Oleaginous Microorganisms for Simultaneous Biodiesel Production and Wastewater Treatment

CHAPTER 8

Oleaginous Microorganisms for Simultaneous Biodiesel Production and Wastewater Treatment: A Review Fatma Arous1, Atef Jaouani1, Tahar Mechichi2 1

Laboratory of Microorganisms and Active Biomolecules, Faculty of Sciences of Tunis, Institut Supe´rieur des Sciences Biologiques Applique´es de Tunis, University of Tunis El Manar, Tunis, Tunisia; 2Laboratoire de Biochimie et Genie enzymatique des lipases, University of Sfax, Sfax, Tunisia

1. Introduction The search for alternative sources of energy to alleviate dependence on petroleum-based fuel has led to the production of biofuels (Cortes and de Carvalho, 2015). Significant studies have been performed on the production of biofuels from renewable sources, usually vegetable oils (palm oil, rapeseed oil and sunflower oil) but also animal fats and waste oils (Liu and Zhao, 2007; Moser, 2009). Nevertheless, this approach can cause food security problems contributing to the rise of food prices (Thompson, 2012). Alternatively, some oleaginous microorganisms have been identified as novel sources to produce biofuels of similar fatty acid composition to that of plant and animal oils. Compared to plant oils, microbial oils have several advantages, i.e., short life-cycle, less labor required, less affected by season and climate, and easier to scale up (Li and Wang, 1997; Liang and Jiang, 2013). Oleaginous microorganisms, when cultivated under appropriate growth conditions, are able to accumulate up to 85% (w/w) lipid of their biomass as a storage compound (Czabany et al., 2007; Li et al., 2007). Lipid accumulation in the oleaginous microorganisms occurs at a surplus of sugars, or similarly metabolized compounds such as glycerol with one limiting element usually nitrogen limitation (Ratledge and Wynn, 2002). However, it has been reported that the limitation of nitrogen is not the only condition that induce lipogenesis. Sulfate limitation in the medium is as well an influencing factor which may affect the biosynthesis of some special amino acids and cellular cofactors (i.e., cysteine, biotin, thiamine and iron-sulfur clusters). Limitation for synthesis of these cellular Microbial Wastewater Treatment. https://doi.org/10.1016/B978-0-12-816809-7.00008-7 Copyright © 2019 Elsevier Inc. All rights reserved.

153

154 Chapter 8 assemblies could influence cell growth significantly and induce lipid accumulation. Exhaustion of some mineral elements such as Magnesium, Phosphorus, Potassium, and Calcium can be also of crucial importance for accumulation of lipids in oleaginous microorganisms (Wu et al., 2010, 2011). Both lipid content and fatty acid composition are highly affected by several environmental factors, including aeration, temperature, pH, incubation period, inoculum size, inorganic salts and the microorganism itself (Moreten, 1988; Subramaniam et al., 2010). In contrast, when growth is conducted on hydrophobic carbon sources (e.g., fats, oils), the accumulation of storage lipid (the so-called “ex novo” lipid accumulation) is totally independent from the nitrogen exhaustion in the medium(Papanikolaou and Aggelis, 2011a, 2011b). A major bottleneck in lipid production by oleaginous microorganisms is the high raw material cost, which accounts for 70% of the total production cost (Azocar et al., 2010). The employment of low-cost substrates like nutrient-rich wastewaters for oleaginous microorganisms (instead of glucose) is a key parameter in order to develop an economically and environmentally viable biodiesel production process (Huang et al., 2013). This chapter is covering the related studies on the performance of different oleaginous microorganisms for simultaneous lipid production and wastewaters treatment. Moreover, the potential of this microbial oil as feedstock for biodiesel production and the possibility of industrial application of this technology are evaluated.

2. Lipid-Accumulating Microorganisms Microbial lipids, also known as single cell oils (SCOs), are produced by some oleaginous microorganisms, such as yeast, fungi, bacteria, and microalgae (Ma, 2006). The eukaryotic yeasts, molds and microalgae are able to synthesize triacylglycerols (TAGs) with similar composition to the plant oils, while prokaryotic bacteria can synthesize specific lipids. A wide range of oleaginous microorganisms can accumulate lipids, especially in the form of TAGs, which are the main materials for biodiesel production. TAGs are transesterified into fatty acid methyl esters (biodiesel) when they are mixed with an alcohol (mostly methanol) in the presence of a base catalyst (commonly potassium hydroxide) (Li et al., 2008) (Fig. 8.1).

Figure 8.1 Transesterification reaction for biodiesel production.

Oleaginous Microorganisms for Simultaneous Biodiesel Production 155 Table 8.1: Oil Content of Some Microorganisms. Microorganisms

Oil content (% dry wt)

Microorganisms

Microalgae Botryococcus braunii Cylindrotheca sp. Nitzschia sp. Schizochytrium sp.

Yeasts 25e75 16e37 45e47 50e77

Candida curvata Cryptococcus albidus Lipomyces starkeyi Rhodotorula glutinis

Bacteria Arthrobacter sp. Acinetobacter calcoaceticus Rhodococcus opacus Bacillus alcalophilus

Oil content (% dry wt)

58 65 64 72 Fungi

>40 27e38 24e25 18e24

Aspergillus oryzae Mortierella isabellina Humicola lanuginosa Mortierella vinacea

