousand doses to many tens of thousands per day. The “French straw,” as it became known worldwide, could at the same time be printed on (for bull identification), filled (diluted semen with extender and glycerol), sealed (to prevent diseases while in LN2 tanks), frozen on LN2

vapors or programmable freezers, preserved for a virtually unlimited time, thawed in a water bath (to prepare the insemination), loaded into a breeding gun, and its volume expelled thanks to a simple plunger system in the breeding gun. All the above advantages yielded more fertility results as well as ease of record keeping (Figs. 6.2.5.3 and 6.2.5.4). A genial multifunction container was born.

FIG. 6.2.5.2

straws.

FIG. 6.2.5.3 Medium and ministraws.

6. AGRICULTURE

IMV IS4 equipment to fill, seal, and print

208

FIG. 6.2.5.4

6.2. VEGETAL PRODUCTION

0% loss IMV Genom’X filling and sealing

equipment.

In 1964, while Nagase of Japan and Dr. Ed Graham from the University of Minnesota were developing a competing system known as the pellet (Nagase and Graham, 1964, Fig. 6.2.5.5), the straw rapidly became the container of choice. Even though the pellet proved to have good fertility results (Maxwell et al., 1980, 1995), its processing, use, and difficulty in labeling were far more cumbersome than the straw. The pellet is today seldom used, mostly only in Russia, Ukraine, and Cuba. More than 50 years after its invention, the straw is still the uncontested container of choice to spread genetics worldwide.

FIG. 6.2.5.5

As mentioned, it was first produced in 1964 in a 1.2-mL format to match the ampule format of the time. Then, in 1968, technological advances in extrusion allowed making the same small tube in 0.5 mL, a similar volume to that used with ampules (1 mL: Curtiss breeders in, 0.75 mL: Tri-State Breeders, then American Breeders Service with 0.5 mL). It was mentioned earlier that the volume/surface ratio is important to freeze living cells more successfully. Eight years after the birth of the French straws and 4 years after the engineering of the medium straw (0.5 mL), Cassou introduced the “ministraw” in 1972 with a total volume of 230 μL (0.23 mL) (Table 6.2.5.1). The thermal exchange during freezing being better than other enclosed systems, it was proven that ministraws (“quarter cc straws”) had better conception rates than medium straws (“.5 cc straws”). Furthermore, the ministraw took less space than any other container previously used. By 1976, most of Europe had switched to the ministraw. TABLE 6.2.5.1 Straw Classification Area (mm2)

Useful Volume (μL)

Volume/ Surface Ratio

Medium (0.5 mL)

1152

500

2304

Mini (0.25 mL)

823

210

3919

Straw Type

Another storage container for semen: pellet making.

6. AGRICULTURE

6.2.5.3 FREEZING TECHNIQUES

6.2.5.3 FREEZING TECHNIQUES 6.2.5.3.1 Semen Cooling in Early Times In the late 1930s and throughout the 1940s, bull sperm was cooled down to 5°C using egg yolk-phosphate, then sodium citrate (Philipps, Lardy, Salisbury), as a sperm membrane protector (Watson and Martin, 1973; Foote, 2002). Diluted semen used for insemination was kept in glass tubes. Balloons containing slushed ice were wrapped around the tube. It was then wrapped in insulating material in order to keep the temperature cool for a day or so (Fig. 6.2.5.6A and B). At that time, most inseminations were carried out the same day or within 3 days at

FIG. 6.2.5.6

209

the latest. Back then, we already knew that colder was better. In 1949, the United Kingdom’s Polge et al. discovered that semen could be successfully frozen with the addition of glycerol in the preserving media. This disruptive finding forever changed the cattle AI industry and to this date, bovine frozen semen represent >98% of all inseminations worldwide. Back in those days, dry ice was used to keep small glass ampules filled with semen frozen at 79°C (Bratton et al., 1955). However at 79°C, biological changes still occur in the sperm cells, making them obsolete after some time. The use of dry ice was also inconvenient due to the required frequent resupply.

