bioenergy production, and bioremediation, advances and prospect

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Mixotrophic cultivation, a preferable microalgae cultivation mode for biomass/bioenergy production, and bioremediation, advances and prospect Jiao Zhan a, Junfeng Rong b, Qiang Wang c,* a

Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China SINOPEC Research Institute of Petroleum Processing, Beijing, China c State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China b

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abstract

Article history:

Microalgae have received much attention in recent years as a feedstock for producing

Received 25 September 2016

renewable fuels. Microalgae cultivation technology is one of the main factors restricting

Received in revised form

biomass production as well as energy fuel production and bioremediation. There are four

7 December 2016

types of cultivation conditions for microalgae: photoautotrophic, heterotrophic, mixo-

Accepted 7 December 2016

trophic and photoheterotrophic cultivation. Though photoautotrophic and heterotrophic

Available online xxx

cultivation are two common growth modes of microalgae, some microalgae can also grow better under mixotrophic condition, which may combine the advantages of autotrophic

Keywords:

and heterotrophic and overcome the disadvantages. This review compared these growth

Microalgae

modes of microalgae and discussed the advantages of mixotrophic mode in bioenergy

Mixotrophic cultivation

production by considering the difference in growth, photosynthesis characteristic and

Bioenergy

bioenergy production. Also, the influence factors of mixotrophic cultivation and the

Biohydrogen

application of mixotrophic microalgae in bioremediation are discussed, laying theoretical

Bioremediation

foundation for large scale microalgae cultivating for biomass production, bioenergy production and environmental protection. © 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

Introduction Microalgae are unicellular photosynthetic microorganisms well-known as a source of several products useful to human beings, ranging from carbohydrates, essential acids, pigments, food supplements, fertilizer and biofuels [1,2]. Especially, microalgae lipids have a great potential to be the raw

material for biodiesel [3]. Because of their widespread availability and higher oil yields than conventional terrestrial plants [4], microalgae have been investigated as feedstock for biodiesel production since the 1970s. Microalgae could also produce biohydrogen as another form of clean energy, unicellular green algae especially Chlamydomonas reinhardtii had been studied extensively in photobiological hydrogen production. Two light-dependent photosynthetic electron

* Corresponding author. Fax: þ86 27 68780123. E-mail address: [email protected] (Q. Wang). http://dx.doi.org/10.1016/j.ijhydene.2016.12.021 0360-3199/© 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Zhan J, et al., Mixotrophic cultivation, a preferable microalgae cultivation mode for biomass/bioenergy production, and bioremediation, advances and prospect, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/ j.ijhydene.2016.12.021

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transport pathways and one light-independent fermentative pathway have been identified as capable of contributing electrons to biohydrogen production in microalgae [5]. Electrons come from photosynthetic electron transport in the two light-dependent pathways, photosystem II (PS II)-dependent and PS II-independent pathways. The PS II-dependent pathway gets electrons from water splitting and O2 is released as by-product. And the PS II-independent pathway gains electrons from NADPH, which in turn comes from the degradation of starch. While the third pathway recruits electrons from the fermentative degradation of endogenous compounds, such as pyruvate [6]. Compared with plants, microalgae grow faster, allowing the use of non-arable land and non-potable water [7]. The ability to use wastewater makes it a useful tool for treating the organic effluent from the industry [8]. Though microalgae have vast potential in large scale production and application, there are also many problems to be solved, e.g. the cultivation of microalgae, to realize economic feasibility and industrial availability [9,10]. Microalgae cultivation technology is one of the main factors restricting new energy fuel production [11]. Autotrophy, heterotrophy, mixotrophy and photoheterotrophy are four main cultivation modes. For autotrophic cultivation, microalgae generate organic matters and energy by fixing inorganic carbon, mainly using CO2 as carbon source and sunlight as energy source [8]. Green alga could also utilize nitrite and NOx, a significant portion of fossil fuel flue gases, as a nitrogen source, providing a new insight into the economically viable application of microalgae [12e15]. For heterotrophic cultivation, microalgae growth depends on the metabolism of organics which provide the carbon source and energy [16]. For mixotrophic cultivation, microalgae grow with both light and organics as energy source, with CO2 and organic carbon as carbon sources, i.e. they have the ability to perform photosynthesis and acquire exogenous organic nutrients [17]. Photoheterophy is a growth mode that the microalgae require light when using organic compounds as the carbon source, similar with mixotrophy. The main difference between mixotrophy and photoheterotrophy is that the latter cannot absorb and metabolize carbon dioxide [18]. As the difference of photoheterotrophy and mixotrophy lies merely in absorbing CO2, in present paper we do not distinguish between these two kinds of culture modes, as well as reported before [19]. There was a review concluded that heterotrophic systems are more suitable for producing high cell densities of microalgae for accumulation of large quantities of lipids when compared with autotrophy [20]. Considering the mixotrophic cultivation conditions, both sunlight and organic material are not the limiting conditions for microalgae growth, and autotrophy and heterotrophy processes are both exiting in the mixotrophy mode [17], mixotrophy may be another useful method for microalgae cultivation and biofuel production. Hydrogen is a clean energy with H2O as the only major byproduct. Microorganisms including photosynthetic and fermentative bacteria, cyanobacteria and green microalgae can produce hydrogen biologically [21]. And among the three bio-hydrogen production pathways, the light-independent pathway generates relatively smaller amounts (traces) of H2