57 86 75 66

Owing to the low lipid yield in most bacteria (Xue et al., 2005), many researchers currently focus mainly on microalgae and fungi (Table 8.1). Microalgae can use carbon dioxide as the carbon source and sunlight as the energy source for photoautotrophic culture, while heterotrophic microalgae can use organic carbon substrates as the sole carbon source instead of using CO2. In mixotrophic growth culturing system, autotrophic photosynthesis and heterotrophic assimilation of organic compounds are combined. Microalgae grow extremely rapidly, doubling in biomass every 24 h, and many are exceedingly rich in oil. The average lipid content of algal cells varies between 1% and 70%, but can reach 90% of dry weight under particular conditions (Spolaore et al., 2006). Microalgae, including Chlorophyta and Bacillariophyceae, are characterized by their high lipid contents, particularly Chlorella, which could be applied for commercial-scale biodiesel production (Xiong et al., 2008). However, the culture of microalgae requires a larger acreages and a longer fermentation period than bacteria. Some bacterial species have the ability to accumulate oil, but the lipid composition is usually quite different from other microbial oils. Most bacteria are generally not lipid-accumulating microorganisms, only few bacteria accumulate complex lipoids (i.e., polyhydroxyalkanoates). The extraction of these compounds is difficult because they are generated in the outer membrane. Biosynthesis of TAG seems to be a common feature of bacteria mostly belonging to the actinomycetes genera, i.e., Mycobacterium (Barksdale and Kim, 1977), Nocardia, Rhodococcus (Alvarez et al., 1997), and Streptomyces (Olukoshi and Packter, 1994). Members of these genera can accumulate up to 70% of their biomass as lipids. Yeasts and fungi (especially molds) are recognized as favorable oleaginous microorganisms since 1980s (Ratledge, 1993). Some yeast strains like Rhodosporidium sp., Rhodotorula sp. and Lipomyces sp. can accumulate significant amounts of lipids as high as 70% of their biomass dry weight. Several mold species, including Aspergillus terreus, Claviceps purpurea, Tolyposporium, Mortierella alpina, Mortierella isabellina (Economou et al., 2011; Chatzifragkou et al., 2011), were found

156 Chapter 8 also to be able to accumulate lipids under some cultivation conditions. Most fungal species are utilized mainly for the production of special lipids, i.e., DHA, GLA, EPA and ARA, while there are few reports on the exploration of fungi lipids for biodiesel production (Yi and Zheng, 2006).

3. Nutrient-Rich Byproducts and Wastewaters for Microbial Lipid Production The use of low-cost carbon sources for SCO production has been extensively investigated as a means of reducing production costs, and thus becoming competitive with conventional energy crops for oil production. A wide range of nutrient-rich byproducts and wastewaters have been used as feedstocks for biodiesel production and some promising results have been achieved (Table 8.2). Molasses, from sugarcane or beet, is considered as one of the most interesting carbon sources, due to its high content of fructose, sucrose and glucose. Although oleaginous microorganisms can grow well on molasses based medium, the high nitrogen content of this waste prevents lipid accumulation. Therefore, when the oleaginous yeast Trichosporon fermentans was cultivated on a mixture of waste molasses and glucose it achieved a biomass yield of 28.1 g L
1 containing 62.4% of lipids (Zhu et al., 2008). In some cases, the increase of molasses concentration causes lower lipid accumulation owing to its toxic effects on the cell growth. For instance, Karatay and Donmez (2010) demonstrated higher lipid accumulation ability of the yeast strains Candida lipolytica, Candida tropicalis and Rhodotorula mucilaginosa when grown on a medium containing 8% molasses compared to that containing 10% molasses. The maximum lipid contents were measured as 59.9%, 46.8% and 69.5% for C. lipolytica, C. tropicalis and R. mucilaginosa, respectively, under optimized conditions. Raw glycerol, a by-product of the biodiesel industry, is contaminated with alkali/acid catalyst and alcohol, and thus, unless purified, its disposal can create serious environmental and economic problems (Papanikolaou, 2008; Singhabhandhu and Tezuka, 2010). Nonetheless, raw glycerol constitutes a versatile carbon source and, therefore, might be regarded as a suitable substrate for many possible applications in industrial fermentations rather than as a waste. Owing to its low-cost and its increasing availability, several studies are presented in literature dealing with the valorization of this by-product through microbial fermentation processes. More specifically, raw glycerol has been successfully used as a carbon source in the production of microbial lipids. Chatzifragkou et al. (2011) performed studies on microbial lipid production by 15 strains of fungi and yeast on waste glycerol derived from the biodiesel industry. The tested yeasts accumulated limited lipid quantities (up to 22%, w/w, in the case of Rhodotorula sp), while fungal strains accumulated higher lipid amounts inside their mycelia (ranging between 18.1% and 42.6%, w/w). More recently, Moustogianni et al. (2015) expanded the application of

Oleaginous Microorganisms for Simultaneous Biodiesel Production 157 Table 8.2: Some Wastewaters and By products Used for Oleaginous Microorganisms.

Feedstock Molasses

Waste glycerol

Cheese whey

Wastewaters from confectionary factories Olive mill wastewater

Palm oil mill effluent Potato processing wastewater Fishmeal wastewater Bioethanol fermentation wastewater Municipal wastewater þ glucose

microorganisms

Total dry weight (g L¡1)

Lipid content (%, w/w)

Lipid yield (g L¡1)

Rhodotorula mucilaginosa Candida lipolytica

e

69.5

e

e

59.9

e

Candida tropicalis

e

46.8

e

Trichosporon fermentans Rhodotorula sp. LFMB 22 Thamnidium elegans CCF 1465 Thamnidium elegans Candida curvata

28.1

62.4

17.5

8.0

22.0

1.76

6.8

42.6

2.9

8.2

48.4

3.95

14.2

51

7.2

Yarrowia lipolytica

7.4

58

4.3

Debaryomyces etchellsii Rhodosporidium toruloides Debaryomyces etchellsii Wickerhamomyces anomalus Lipomyces starkeyi