(A) Fresh semen tubes (Cornell, 1941). (B) Semen tubes being prepared for dispatch (Cornell, 1941).

6. AGRICULTURE

210

6.2. VEGETAL PRODUCTION

Scientists had shown that keeping cells at ultralow temperatures, as with liquid nitrogen (LN2), stopped all metabolism. Although LN2 was first liquefied in 1883 by Polish physicists Wroblewsi and Olszewski (Sarangi, 1987) and the “dewar” (a vacuum flask) invented by James Dewar from the United Kingdom in 1892 (Freiman and Bouganim, 2005), it is not until the mid-1950s that large, efficient LN2 containers were made available to the AI world. J. Rockefeller Prentice, owner of American Breeders Service, personally funded the American Cyanamid Corporation (a division of Linde) to develop an efficient insulated container, which they supplied to him in 1952. LN2, with a boiling temperature of 196°C/ vapor of 140°C showed that sperm survival, at this ultralow temperature, could be essentially indefinite. In 1952, American Breeders Service of De Forest, Wisconsin, collected and froze the first bull semen in liquid nitrogen. “Cottonade Emmet” produced offspring well into the 1980s. The combination of being able to successfully freeze semen and the availability of LN2 storage containers was the cornerstone of the development of the AI industry.

6.2.5.3.2 Generalities About Reproductive Tissue Freezing (Saragusty and Arav, 2011 for Review) Cryopreservation is the process of preserving living cells by controlled cooling to cryogenic temperatures where chemical reactions are slowed down to an extent that cells can be preserved for a very long period. An integral part of cryopreservation is the warming of the cells to bring them back to physiological temperature and to normal functioning. The cryopreservation process is characterized by two phase transition temperatures (Fig. 6.2.5.7). The first one is the freezing temperature (Tf). Above this temperature, the cryopreservation medium is still liquid. When cooling under the Tf, ice crystals appear and grow until

FIG. 6.2.5.7 Phase transitions during the cryopreservation process. Tf, freezing temperature; Tg, glass transition temperature, Tg 0 , amorphous glassy state.

the second phase transition temperature, the glass transition temperature (Tg). At this stage, the freezing medium is in a rubbery state (ice and cryo concentrate solution). When reaching the Tg, the growth of ice crystals stops and the sample turns into an amorphous glassy state (Tg ’ ), in which no change occurs anymore. Living cells contain large quantities of water and water molecules tend to form ice crystals when cooled to subzero temperatures. To avoid damaging ice crystal formation in freezing cells, the cells are dehydrated before and in the cooling process. To avoid damage from dehydration—the so-called osmotic stress— cryoprotectants are used to replace the water in the cells and to bind the water molecules. Much research has been done on the development of cryopreservation techniques for different types of cells to find the right balance between ice crystal formation and osmotic stress. Cryoprotectants are chemicals and may also affect cells when exposed at higher concentrations and temperatures. The development of artificial insemination techniques has become possible with the development of cryopreservation of spermatozoa.

6. AGRICULTURE

211

6.2.5.3 FREEZING TECHNIQUES

As mentioned earlier, Polge, Smith, and Parkes described the use of glycerol to freeze spermatozoa (Polge et al., 1949). Also, offspring by artificial insemination using frozen-thawed semen was reported in 1951 for cows (Stewart, 1951,) and in 1957 for pigs (Hess et al., 1957) and horses (Barker and Grandier, 1957). The use of frozen semen was developed for the species where the results were at least comparable with those achieved by natural mating and where commercial advantages existed. The development was also made possible in regions where cryogenic gasses were available and transport systems adapted.