in the microalgal cells [22]. Thus the cultivation mode should also affect the efficiency of microalgae biohydrogen production. In this review, by comparing with autotrophy and heterotrophy, we discussed mixotrophy by summarizing the growth characteristics, photosynthetic parameters, biomass accumulation, biolipid as well as biohydrogen production, and the influence factors of microalgae cultivated on mixotrophic mode. We propose that mixotrophic culture mode also has preferable potential for the application of microalgae in biomass and bioenergy production, and bioremediation.

Overview of three different cultivation modes Autotrophic cultivation Autotrophy is the most primitive way of cultivating microalgae, and open pond system and closed photo bioreactor system are two main autotrophic cultivating methods [23]. Algae cultivation in open pond production system has been used since the 1950s [24]. The system can be categorized into natural waters (lakes, lagoons, and ponds), artificial ponds and containers according to the water used. The CO2 requirement of microalgae is usually satisfied from the surface air, and submerged aerators may be installed to enhance CO2 absorption [9]. For its convenient resource and simple operation, open pond system may be a promising method for new fuel production. However, it was reported that the cost of cultivating microalgae (e.g. Dunaliella salina) in open pond was 2.55 V every kilogramme, which was too expensive for producing biodiesel [25]. Moreover, the easily being polluted by other algae and bacteria [26] and the high temperature difference between day and night [27] make the open pond system not be the optimal method for large scale microalgae culture. In order to overcome the weakness of open pond system, closed photo bioreactor system has been developed [28]. Plate photo bioreactor, tubular photo bioreactor and verticalcolumn photo bioreactor are three main closed photo bioreactor culture systems and it was proposed that tubular photo bioreactor could be more suitable for large-scale cultivation [29] because of its higher surface to volume ratio. However, the costs of closed systems are substantially higher than open pond systems [30]. The drawbacks of difficult to achieve aseptic, high cost of open system and close bioreactor microalgae cultivation and the dependence on weather condition (such as, temperature and light) make the autotrophic cultivation being not suitable for large-scale microalgae cultivating for industry use.

Heterotrophic cultivation Compared with autotrophy, heterotrophy doesn't need light, which reduces the require of surface to volume ratio of bioreactor [23], thus makes the design of bioreactor easier. The growth rate of microalgae under heterotrophic conditions increased and the harvesting cost decreased because of the high cell density [31]. Moreover, heterotrophic culture can be combined with plant fermentation technology and facilities,

Please cite this article in press as: Zhan J, et al., Mixotrophic cultivation, a preferable microalgae cultivation mode for biomass/bioenergy production, and bioremediation, advances and prospect, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/ j.ijhydene.2016.12.021