4.3e5.6

0.8

4.4

14.7 e19.3 51.7

2.1

4.2

17.9

0.7

3.3

23

0.8

e

29.9

e

Yarrowia lipolytica TISTR 5151 Aspergillus oryzae

5.68

e

1.64

e

e

3.5

Lipomyces starkeyi HL Rhodosporidium toruloides

5.34

20.8

n.m

3.8

34.9

e

Rhodotorula glutinis Yarrowia lipolytica

e

21.4

e

e

15.3

e

Culture mode

References

Batch shake flasks Batch shake flasks Batch shake flasks Batch shake flasks Batch shake flasks Batch shake flasks

Karatay and Donmez (2010) Karatay and Donmez (2010) Karatay and Donmez (2010) Zhu et al. (2008) Chatzifragkou et al. (2011) Chatzifragkou et al. (2011)

Fed-batch culture Continuous culture Batch shake flasks Batch shake flasks Batch shake flasks Batch shake flasks Batch shake flasks Batch shake flasks Batch shake flasks Batch shake flasks

Moustogianni et al. (2015) Floetenmeyer et al. (1985) Taskin et al. (2015) Arous et al. (2016) Ling et al. (2013) Arous et al. (2016) Arous et al. (2017) Yousuf et al. (2010) Louhasakul et al. (2015) Muniraj et al. (2013)

Batch shake flasks Batch shake flasks

Huang et al. (2011) Zhou et al. (2013)

Batch shake flasks Batch shake flasks

Chi et al. (2011) Chi et al. (2011)

158 Chapter 8 glycerol to be used as feedstock to produce single cell oil by oleaginous Zygomycetes under nonaseptic conditions on selective (i.e., containing essential oils and/or antibiotics) nitrogen limited media. Specifically, when growth of Thamnidium elegans was performed under aseptic conditions (first stage) followed by lipid accumulation performed under nonaseptic conditions (second stage), 8.2 g L
1 of biomass was produced containing 48.4%,w/w lipids, which are comparable to those obtained under aseptic conditions. Cheese whey is a green-yellowish liquid resulting from the precipitation and removal of milk casein during the cheese-making process. The principal whey components, in addition to water, are, lactose (44e52 gL
1), proteins (6e8 gL
1) and minerals (4.3e9.5 gL
1) (Jelen, 1992). Although numerous possibilities of cheese whey utilization have been developed, major part of world cheese whey production is wasted rather than used, causing a significant loss of resources and serious environmental pollution. Up to now, several studies have showed that cheese whey can be used as growth substrate for the production of a wide range of valuable microbial products (i.e., carotenoid, polysaccharides, ethanol, lactic acid, and hydrogen) (Roukas, 1999; Azbar et al., 2009; Christensen et al., 2011; Nasrabadi and Razavi, 2011; Cui et al., 2012). In addition, some studies reported that whey could be utilized as a substrate for lipid production. Floetenmeyer et al. (1985) reported lipid content of 51%, w/w during growth of Candida curvata on whey permeate in bioreactor under continuous cultivation, while Taskinet al. (2015) reported lipid production of 58%, w/w during growth of Yarrowia lipolytica on lactose-enriched deproteinized whey-based medium in batch shake flasks. Nevertheless, Ahmed et al. (2006) reported the inability of several Zygomycetes to grow sufficiently well on lactose based media. The utilization of agroindustrial wastewaters has also been widely investigated for microbial oil production. For instance, Arous et al. (2016) showed the ability of the oleaginous yeast Debaryomyces etchellsii to grow and to accumulate significant lipid quantities on different agroindustrial wastewaters, i.e., olive mill wastewater (OMW), wastewaters from confectionary industries, and expired soft drinks. In this study, D. etchellsii was cultivated on wastewaters from two confectionary factories. Results showed that sucrose-rich confectionary wastewater was less adequate for SCO production, as maximum lipid accumulation was 14.7% compared to 19.3% obtained in confectionary wastewater-based medium containing a mixture of sugars. In another study, milk candy wastewater was successfully used for the cultivation of a strain of Rhodosporidium toruloides resulting in a moderate biomass concentration (4.4 g L
1) containing significant quantities of reserve lipids (51.7% w/w) (Ling et al., 2013). Owing to its high content of soluble and insoluble carbohydrates, OMW should be considered as a suitable substrate for SCO production. The use of OMW as a rich organic substrate for lipid production was properly investigated by Arous et al. (2016) and Arous et al. (2017) for the strains D. etchellsii and Wickerhamomyces anomalus which were able to accumulate up to