2.5% to 10%. According to the type of tissue, the cryopreservative solution can be added to the semen at 34°C just after the collection (cattle), at room temperature (embryos), or at 4°C (cattle and swine). Germplasms are then kept at 4°C equilibration temperature (from a few minutes to 24 h) to allow osmotic exchanges between the cells and the freezing medium, then filled in straws before freezing. The freezing process traditionally follows three phases. First, the sample undergoes a slow cooling rate (3–60°C/min, respectively, for embryos and equine semen) from the equilibration to the supercooling temperature (approximately minus 7–10°C). At this stage, the solution, under the freezing temperature, is still in a liquid state. To limit the supercooling amplitude, crystallization can be induced to initiate the first ice crystal. The “seeding” can either be controlled at a set temperature by contact with a colder surface, shock, or chemical additive or uncontrolled to occur spontaneously. The cooling rate applied is then from 0.3°C/min for embryos to 60°C/min for certain spermatozoa, inducing a rubbery state. The slope allows the control of the size and the shape of ice crystals until 35°C (embryos) or 110 °C (semen).

6.2.5.3.3 Slow Freezing and Programmable Freezers (For Example, Holt, 2000; Pegg, 2007; Saragusty and Arav, 2011) 6.2.5.3.3.1 Slow Freezing (See Fig. 6.2.5.8) Germplasms are extended in cryopreservative solution to be gradually exposed to cryoprotectant agents and to reach the requested number of spermatozoa per dose. With the slow freezing technique, the concentration of cyoprotectant agent varies from

Time (s) 0 20

100

200

300

Liquid state

0

Temperature (°C)

–20

Super cooling

–40 –60 –80

Rubbery state /ice crystal grows

–100 –120 –140

Glassy state/ solid state

–160

FIG. 6.2.5.8

400

Freezing temperature/nucleation

Slow cooling slope.

6. AGRICULTURE

500

212

6.2. VEGETAL PRODUCTION

This phase is crucial to maintain cell survival. In the sample, only pure water will turn into ice. The cells and all solutes will form the “unfrozen fraction.” The concentrations of cryoprotectants, sugar, and salt will increase while the volume of unfrozen fraction decreases. Finally, the sample is transferred into liquid nitrogen to stop ice crystal growth and maintain the sample under the glass transition temperature (130°C). 6.2.5.3.3.2 Programmable Freezers The cryopreservation process has to follow a very precise and controlled succession of events to prevent cell death. To control precisely the cryopreservation process, IMV Technologies has designed special programmable freezers

FIG. 6.2.5.9

(Digitcool, capacity of 45–5300 straws according to the size), composed of four different parts: a temperature regulator, software to program the regulator and monitor the freezing, an isolated chamber, and an autopressurized tank. According to the wanted slope, the regulator controls the quantity of liquid nitrogen injected from the autopressurized tank into the chamber, thanks to a special solenoı¨d valve. The control of the temperature inside the isolated chamber and inside the sample is managed via two temperature probes (Figs. 6.2.5.9 and 6.2.5.10). Liquid nitrogen is injected on a squirrel cage fan and pulverized on the side. The cooled air flow is pushed along the sides of the chamber

Digitcool programmable freezer (IMV technologies) functioning.

6. AGRICULTURE

6.2.5.3 FREEZING TECHNIQUES

FIG. 6.2.5.10

213

IMV Digitcool programmable freezer. Up to 5250 straws frozen in LN2 per cycle.

to the top and then downward through the sample racks (inversed chimney effect).

6.2.5.3.4 Vitrification (Pegg, 2007; Sakai and Engelmann, 2007; Saragusty and Arav, 2011) Vitrification is the transformation of a liquid to a glass state. For liquid biological samples, it means a transformation into a solid without formation of ice crystals. It can be obtained by ultrafast cooling and/or chemical reactions. The water present in germplasm cells and embryos will be transformed into ice crystals when cooled to subzero temperatures. As indicated before, these ice crystals may lead to potentially lethal mechanical and osmotic damage to the cells. To avoid formation of ice crystals, it is therefore necessary to block the regrouping of water molecules in crystals. It can be done by ultrafast cooling—molecules do not have time to move—or by increasing the viscosity of the liquid so that molecules cannot move easily. Ultrafast cooling can be obtained by plunging small volumes of liquid directly into liquid nitrogen. The glass state is unstable and water molecules may form ice crystals in the warming process as soon as the temperature allows