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such as beverage, pharmaceutical and nutraceutical industries, dramatically reducing the production cost [32,33]. The main carbon source of heterotrophy has been glucose. However, because of the high cost of glucose, some researches use the cellulose hydrolysis product to replace glucose as carbon source [34]. In addition, some algae can also grow with glycerol as carbon source [35], which can serve as an osmolyte to maintain cell osmotic balance and is not harmful for microalgae even in high concentration. The type of organic carbon sources may affect the growth of microalgae, when Chlorella vulgaris was cultivated with glucose, glycerol and acetate as carbon source, a higher biomass and lipid accumulation was obtained under lower concentration of glucose and glycerol [36]. It was also reported that some microalgae could grow with composite carbon sources such as sweet sorghum and yams to produce lipid [36]. A disadvantage caused by addition of organic carbon source is that the media is easy to get contaminated by Escherichia coli and other bacterias [18]. Considering the cost of organic nutrients supplement, some researchers combined microalgae cultivation with factory effluent and waste co-productions and even factory/ domestic wastewater. A large number of surplus nutrients in life or factory sewage can reuse to provide the substrate for microalgae growth, dramatically reducing the cost of microalgae cultivating. It was reported that some microalgae could grow in carbon-rich sewage under alternating light or dark condition [37], or in the wastewater with additional organic nutrients [38]. Chlorella protothecoides cultivated with waste activated sludge (WAS) hydrolysate could grow well and produce lipid [39]. During the cultivation of three newly isolated microalgae (Scenedemus sp. ZTY2, Scenedesmus sp. ZTY3 and Chlorella sp. ZTY4) in domestic wastewater without illumination, both algal densities and lipid contents increased dramatically, which suggested that these three strains could be heterotrophically cultivated for domestic wastewater treatment and lipid production under dark condition [8]. In summary, heterotrophic culture can accelerate cell growth rate, increase biomass and lipid accumulation compared with autotrophic culture and even can integrated with wastewater treatment reducing the cost. A previous review has summarized the heterotrophic cultivation of microalgae for biodiesel product and concluded that heterotrophy is a more suitable system for microalgae lipid accumulation [20].

Mixotrophic cultivation A “two-stage” mode was summarized for the growth regime of microalgae mixotrophy. In which the first stage was described as heterotrophy due to high content of initial organic carbon. When the organic carbon was drained to a certain level, photosynthesis was induced and the algae began to assimilate CO2 photoautotrophically which indicated the onset of the second stage. If integrateds with darkelight cycle, which allows the growth of autotrophy and heterotrophy under their optimum conditions, the biomass and lipid content of the mixotrophic cultivation will be not merely a sum of autotrophy and heterotrophy.

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Mixotrophy combines the advantages of autotrophy and heterotrophy and overcomes the disadvantages of autotrophy. Earlier in 1977, it was reported that cell growth rate of Chlorella regularis was almost the sum total of autotrophy and heterotrophy when cultivated under mixotrophic mode with light and acetate as carbon source [25]. Under mixotrophic cultivating condition, microalgae can not only grow heterotrophically with organic carbon, increasing the growth rate and improving biomass and lipid accumulation, but also consume inorganic carbon (CO2) and produce oxygen through photosynthesis [40], which makes the overall CO2 emission lower under mixotrophic cultivation mode than under heterotrophic cultivation mode. Different from heterotrophy, the valuable pigments and photosynthetic carotenoids such as bcarotene could be preserved in illuminated conditions under mixotrophic cultivation mode [41], although the mixotrophic energy conversion efficiency is lower because of photosynthetic losses [42]. The overall differences of the three cultivation modes are summarized in Table 1.

Advantages of mixotrophic cultivation Photosynthetic parameter The photosynthetic characteristics of mixotrophy are different from those shown in autotrophy and heterotrophy [43,44]. It was reported that the net photosynthetic rate of Nannochloropsis sp. did not change but the respiratory rate increased in the mixotrophic cultivation comparing to those in autotrophic conditions. The photosynthetic parameter was also presented in two stages cultivation of Arthrospira platensis under autotrophic and mixotrophic culture conditions [44] that the maximum instantaneous growth rate, net photosynthetic rate and respiratory rate of mixotrophy was almost 1.5 times higher than autotrophic cultivation at the first stage (the first 3 days). The study of Synechococcus sp. PCC 7002 in autotrophic and mixotrophic conditions concluded that the net photosynthetic rate (263 mmol O2 mg Chl1 h1) of mixotrophy was much higher than that of autotrophy, and the growth rate was rapider than autotrophy [45]. The photosynthetic pigment content and photosystem activity of algae under mixotrophic condition are also different from those under autotrophic condition. When Anabaena sp. PCC 7120 was cultivated under mixotrophic condition, the chlorophyll a content and the fluorescence emission ratio of PS ӀӀ to PS Ӏ were improved compared with autotrophy, but the carotenoid and phycobilisome to chlorophyll a ratio was decreased. Moreover, the change of photosynthetic pigments was dependent on the microalgae species and the organic carbon source [23,46]. When Platymonas subcordiformis was cultivated with acetate as organic carbon source in mixotrophic condition, it was reported that the contents of Chlorophyll a and Chlorophyll b decreased dramatically [47]. However, the photosynthetic pigment contents of some algae such as Spirulina platensis [47] have little change. When cultivated Phaeodactylum tricornutum in mixotrophic condition, the activity of PS ӀӀ decreased, suggesting that in presence of organic carbon source the photosynthetic efficiency decreases, although biomass increase [48]. And with