Oleaginous Microorganisms for Simultaneous Biodiesel Production 159 17.9% w/w and 23% w/w on diluted OMW, respectively. Furthermore, Yousuf et al. (2010) reported a lipid content of 29.9% w/w during growth of Lipomyces starkeyi on OMW. The palm oil industry generates a large volume of effluents with high chemical oxygen demand (COD) which make its disposal a pollution problem. Palm oil mill effluent (POME) contains high concentrations of carbohydrates, proteins, and nitrogenous compounds. It is also rich in minerals and vitamins that stimulate cell growth and SCO production by aerobic microorganisms (Habib et al., 1997; Marjakangas et al., 2015). Some studies have focused on this application in the recent years. For instance, Louhasakul et al. (2015) investigated the lipid accumulation potential of several strains of Yarrowia lipolytica grown on POME. Results showed that all strains grew well on POME and accumulated lipids more than 33%, w/w. The use of starch-rich industrial wastewater for microbial lipid production by oleaginous microorganisms was studied with the purpose of recycling this wastewater for biodiesel production. Potato processing wastewater contains high concentrations of solids, starch, and nutrients. When it was used at the dilution ratio of 25% (v/v), the oleaginous fungus Aspergillus oryzae provided the highest lipid yield estimated at 3.5 g L
1 (Muniraj et al., 2013). Due to its high organic compounds content, fishmeal wastewater (FW) was first used to produce microbial lipids by the oleaginous yeast Lipomyces starkeyi HL without any nutrient supplementation. The exclusive use of FW resulted in a biomass yield of 5.34 g L
1 containing 20.8% of lipids. The addition of 20 g L
1 glucose at initial pH 4.0 led to a significant improvement of biomass and lipid yields (17.6 g L
1 and 2.7 g L
1, respectively) (Huang et al., 2011). Fermentation is among one of the most important biological technologies that can synthesize a variety of valuable compounds in order to satisfy the requirement of chemical industry, energy, food, etc. Nevertheless, a wide range of wastewaters is usually produced after fermentation. For instance, bioethanol fermentation wastewater was used as fermentation substrate to produce SCO by the oleaginous yeast Rhodosporidium toruloides (Zhou et al., 2013). A maximum biomass yield of 3.8 g L
1 containing 34.9% w/w was recorded. Organic carbon from municipal wastewater has as well been investigated to produce SCO for the biodiesel industry. The oleaginous consortium of yeasts consisting of Cryptococcus curvatus, Yarrowia lypolitica and R. glutinis was inoculated into wastewater without any additives adding. Results showed that existing nutrients were insufficient and the oleaginous consortium could not compete with the indigenous microorganisms (Hall et al., 2011). Supplementing municipal wastewater with an organic carbon source provided higher cell growth and oil yield (Chi et al., 2011).

160 Chapter 8

4. Oleaginous Microorganisms Used for Simultaneous Lipid Production and Wastewater Treatment 4.1 Oleaginous Yeasts Yeasts are known for their higher growth rate, when compared to filamentous fungi, as well as for their excellent capacity for substrate consumption and their more appropriate morphology for large-scale cultivation and SCO production (Papanikolaou et al., 2011a, 2011b; Arous et al., 2015). Recently, several oleaginous yeasts from the genus of Rhodotorula have been applied for wastewater treatment and lipid production (Table 8.3). For instance, when Rhodotorula glutinis TISTR 5159 was cultivated on POME in semicontinuous fermentation conditions, produced up to 34.15%, w/w lipids and the COD removal was close to 65% (Saenge et al., 2011). The bioconversion of corn starch wastewater into lipids by the oleaginous yeast Rhodotorula glutinis was performed by Xue et al. (2010) in a 300-L fermenter. After 30e40 h of incubation period, around 40 g L
1 biomass containing significant quantities of reserve lipids (35%, w/w) were produced and around 80% COD removal efficiency was achieved. In addition, oleaginous yeasts from genus of Trichosporon showed ability to remove pollutants from a wide range of wastewaters. For example, the oleaginous yeast Trichosporon cutaneum ACCC 20271 was able to accumulate significant lipid quantities (2.16 g L
1) and to remove up to 55% of COD from cellulosic ethanol fermentation wastewater-based medium (Wang et al., 2017), while the oleaginous yeast Trichosporon dermatis has the potential to remove about 68% of COD from butanol fermentation wastewater-based medium and to produce around 7.4 g L
1 of biomass containing 13.5%, w/w of lipids (Peng et al., 2013). On another hand, several yeasts from the genus of Cryptococcus have been used for simultaneous wastewater treatment and SCO production such as Cryptococcus curvatus (GonzalezGarcia et al., 2011), Cryptococcus laurentii (Santos et al., 2014), and Cryptococcus podzolicus (Fernandes et al., 2014). Some encouraging results have been achieved with the above-mentioned yeast species. Gonzalez-Garcia et al. (2011) reported the treatment of tequila’s vinasse by means of a strain of Cryptococcus curvatus, which showed more than 78.98% removal value for COD and up to 25.2%, w/w lipids. Several authors have reported the use of some other kinds of oleaginous yeasts for wastewater treatment e.g., Rhodosporidium toruloides (Zhou et al., 2013), Lipomyces starkeyi (Liu et al., 2012) and Yarrowia lipolytica (Louhasakul et al., 2015). When the oleaginous yeast Rhodosporidium toruloides Y2 was used to treat bioethanol wastewater, a maximum biomass yield of 3.8 g L
1 containing 34.9% w/w lipids was recorded and 72.3% COD removal efficiency were achieved (Zhou et al., 2013). A strain of Lipomyces starkeyi was able to remove up to 74.96% COD from monosodium glutamate wastewater and to produce up to 4.61 g L
1 of biomass containing 24.73% of lipids (Liu et al., 2012). Louhasakul et al. (2015) obtained up to 5.68 g L
1 of biomass, 1.64 g L
1 of lipid yield and 93.4% COD removal efficiency when Y. lipolytica TISTR 5151 was cultivated in POME.

Oleaginous Microorganisms for Simultaneous Biodiesel Production 161 Table 8.3: Oleaginous Microorganisms Used for Simultaneous Lipid Production and Wastewater Treatment.

Microorganism

Substrate

Biomass (g L¡1)

Lipid content (%)

Lipid yield (g L¡1)

COD removal (%)

References

Oleaginous Yeasts Rhodotorula glutinis Rhodotorula glutinis Trichosporon cutaneum

Trichosporon dermatis Cryptococcus curvatus Lipomyces starkeyi Yarrowia lipolytica

Palm oil mill effluent Corn starch wastewater Cellulosic ethanol fermentation wastewater Butanol fermentation wastewater Tequila’s vinasses

6.64

34.15

e

65

Saenge et al. (2011) Xue et al. (2010) Wang et al. (2017)

40

35

e

80

e

e

2.16

55

7.4

13.5

e

68

Peng et al. (2013)

5.19

25.2

1.49

78.98

Monosodium glutamate wastewater Palm oil mill effluent

4.61

24.73

1.14

74.96

GonzalezGarcia et al. (2011) Liu et al. (2012)

5.68

e

1.64

93.4

Louhasakul et al. (2015)

Oleaginous Microalgae Chlorella vulgaris

Chlorella pyrenoidosa Scenedesmus dimorphus Scenedesmus quadricauda Scenedesmus obliquus Scenedesmus sp.