molecules to move—above the glass transition temperature of water, 130°C. Therefore, it is very important to maintain constant cryogenic temperatures. Storage in liquid nitrogen is the best way to maintain samples at stable cryogenic temperatures. When the biological sample is to be recovered, the warming process has to be very fast in order to avoid formation of ice crystals (devitrification) during the warming up from the cryogenic to the positive temperatures. Vitrification has developed in cryopreservation of embryos and oocytes because of better survival, fertilization, and reimplantation rates, first in human IVF but more and more in veterinary applications. Embryos and oocytes are prepared with stepwise baths of increasing concentrations of cryoprotectants to diminish the concentration of free water in the cells and therefore the risk of icecrystal formation. Vitrification straws (Cryo Bio System) are composed of a very thin carrier for a droplet of a maximum 0.5 μL that is placed in a very thin straw. The cell is placed on the carrier in the straw and vitrified by plunging it directly in liquid nitrogen. For warming, the carrier is withdrawn from the straw and immediately plunged in a warmed recovery fluid that brings the cell from the glass state to the liquid state and washes out progressively the cryoprotectants.

6. AGRICULTURE

214

6.2. VEGETAL PRODUCTION

6.2.5.3.5 Storage Germplasms are stored under glass transition temperature (<130°C) to prevent any modification of the ice crystals’ structure and maintain the stability of the sample over time. Cryopreservation allows a delay in the use of germplasms and thus, the respect of the sanitary quarantine period while preserving the semen viability. Depending on the purpose and product, germplasm samples are stored in tanks (Fig. 6.2.5.11) of various sizes from a few liters for shipment and distribution (45 L) to large containers up to 1000 L (capacity: 500,000 straws) for long-term storage. The advantage of liquid nitrogen storage systems is the energetic autonomy (no need for electricity) and the low rate of evaporation, leading to reduced costs and easy handling of the materials. In long-term storage tanks, an automatic refilling line maintains the desired level of liquid nitrogen. With a small container, weekly or monthly refilling is needed according to the device and the frequency of opening.

6.2.5.3.6 Thawing The thawing phase is as important as the freezing phase in regard to cell survival. The thawing rate is correlated to the freezing rate

FIG. 6.2.5.11

and cryoprotectant concentration: samples frozen using the slow freezing technique have to follow a slow thawing rate (embryos thawing at ambient air for 20 s and plunged in a water bath at 20°C), whereas quickly frozen samples have to be quickly (semen thawing in water bath at 37°C for 30 s) and slow thawing for slow freezing.

6.2.5.4 FREEZABLE TISSUES 6.2.5.4.1 Gem Cells 6.2.5.4.1.1 Semen (Bailey et al., 2000; Curry, 2000) Because of their small size, the spermatozoa are the easiest germplasm to cryopreserve (Bailey et al., 2000; Curry, 2000), but the risk of consequences on the reproductive efficiency is real. The osmotic pressure during freezing and the mechanical stress following volume modifications inside the cell may strongly alter the plasma membrane integrity and subsequent sperm viability. Spermatozoa are also very sensitive to cold shock (rapid cooling to 0°C), resulting in cell-reduced motility and necrosis. The repercussions may differ according to the target

Storage liquid nitrogen tanks. Each 1000-L container can hold >920,000 ministraws. 6. AGRICULTURE