Please cite this article in press as: Zhan J, et al., Mixotrophic cultivation, a preferable microalgae cultivation mode for biomass/bioenergy production, and bioremediation, advances and prospect, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/ j.ijhydene.2016.12.021

(1) Higher growth rate, higher biomass and lipid accumulation (2) Bioreactors design with little constraints

(1) Higher growth rate, higher biomass and lipid accumulation (2) Sustain of pigmentation and phytochemicals production (3) Decreased production of CO2

Organic carbon source

Light and organic carbon source

Organic carbon source (eg. Glucose, glycerol, acetate)

CO2 and organic carbon source

Heterotrophic

Mixotrophic

(1) Low growth rate, low biomass and lipid accumulation (2) Need special bioreactors or high dependence of weather condition (1) Higher cost (2) Need for sterile media and easy to be contaminated (3) Production of CO2 (4) Dark conditions diminish the pigmentation and production of phytochemicals (1) Higher cost (2) Need for sterile media and easy to be contaminated (3) Reduced energy conversion efficiency (1) Low cost (2) High production of pigmentation and phytochemicals Light CO2

Carbon source

Energy supply Autotrophic

Disadvantage Advantage

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 6 ) 1 e1 3

Cultivation mode

Table 1 e The overall differences among the three cultivation modes.

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the addition of inhibitor of photosynthetic electron transport chain DCMU, the cell growth of autotrophy of Nostoc flagelliforme was completely suppressed, but cells could still grow under mixotrophic condition [49].

Biomass production Cell density and biomass are important factors for microalgae large-scale production and application, so the study of cultivation methods for high biomass production is one strategy for decreasing the cost of microalgae utilization. For some microalgae that can grow under mixotrophic condition, the biomass accumulation is improved by mixotrophic cultivation. It was reported that the cultivation of Chlorella vulgaris under mixotrophic condition significantly improved the biomass compared with the autotrophy and heterotrophy [50]. As for Nannochloropsis sp., the largest biomass under mixotrophic condition was 1.6 times of that of autotrophic cultivating [43,47]. A research on Neochloris oleoabundans showed that when cultivated with carbon-rich manure derived from the apple vinegar production, cell density of the algae was 150% higher than that in autotrophic culture condition, which concluded mixotrophy was one useful cultivation method to increase microalgae biomass [51]. This result was consistent with the report that Nannochloropsis salina and Chlorella protothecoides in mixotrophic condition grows faster than those in autotrophy and heterotrophy [52]. When compared the cultivation of N. flagelliforme in autotrophic, heterotrophic and mixotrophic conditions and it was found that the cell density of mixotrophy was the highest at any time and the largest biomass of mixotrophy was 4.98 times of that of autotrophy and 2.28 times of that of heterotrophy at the harvest time [49].

Lipid accumulation Many microalgae can produce lipid as raw materials for biodiesel production. Some studies had shown that the lipid content of microalgae cells under mixotrophic condition is much higher than that of autotrophy and heterotrophy [53,54], which has been verified in many different microalgae. The biomass and lipid productivity of various microalgae strains under the three different cultivation modes with common organic carbon sources are listed in Table 2. The cultivation of Chlorella protothecoides under mixotrophic condition increased the content of biomass and lipid [47,55]. Both the largest lipid productivity and the highest lipid content were got in Chlorella vulgaris ESP-31 under mixotrophic culture condition [56]. The mixotrophic cultivation mode was reported to be able to increase lipid accumulation of N. salina and Chlorella protothecoides in comparison with autotrophic cultivation [52]. There was a similar report that the largest lipid productivity of Chlorella vulgaris was acquired in mixotrophic condition with glucose as carbon source [37,57]. Day and Tsavalos [58] got a conclusion that the lipid content of Tetraselmis in mixotrophic/photoheterotrophic condition was 5.8 times higher than that of heterotrophy.