Artificial wastewater medium Soybean processing wastewater Synthetic secondary effluent Synthetic secondary effluent Synthetic secondary effluent Synthetic secondary effluent

e

42

e

86

Feng et al. (2011)

e

37

e

77.8

Hongyang et al. (2011)

e

30.59

1.30

e

Zhan et al. (2016)

e

66.05

1.71

e

Zhan et al. (2016)

e

17.03

1.36

e

Zhan et al. (2016)

e

12.75

0.96

e

Zhan et al. (2016) Continued

162 Chapter 8 Table 8.3: Oleaginous Microorganisms Used for Simultaneous Lipid Production and Wastewater Treatment.dcont’d

Microorganism Neochloris oleoabundans Botryococcus braunii

Substrate Anaerobic effluents from pig waste Molasses

Biomass (g L¡1)

Lipid content (%)

Lipid yield (g L¡1)

COD removal (%)

References

0.63

22.4

e

e

Olguı´n et al. (2015)

3.05

36.9

e

e

Yeesang and Cheirsilp (2014) Yeesang and Cheirsilp (2014) Hena et al. (2018) Hena et al. (2015)

Botryococcus braunii

Nitrate-rich wastewater

2.26

30.3

e

e

Arthrospira platensis Oleaginous consortium

Dairy farm wastewater Dairy farm treated wastewater

4.98

30.23

e

98

e

16.89

e

98.8

Oleaginous Fungi Aspergillus oryzae Mucor circinelloides

Trichoderma reesei

Potato processing wastewater Wastewaters from equalization tank Wastewaters from equalization tank

e

e

3.5

91

Muniraj et al. (2013)

0.60

22.11

e

88.72

Bhanja et al. (2014)

0.68

9.82

e

86.75

Bhanja et al. (2014)

Kumar et al. (2015) Gupta et al. (2017)

Oleaginous Bacteria Rhodococcus opacus Rhodococcus opacus

Dairy wastewater Dairy wastewater

e

33

e

62

e

53.26

1.8

100

4.2 Oleaginous Microalgae Some oleaginous microalgae are not only capable of producing microbial lipids, but also remove pollutants (Perez-Garcia et al., 2011). The cultivation of microalgae offers significant advantages over yeasts. First, they can grow in wastewaters containing low nutrient concentrations owing to their autotrophic character (Cai et al., 2013).

Oleaginous Microorganisms for Simultaneous Biodiesel Production 163 Furthermore, they can be cultivated under nonaseptic conditions (Ratledge and Cohen, 2008). Another advantage is that many byproducts like carotenoids and polysaccharides with high value can be produced by different microalgae (Gong and Bassi, 2016; Behera et al., 2014). For simultaneous lipid production and wastewater treatment, oleaginous microalgae from the genus Chlorella were investigated in several studies (Table 8.3). For example, the oleaginous microalga Chlorella vulgaris cultivated on an artificial wastewater medium was able to accumulate up to 42%, w/w of lipids and to remove up to 86% of COD, 97% of NH4þ and 96% of total phosphate (Feng et al., 2011), while the oleaginous yeast Chlorella pyrenoidosa was able to produce around 37% w/w of lipids and to remove 77.8%, 88.8%, 89.1% and 70.3% of COD, total nitrogen, NHþ 4 and total phosphate, respectively from soybean processing wastewater-based medium (Hongyang et al., 2011). Strains from the genus of Scenedesmus showed as well great ability for synchronous wastewater treatment and SCO production. Recently, four algal species from the genus Scenedesmus, i.e., Scenedesmus dimorphus, Scenedesmus quadricauda, Scenedesmus obliquus and Scenedesmus sp. LX1were used for synthetic secondary effluent treatment. The tested microalgae were able to remove 40.43%e69.58% of total nitrogen and 50.14% e88.42% of total phosphate with simultaneous production of significant lipid quantities (ranging between 12.75% and 66%, w/w) (Zhan et al., 2016). Some promising results have been achieved using other genus of oleaginous microalgae such as Neochloris, Botryococcus, Arthrospira, and so on. Olguı´n et al. (2015) proposed a dual purpose system for the treatment of the anaerobic effluents from pig waste utilizing the oleaginous microalga Neochloris oleoabundans and for the production of lipid rich microalgae biomass. This strain was very efficient at nutrient removal and lipid accumulation since 3
98% of NeNHþ 4 and 98% of PO4 have been removed with production of nonnegligible lipid amounts (up to 22.4%, w/w). Yeesang and Cheirsilp (2014) proposed two strategies for low-cost production of Botryococcus braunii biomass with high lipid content, i.e., the mixotrophic cultivation using molasses as a carbon source and the photoautotrophic cultivation using nitrate-rich wastewater supplemented with CO2 as a carbon source. The mixotrophic cultivation added with molasses produced a high amount of biomass of 3.05 g L
1 with a high lipid content of 36.9%, while the photoautotrophic cultivation in nitraterich wastewater supplemented with 2.0% CO2 produced a biomass of 2.26 g L
1 containing 30.3% of lipids with high nitrate removal efficiencies (more than 90%). A recent study conducted by Hena et al. (2018) found that the oleaginous microalga Arthrospira platensis cultivated in dairy farm wastewater is able to produce a moderate biomass yield (4.98 g L
1) containing 30.23%, w/w of lipids and to remove more than 98% COD and nutrients. Co-culture of oleaginous microalgae has been applied efficiently for lipid accumulation and wastewater treatment. For instance, co-culture of three species of microalgae, i.e., Chlorella vulgaris, Scenedesmus obliquus, and Ourococcus multisporus was used to treat