6.2.5.5 TARGET ANIMAL SPECIES

species, but the spermatozoa modifications are never fully reversible and the reproductive efficiency of the frozen semen remains below that of the fresh one. 6.2.5.4.1.2 Oocyte (See Saragusty and Arav, 2011 for Review) Techniques for oocyte cryopreservation are much less developed than for semen or embryos (FAO Guidelines, 2012). Oocytes are, by nature, less prone to cryopreservation than spermatozoa: their larger size makes them more sensitive to chilling and intracellular ice crystallization, and the presence of the Zona Pellucida (protection envelop of the oocyte), acting as a supplementary barrier, may interfere with the diffusion of cryoprotectants. In addition to this simple physics phenomenon, the cryopreservation process may impact the oocyte’s structure in various ways: (1) The Zona Pellucida can be hardened during the freezing process, making it impossible for the spermatozoa to fertilize the oocyte. (2) The maturation process is interrupted by cryopreservation, altering the quality of the oocyte and compromising its viability and fertility after thawing. Despite these difficulties, successful freezingthawing processes exist in a great number of animal species (cattle and pigs: Critser et al., 1997 for review; goats: Le Gal, 1996), leading to viable birth of progeny in cattle (for example, Abe and Hoshi, 2005; Otoi et al., 1995) and horses (Maclellan et al., 2002).

6.2.5.4.2 Embryos (Saragusty and Arav, 2011 for Review) An embryo is a pluricellular structure, increasing the risk of ice formation during the freezing process. The main advantage of embryo cryopreservation is that it allows the dispersion of both male and female genetics. Two methods that reduce this risk are used for embryo cryopreservation: the embryos may be exposed to a high concentration of

215

cryoprotectant and submitted to a fast cooling rate (vitrification), or to a low concentration of cryoprotectant and submitted to a slow cooling rate, allowing the dehydration of cells during the process and leading to the formation of ice in the extracellular matrix only. The choice of the freezing process depends on the species’ characteristics. Along with the intrinsic characteristics of the species, the stage of the embryo’s development has an impact on its resilience to cryopreservation; morulae or young blastocysts resist better than older embryos.

6.2.5.5 TARGET ANIMAL SPECIES 6.2.5.5.1 Bovine The bovine species is the primary target of germplasm and embryo cryopreservation (the first calf produced using cryopreserved semen was born in 1951, Curry, 2000). The artificial insemination using frozen semen represents nowadays >98% of the estimated 300 million yearly cattle AI in the world. More than 500 million semen doses are produced each year. For each collected ejaculate, an average of 400 straws is filled in, meaning potentially 400 cows inseminated. When using frozen semen, the pregnancy rate varies generally from 40% to 65%, depending on several parameters including the breed, age, feeding, management, and geographic location of the cow. In order to maximize the chance to obtain heifers (optimization of genetic performance and interval between generations), it is now possible for breeders to access frozen sexed (sorted) semen (8% of the insemination in 2014) and embryos (350,000 transfers/year). Two main techniques are used in bovine embryo production. With in vivo embryo production, heifers are superovulated and inseminated. After 7 days, uterine horns are “washed” (flushing techniques) and embryos collected. If the cow was inseminated with conventional (nonsorted) semen, embryos can be sexed and

6. AGRICULTURE

216

6.2. VEGETAL PRODUCTION

genotyped before freezing. The second technique, in vitro production, is mainly used in South America (Brazil, Argentina, Japan). Oocytes of cows with high genetic value are collected and fertilized in vitro. Embryos are then transferred in recipient cows (milking cow or lower genetic heifer). In bovine embryo cryopreservation, slow freezing is less constraining than vitrification. With slow freezing, the embryo can be directly transferred from the straw to the cow, whereas vitrification requires the removal of the cryoprotectants, which are toxic for the embryo.

6.2.5.5.2 Fish Use of cryopreservation in aquatic species started in the 1980s and rapidly spread to more than 200 species. Today, the largest volume of freezing comes from (1) reproductive practices in aquaculture, (2) breeding program management in aquaculture, and (3) conservation of genetic resources and endangered species. Milt cryopreservation is now fully integrated into reproductive practices of cultured marine and freshwater aquatic species by simplifying broodstock management (Martı´nez-Pa´ramo et al., 2016) and is used to: • Get milt available all-year round in nitrogen tanks, independently of male maturation control through hormone induction or photoperiod control. • Transport genetic resources in straws between hatcheries or from the wild. • Optimize the fertility of sperm when milt can be collected at its highest quality and limit risk of senescence. With the fast development of salmonid breeding programs, cryopreservation became a classical tool for aquaculture geneticists. The technique is used to: • Optimize the mating plans when male genetics can be repeatedly used in different crosses. • Improve the accuracy of estimated breeding values through progeny testing, when the