164 Chapter 8 tertiary municipal wastewater amended with 15% CO2. This microalgae consortium showed ability to remove almost all the nitrogen and phosphorus from municipal wastewater within 4 days with a maximum lipid productivity of 0.164 g-lipids g-cell
1 day
1 (Ji et al., 2013). In another study, a consortium of native strains was found to be capable of the removal of more than 98% nutrients from dairy farm treated wastewater and to accumulate around 16.89% of lipids (Hena et al., 2015).

4.3 Oleaginous Fungi and Bacteria Fungi and bacteria were applied somewhat less than microalgae and yeasts for synchronous microbial lipid production and wastewater purification. Up to date, some results have been reported using oleaginous fungi namely Aspergillus oryzae (Muniraj et al., 2013), Mucor circinelloides and Trichoderma reesei (Bhanja et al., 2014) (Table 8.3). When potato processing wastewater was used at the dilution ratio of 25% (v/v), the oleaginous fungus Aspergillus oryzae provided the highest lipid yield estimated at 3.5 g L
1 (Muniraj et al., 2013). In addition to lipid production, removals of COD, total soluble nitrogen and total soluble phosphorus up to 91%, 98% and 97% were achieved, respectively. Bhanja et al. (2014) reported the treatment of wastewaters from equalization tank by means of Mucor circinelloides MTCC1297 and Trichoderma reesei NCIM992 strains. The observed reductions in COD were 88.72% and 86.75% and the resulted lipid contents were 22.11% and 9.82% for M. circinelloides reactor and T. reesei reactor, respectively. Basically, bacterial system is ideal for wastewater treatment due to its high rate of organic degradation. However, few studies investigated the ability of oleaginous bacterial species to treat wastewaters with simultaneous lipid production (Table 8.3). For example, the oleaginous bacteria Rhodococcus opacus showed great potential in the treatment and valorization of dairy wastewater into lipid (Kumar et al., 2015). Using only the raw dairy wastewater, the bacterium accumulated up to 14.28% w/w lipid and reduced the initial COD by 30%, while the addition of mineral salts to the dairy wastewater led to an increase of the lipid content and the COD removal efficiency to up to 33% and 62%, respectively. More recently, Gupta et al. (2017)proved that R. opacus PD630 can be successfully scaled up for efficient dairy wastewater treatment along with lipid accumulation. This strain was cultivated in bioreactor under three different culture modes: fed-batch, continuous and continuous cell recycling. Results from the fed-batch operated bioreactor showed a maximum COD removal efficiency of 93% with 1.1 g L
1 of lipid accumulation. Continuous mode experiments provided up to 100% COD removal efficiency and a maximum lipid yield of 1.8 g L
1 at an hydraulic retention time of 6.6 h. However, the highest lipid yield (3.4 g L
1) has been achieved under continuous cell recycle mode.

Oleaginous Microorganisms for Simultaneous Biodiesel Production 165

5. Biodiesel Predicted Properties Based on the Fatty Acid Composition of Microbial Lipids The influence of the chemical structure of biodiesel fatty acids on fuel physical and chemical properties has been studied in many papers (Canakci and Sanli, 2008; Ramos et al., 2009; Knothe et al., 2008; Pinzi et al., 2011). Heating value (Mehta and Anand, 2009), cetane number (Lapuerta et al., 2009) and oxidation stability (Knothe, 2007) increase with the chain length and decrease with the unsaturation degree, while low temperature behavior properties (Sarin et al., 2010) and viscosity (Knothe and Steidley, 2005) improve with shorter and more unsaturated fatty acid chains. Mono-unsaturated fatty acids are considered ideal for biodiesel production due to their ability to improve the engine behavior under cold weather conditions without losses to oxidative degradation (Mittelbach and Remschmidt, 2004). On another hand, saturated medium chain acids show excellent oxidative stability owing to the absence of double bonds. Based on European Biodiesel Standards EN14214 for vehicle use, content of alpha-linolenic acid (18:3) should not exceed 12% (Knothe, 2005). According to Meng et al. (2009), oleic acid (C18:1) is the principal fatty acid accumulated by the yeast cells followed by linoleic acid (C18:2) and palmitic acid (C16:0). However, the accumulated oil in almost all microalgae is mainly triglyceride (>80%) with a fatty acid profile rich in C16 and C18, showing D9, D12 and D15 desaturation. Oleaginous fungi accumulate triacylglycerol rich in polyunsaturated fatty acids. Oleic (18:1), linoleic (18:2), linolenic (C18:3) and palmitic (16:0) acids are the most frequently found fatty acids, and nearly all of them are unsaturated fatty acid (see Table 8.4). The effect of the feedstock on the fatty acid composition of microbial lipids is shown in Table 8.5. Most feedstocks lead to the production of more highly saturated fatty acids compared with rapeseed oil, the most commonly used oil for biodiesel production in Europe. The increase of the saturated fat content may improve the cetane number and the oxidation stability and decrease NOx emissions, although it may has a detrimental influence on cold behavior properties and viscosity. Yeast lipids present a quite similar fatty acid composition to that of palm oil, which has a higher saturation degree than

Table 8.4: Lipid Composition of Some Microorganisms (Meng et al., 2009). Lipid composition (w/total lipid) Microorganisms Microalga Yeasts Fungi Bacteria

C16:0 12e21 11e37 7e23 8e10

C16:1 55e57 1e6 1e6 10e11

C18:0 1e2 1e10 2e6 11e12

C18:1 58e60 28e66 19e81 25e28

C18:2 4e20 3e24 8e40 14e17

C18:3 14e30 1e3 4e42 e

Table 8.5: Fatty Acid Composition considering Different Culture Media and Oleaginous Microorganisms.