male genetics of the father are accessible for years. • Control of inbreeding in a selected population. • Conserve valuable genetic candidates. • Estimate the yearly cost effectiveness of a breeding program, when ancestor milt has been successfully frozen. Finally, conservation of aquatic genetic resources offers a chance to preserve species listed as threatened or endangered. With the help of reproductive biotechnologies, cryopreservation would help to further reconstruct the original strain, population, or diversity.

6.2.5.5.3 Porcine In swine species, cryopreservation is mostly used in the context of genetic line preservation, international breeding exchanges, and preservation of rare specimens. Considering the decreased number of doses per ejaculate (15 versus 30–50 in fresh semen), the lower farrowing rate and litter size with frozen semen, and the relatively long preservation of fresh semen in boars (up to 7 days), semen freezing is mostly used on high-value sows or species/breed preservation (Curry, 2000). Countries such as Vietnam, Laos, and Indonesia receive a rather important ratio of their genetic replacement via frozen semen coming mainly from the United States or Canada. South Africa routinely receives high-value genetics in frozen straws as well. Pig embryos have long been the most difficult embryos to cryopreserve because of their extreme sensitivity to chilling and high lipid content. Some studies focused on overcoming these difficulties, leading to live piglets derived from cryopreserved embryo transplantation (for example, Bertelot et al., 2000; Dobrinsky et al., 2000; Nagashima et al., 2007). Swine embryos remain, however, very difficult to cryopreserve, and regarding the cost/benefit balance, cryotransfer is not recommended (FAO Guidelines, 2015).

6. AGRICULTURE

6.2.5.7 CONCLUSION/PERSPECTIVES

6.2.5.5.4 Caprine Domesticated goats have been associated with mankind for more than 10,000 years (Ensminger and Parker, 1986), in particular in poor rural areas. Because of its frugal needs in terms of water and food and its efficiency in absorbing poor quality roughage, goats are particularly appreciated and valuable in developing areas with harsh environments (Table 2). Goats are used in meat and fiber (Mohair, Cashmere) production, but mainly for milk. The total goat milk production approaches 15 million metric tons (MT), representing only 2% of the total amount of milk production (cows, buffaloes). Of this production, 83% comes from developing countries and only 3% from Europe (mostly for cheese production). As a species of interest mostly in developing countries (table 1, FAOSTAT, 2008), goats have not been subjected to the same genetic improvement pressure as cattle and no strategy has yet been organized to solve the seasonality problem of reproduction to ensure a regular milk supply (Dubeuf and Bozayoglou, 2009). However, given the increased interest for milk production in developed countries (France principally), a genetic improvement program using frozen semen AI has been settled, especially for the Alpine and Saanen breeds. France was the pioneer country in the field of goat breeding technologies, with the first artificial insemination using fresh semen in 1954 and the first use of frozen semen for AI as soon as 1968 (CAPGENES). Nowadays, 400,000 AIs using frozen semen are conducted every year in goats, with 80,000 of them in France.

217

Of the bovine straw production, 80% is made in ministraws and 20% in medium straws. There is a continual propensity to go from medium to mini in all countries. This trend that started in 1972 continues. Today, only a dozen countries are using medium straws for bovine semen. These countries include the United States (partial), Japan, Pakistan, Argentina, and the Philippines. More than 90% of all straws sold and frozen in the world are for bovine, because not all species are kept frozen. Furthermore, due to suboptimal results, physiology, economics, or logistics, straws are still not used on some species. For example, the swine industry where AI is used extensively uses fresh semen in bags of toothpaste-type tubes. Frozen swine semen is done in medium straws or in larger straws, but freezing swine semen remains very selective and rare. When done, it is usually for foreign exports where live animal cannot be imported. Equine semen is mostly used fresh. Frozen equine semen, only exploited for sport horses, is processed in medium straws outside the reproductive season. While ovine is mostly fresh semen in straws, caprine is frozen. Rabbit is also a species where mostly fresh semen is used. Other species that are routinely frozen are camelids, fish milt, or canines and felines. Poultry semen is used fresh as frozen semen results are poor. In addition to the above, more than 500,000 bovine embryos are frozen yearly using specific ministraws and 500,000 are transferred fresh.