Lipid source

Feedstock

C16:0

C18:0

C16:1

C18:1

C18:2

C18:3

Others

References

Oleaginous Yeasts Rhodotorula glutinis TISTR 5159 Trichosporon cutaneum ACCC 20271 Trichosporon dermatis Rhodosporidium toruloides Y2

Palm oil mill effluent

20.37

10.33

0.83

47.88

7.31

0.85

12.43

Saenge et al. (2011)

Ethanol fermentation wastewater of corn stover Butanol fermentation wastewater Bioethanol wastewater

14.21

10.12

0.55

67.81

0.36

e

5.08

Wang et al. (2017)

19.4

10.6

e

39.9

21.7

e

8.4

Peng et al. (2013)

0.9

16.9

11.2

49.9

13.6

e

7.7

Zhou et al. (2013)

Oleaginous Microalgae Neochloris oleoabundans Arthrospira platensis Chlorella vulgaris Scenedesmus quadricauda

Anaerobic effluents from pig waste Dairy farm wastewater Municipal wastewater Primary settled and filtrate wastewater

23.87

3.18

0.78

35.77

25.05

5.46

5.88

Olguı´n et al. (2015)

48.37 19.0 55.6

2.09 0.69 1.5

3.01 9.40 2.4

10.88 19.62 3.3

12.26 11.2 19.9

19.19 22.12 14.4

e 18.20 3

Hena et al. (2018) Ahmad et al. (2013) Wong et al. (2015)

Oleaginous Fungi Aspergillus oryzae Mortierella isabellina Thamnidium elegans CCF 1465 Zygorhynchus moelleri MUCL 1430

Potato processing wastewater Rice hulls hydrolysate

11.6

19.3

15.6

30.3

6.5

5.5

8.3

Muniraj et al. (2013)

22.6

2.9

2.9

50.7

16.1

3.4

1.5

Raw glycerol

19.7

9.6

1.4

52.9

11.3

3.6

e

Raw glycerol

15.1

5.5

1.4

21.9

47.5

3.7

e

Economou et al. (2011) Chatzifragkou et al. (2011) Chatzifragkou et al. (2011)

Oleaginous Bacteria Rhodococcus opacus PD630 Rhodococcus opacus (MITGM-173)

Petroleum processing wastewater Glycerol

44.32

5.06

1.25

26.21

0.42

0.21

e

24.8

2.0

16.1

17.5

0.9

0

e

Saisriyoot et al. (2016) Kurosawa et al. (2015)

Plant Species Rapeseed

e

3.89

2.14

e

65.13

17.14

9.79

1.91

Palm

e

36.7

6.6

0.1

46.1

8.6

0.3

1.8

Leiva-Candia et al. (2013) Ramos et al. (2009)

166 Chapter 8

Fatty acids composition (w/total lipid)

Oleaginous Microorganisms for Simultaneous Biodiesel Production 167 rapeseed oil. However, Microalgal and fungal lipids differ from most vegetable oils in being quite rich in polyunsaturated fatty acids with four or more double bonds, which make it susceptible to oxidation during storage and decrease their acceptability for biodiesel production purposes (Chisti, 2007). The fatty acid composition was used to predict several important properties of the biodiesel produced from microorganisms grown on various substrates following a similar methodology used for estimating vegetable biodiesels (Arous et al., 2016, 2017). The estimated properties of microorganisms biodiesels, i.e., cetane number (CN), iodine value (IV) and cold-filter plugging point (CFPP), are presented in Table 8.6 and compared with those produced from most commonly used vegetable oil-based biodiesels (rapeseed and palm oil biodiesels). CN is widely used as biodiesel quality parameter related to the ignition and combustion quality of the fuel. High CN (above 50) guarantees good cold start properties and reduces the formation of white smoke (Balat and Balat, 2010). The IV, that reflects the stability of biodiesel to oxidation, depends on both the number and position of the double bonds in the fatty acid methyl esters. High number of double bonds decreases the stability of fuel. The maximum IV accepted in the European Standards EN 14214 is 120 I2/100 g. The CFPP is one of the representative parameters, typically used for the prediction of the biodiesel behavior at low temperatures (Knothe et al., 2005). According to cetane number, most lipid-based biofuels from oleaginous microbes showed significantly higher CN values compared to that found in rapeseed oil biodiesel and somewhat similar to that of palm oil biodiesel. In any case, microbial lipids fulfilled European standard for biodiesel EN 14214. All samples were found to have a moderate degree of unsaturation, since the estimated IV satisfied the limits imposed by EN 14214 standards. With respect to CFPP, predictive models for microbial oil biodiesel showed large differences among them and comparing with the traditional biodiesel. As CFPP values of most of them would not be advisable for cold climates, we can conclude that lipid-based biofuels from some oleaginous microbes could be used as fuel for diesel engines only when they are blended with diesel fuel.

6. Industrial Application of Oleaginous Microorganisms: Issues and Outlooks As mentioned above, the use of “zero”-cost carbon sources for SCO production can reduce significantly the production costs, but for industrial application, more factors on the practical cost should be considered. In fact, the optimization of medium composition and fermentation conditions are crucial components to increase both COD removal and lipid production and to make this technology more competitive. Indeed, high initial COD concentration might inhibit the COD removal efficiency possibly due to the “substrate inhibition”. In order to overcome this issue, several studies reported the effect of

Table 8.6: Predicted Properties for Microbial Oil Biodiesel. Feedstock

SV

IV (g I2/100 g)

CN

LCF

CFPP ( C)

References

Oleaginous Yeasts Rhodotorula glutinis TISTR 5159 Trichosporon cutaneum ACCC 20271 Trichosporon dermatis Rhodosporidium toruloides Y2

Palm oil mill effluent Ethanol fermentation wastewater of corn stover Butanol fermentation wastewater Bioethanol wastewater

183.29 192.05

58.70 62.14

61.11 58.87

11.51 10.25

19.69 15.71

Saenge et al. (2011) Wang et al. (2017)

180.41

73.31

57.86

7.24

6.27

Peng et al. (2013)

188.86

79.51

54.92

10.79

17.42

Zhou et al. (2013)

Oleaginous Microalgae Neochloris oleoabundans Arthrospira platensis Chlorella vulgaris Scenedesmus quadricauda

Anaerobic effluents from pig waste Dairy farm wastewater Municipal wastewater Primary settled and filtrate wastewater

189.06

90.37

52.12

5.36

0.35

Olguı´n et al. (2015)

199.49 168.85 197.25

83.92 103.97 77.20

52.26 52.11 54.28

6.18 2.25 6.31

2.94
9.42 3.35

Hena et al. (2018) Ahmad et al. (2013) Wong et al. (2015)

Oleaginous Fungi Aspergillus oryzae

181.15

68.36

59.00

14.26

28.32

Muniraj et al. (2013)

Mortierella isabellina Thamnidium elegans CCF 1465

Potato processing wastewater Rice hulls hydrolysate Raw glycerol

196.25 195.13

85.11 77.83

52.41 54.42

3.71 6.77


4.82 4.79

Zygorhynchus moelleri MUCL 1430

Raw glycerol

185.48

112.66

47.00

4.26


3.09

Economou et al. (2011) Chatzifragkou et al. (2011) Chatzifragkou et al. (2011)

Oleaginous Bacteria Rhodococcus opacus PD630 Rhodococcus opacus (MITGM-173)

Petroleum processing wastewater Glycerol

158.40

26.09

74.11

6.96

5.40

Saisriyoot et al. (2016)

148.14

33.36

74.64

3.48


5.54

Kurosawa et al. (2015)

45.68 59.14 53 50 51 min

1.46 7.70 Nd Nd NS


11.89 10.00
13
6 NS

Leiva-Candia et al. (2013) Ramos et al. (2009)

Plant Species Rapeseed Palm Conventional biodiesel Petro-diesel Limits

e e

Biodiesel standard EN 14214

192.52 199.09 Nd Nd NS

113.63 57.17 Nd Nd 120 max

CFPP, cold-filter plugging properties; CN, cetane number; EN14214, European Standard for Biodiesel; IV, iodine value; LCSF, long-chain saturation factor; max, maximum; min, minimum; Nd, not determined; NS, not specified; SV, saponification value.

168 Chapter 8

Biodiesel source

Oleaginous Microorganisms for Simultaneous Biodiesel Production 169 wastewater dilution on nutrients removal efficiency and lipid production for biological treatment by oleaginous microorganisms (Ji et al., 2013; Arous et al., 2016). On another hand, the unsuitable C/N ratio of most of wastewaters may lead to low biomass and lipid content. More exogenous carbon (mainly glucose) or nitrogen sources (i.e., ammonium sulfate, ammonium chloride, peptone, yeast extract, malt extract and so on) could be added into different substrates (Karatay and Donmez, 2010; Chi et al., 2011; Thiru et al., 2011; Saenge et al., 2011), but this could as well increase the cost of production. As a solution, Arous et al. (2016) proposed the use of a mixture of two or more substrates of different C/N characteristics, instead of a single substrate, for ensuring an adequate C/N ratio (C/N > 50) (Arous et al., 2016). In addition, the cost of transportation of wastewaters used for lipid production and treatment by oleaginous microorganisms should be added into the total cost of this technology. In order to save the transportation costs, the bioreactor for oleaginous microorganisms should be set up near the wastewaters source. Considering the equipment building cost, the bioconversion process requires a basic fermentation equipment with low energy consumption owing to the demand of a short fermentation period and a moderate temperature. Besides above factors, the pretreatment step of wastewaters like saccharification process could increase as well the cost of this technology and thus should be considered in the industrial application (Leiva-Candia et al., 2014). Overall, if we could handle the above-mentioned problems, the possibility of SCO industrialization is high and could be fulfilled. Although the potential of using oleaginous microorganisms for simultaneous wastewaters treatment and SCO production have been proven, and some scale up attempts of this technology have been performed as well (Fernandes et al., 2014; Xue et al., 2010), most investigations on this field are still in laboratory scale. Accordingly, further researches should be emphasized on the scale up of this technology at industrial level.

7. Conclusion The use of different nutrient-rich wastewaters to produce microbial oil from oleaginous microorganisms has great potential for industrial application owing to its advantages of both removal of COD or nutrients from wastewaters and production of completely renewable oil for biodiesel production. The predicted biodiesel properties based on fatty acids methyl esters composition of the lipids produced by oleaginous microorganisms grown on different raw wastewaters were found to lie within the range specified by international biodiesel standard specifications, which may suggest that wastewaters-based media could be used without any compromise on the quality of the microbial oil. Future researches should focus more on the improvement of the cost effectiveness of this technology accompany with the effort to its scale up at industrial level.

170 Chapter 8

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