6.2.5.7 CONCLUSION/ PERSPECTIVES

6.2.5.6 ECONOMICAL STAKE Today, more than 500 bovine semen collection centers in more than 110 countries process hundreds of millions of straws yearly for bovine insemination. In more than 98% of the cases, the doses are frozen in LN2 vapor or in programmable freezers, using liquid nitrogen.

Today, the bovine semen industry is a multibillion euro business and it is growing, largely in Asia (India) or on the African continent. Moreover, thanks to heat synchronization protocols, the beef industry—much larger than the dairy cow business—is using more and more AI frozen in liquid nitrogen.

6. AGRICULTURE

218

6.2. VEGETAL PRODUCTION

AI with frozen semen is the single main genetic transfer system that helps poorer populations access descript genetics. Using superior semen can double the milk production of a sub-Saharan cow breed. AI with frozen semen helps feed the world by unlocking the potential of small dairy stakeholders.

References Abe, H., Hoshi, H., 2005. Evaluation of bovine embryos produced in high performance serum-free media. J. Reprod. Dev. 49, 193–202. Bailey, J., Bilodeau, J.F., Cormier, N., 2000. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J. Androl. 21, 1–7. Barker, C., Grandier, J.C., 1957. Pregnancy in a mare resulted from frozen epididymal spermatozoa. Can. J. Comp. Med. Vet. Sci. 21, 45–51. Bartlett, J., 1946. Artificial insemination of dairy cattle. In: Problem of Fertility: Proceedings of the Conference on Fertility. Princeton University Press, p. 206. Bratton, R., Foote, R., Cruthers, J., 1955. Preliminary fertility results with frozen bovine spermatozoa. J. Dairy Sci. 38, 40–46. Cassou, R., Cassou, M., Cassou, B., 1994. Tube known as straw, for cryogenically preserving biological samples. Biotechnol. Adv. 12, 156. Critser, J., Russel, R., 2000. Genome resource banking of laboratory animal models. Inst. Lab. Anim. Res. J. 41, 183–186. Critser, J.K., Agca, Y., Gunasena, K.T., 1997. The cryobiology of mammalian oocytes. In: Karow, A.M., Critser, J.K. (Eds.), Reproductive Tissue Banking: Scientific Principles. Academic Press, New York, pp. 329–357. Curry, M., 2000. Cryopreservation of semen from domestic livestock. Rev. Reprod. 5, 46–52. Dubeuf, J.P., Bozayoglou, J., 2009. An international panorama of goat selection and breeds. Livest. Sci. 120, 225–231. FAO, 2012. Cryoconservation of Animal Genetic Resources. FAO Animal Production and Health Guidelines No. 12, Rome. FAOSTAT, 2008. http://www.fao.org/faostat/en/#home. Foote, R.H., 2002. The history of artificial insemination: selected notes and notables. J. Anim. Sci. 80, 1–10. Freiman, A., Bouganim, N., 2005. History of cryotherapy. Dermatol. Online J. 11, 9. Heape, W., 1897. The artificial insemination of mammals and subsequent possible fertilisation or impregnation of their ova. Proc. R. Soc. Lond. 61, 52–63. Hess, E.A., Teague, H.S., Ludwick, T.M., Martig, R.C., 1957. Swine can be bred with frozen semen. Ohio Farm Home Res. 42, 100.

Holt, W., 2000. Basic aspects of frozen storage of semen. Anim. Reprod. Sci. 62, 3–22. Knight, J., Abbott, A., 2002. Mouse genetics: full house. Nature 417, 785–786. Kuczynski, W., Dhont, M., Grygoruk, C., Grochowski, D., Wolczynski, S., Szamatowicz, M., 2001. The outcome of intracytoplasmic injection of fresh and cryopreserved ejaculated spermatozoa—a prospective randomized study. Hum. Reprod. 16, 2109–2113. Le Gal, F., 1996. In vitro maturation and fertilization of gaoat oocytes frozen at the germinal vesicle stage. Theriogenology 45, 1177–1185. Maclellan, I., Carnevale, E., Coutinho da Silva, M., Scoggin, C., Bruemmer, J., Squires, E., 2002. Pregnancies from vitrified equine oocytes collected from superstimulated and nonstimulated mares. Theriogenology 58, 911–919. ´ ., Labb, C., Zhang, T., Martı´nez-Pa´ramo, S., Horva´th, A Robles, V., Herra´ez, P., Suquet, M., Adams, S.L., 2016. Aquaculture 462, 1–9. Maxwell, W., Curnock, R., Logue, D., Reed, H., 1980. Fertility of ewes following artificial insemination with semen frozen in pellets or straws, a preliminary report. Theriogenology 14, 83–89. Maxwell, W., Landers, A., Evans, G., 1995. Survival and fertility of ram spermatizia frozen in pellets straws and minitube. Theriogenology 43, 1201–1210. Nagase, H., Graham, E.F., 1964. Pelleted semen: comparison of different extenders and processes on fertility of bovine spermatozoa. In: VICAR Meeting. International Congress on Animal Reproduction and Artificial Insemination, Trento, pp. 387–389. Nalesnik, J., Sabanegh, E., Eng, T., Buchholz, T., 2004. Fertility in men after treatment for stage 1 and 2A seminoma. Am. J. Clin. Oncol. 27, 584–588. Ombelet, W., Robays, J., 2015. Artificial insemination history: hurdles and milestones. Facts Views Vis. ObGyn 7, 137–143. Otoi, T., Yamamoto, K., Koyama, N., Suzuki, T., 1995. In vitro fertilization and development of immature and mature bovine oocytes cryopreserved by ethylene glycol with sucrose. Cryobiology 32, 455–460. Pegg, D., 2007. Principles of cryopreservation. In: Day, J.G., Stacey, G. (Eds.), Cryopreservation and Freeze-Drying Protocols, second ed. Humana Press Inc., Totowa, USA, pp. 39–57. Polge, C., Smith, A., Parkes, A., 1949. Revival of spermatozoa after vitrification and dehydration at low temperature. Nature 164, 666. Sakai, A., Engelmann, F., 2007. Vitrification, encapsulationvitrification and droplet-vitrification: a review. Cryoletters 28, 157–172. Saragusty, J., Arav, A., 2011. Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction 141, 1–19.

6. AGRICULTURE

FURTHER READING

Sarangi, S., 1987. Cryogenic storage of hydrogen. In: Dahiya, R.P. (Ed.), Progress in Hydrogen Energy. Reidel Publishing Company, pp. 123–132. Spallanzani, L., 1785. Experiences pour servir a` l’histoire de la g
en
eration des animaux et des plantes (etc.). Chirol. Stewart, D., 1951. Storage of bull spermatozoa at low temperatures. Vet. Rec. 63, 65–66. Walters, E., Benson, J., Woods, E., Crister, J., 2009. The history of sperm cryopreservation. In: Pacey, A., Tomlinson, J. (Eds.), Sperm Banking: Theory and Practice. Cambridge University Press, pp. 1–10.

219

Watson, P., Martin, C., 1973. Artificial insemination of sheep: the effect of semen diluent containing egg yolk on the fertility of ram semen. Theriogenology 6, 559–564.

Further Reading Webb, D.W., 1992. Artificial insemination in dairy cattle. University of Florida Cooperative Extension Service, Institute of Food and Agriculture Sciences, EDIS.

6. AGRICULTURE