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Table 2 e Biomass and lipid productivity of various microalgae strains under the three different cultivation modes. Carbon source used are common organic carbon sources and/or CO2. Microalgae

Autotrophic

Chlorella sp. Y8-1 Chlorella vulgaris Chlorella protothecoides Chlamydomonas sp. BTA 9032 Chlorella sp. BTA 9031 Chlorella vulgaris Chlorella vulgaris ESP-31 Nannochloropsis sp. Chlorella sp. N. oleoabundans UTEX 1185

Heterotrophic

Mixotrophic

Ref

Lipid content (%)

Biomass (g/L)

Lipid content (%)

Biomass (g/L)

Lipid content (%)

Biomass (g/L)

16.5 38 ± 2 ~23 15.12 14 NA 20.0 28 13.5 20.3

0.22 0.31 ~1 0.95 0.93 NA 0.8 0.37 0.60 0.25

5.9 NA 15.7 ± 4 NA NA 2.0 16.0 20.0 13.0 NA

0.17 NA 4.00 ± 1.3 NA NA 3.00 0.2 0.38 0.75 NA

35.5 21 ± 1 ~25 38.6 30 0.8 53.0 27.5 15.0 27.1

0.45 1.696 4.07 ± 0.2 1.15 1.55 4.00 3.0 1.2 1.40 1.8

[139] [57] [140] [141] [141] [142] [89] [89] [143]

“NA” means corresponding data is not supplied in the paper. “~” means the data is not supplied directly in the paper bur is calculated or estimated based on the related references.

Hydrogen production

Influence factors of mixotrophy

In comparison with those under autotrophic culture, a higher H2 photoproduction can be obtained by C. reinhardtii under mixotrophic culture [59]. The rates of biohydrogen production of C. reinhardtii under mixotrophic cultivation mode from various studies are listed in Table 3. There are two causes why a mixotrophic cultivation mode is benefit to hydrogen production. On one hand, C. reinhardtii is capable of efficient heterotrophic growth in the presence of acetate [60], the acetate can increase growth rate leading to the accumulation of carbohydrates whose successive degradation can participate in H2 production. On the other, the hydrogenase is sensitive to oxygen and the presence of acetate could stimulate respiration and thus help to establish and maintain anoxia [61]. Glycerol is the primary energy source during fermentation for many cyanobacteria [62], and the Cyanothece 51142, a diazotrophic cyanobacterium, can produce hydrogen at rates as high as 465 mmol per mg of chlorophyll per hour under aerobic conditions [63]. Besides the traditional mixotrophic culture, a two-phase incubation protocol combined dark fermentation with mixotrophic culture was constructed for hydrogen production using the marine green algae P. subcordiformis, in which anaerobic incubation in the dark was followed by the exposure to light illumination and the first step induced hydrogenase activity to catalyse H2 evolution in the second phase [64].

Light Since the process of mixotrophy includes autotrophy and heterotrophy, light intensity is an important influencing factors. Light intensity affects the synthesis of chlorophyll and then the growth of microalgae and the lipid accumulation. In the bioreactor, cells with large-size antenna often shield the light of the surface [65,66] and with the increasing of cell density, self-shading occurs and this leads to low light penetration, slow algal growth, and low production. It was reported that, under low light intensity, diatom tend to increase their photosynthetic pigment (e.g. Chl a) and antenna pigments to maximize their ability to harvest light for normal growth [67]. Xie, Zhang [68] suggested that light intensity could significantly affect the cell specific growth rate of P. subcordiformis under mixotrophic condition. In mixotrophy culture, it is very important to optimize the balance between the relative heterotrophic and photoautotrophic metabolic activities. Lightedark cycle regime is one of the strategies to realize this purpose. Under light period, microalgae perform photoreduction that absorbs light energy and stores it in energy-carrying molecules such as ATP and NADPH [73]. Under dark period, carbon dioxide was fixed via Calvin cycle using ATP and NADPH from the photoreduction

Table 3 e Maximum hydrogen production rates of Chlamydomonas reinhardtii from different report. Microalgae strains

Organic carbon

C. C. C. C. C. C. C. C.

17.4 mM 17.4 mM 17.4 mM Glucose 17.4 mM 17.4 mM 17.4 mM 17.4 mM

reinhardtii reinhardtii reinhardtii reinhardtii reinhardtii reinhardtii reinhardtii reinhardtii

Dang 137c 137c 704 ATCC 824 CC124 137c CC-124 CC124

acetate acetate acetate acetate acetate acetate acetate

Light intensity (mE m2 s1)

Maximum H2 production rate (mL/L/h)

Reference

25 110 12 NA 70 110 140*2 70*2

4.5 ± 1.6 1.4 ± 0.1 25 ± 7.2 6 0.5 2.5 7.5 170

[144] [73] [77] [145] [146] [147] [74] [148]

“NA” means corresponding data is not supplied in the paper.

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and microalgae are stimulated to oxides supplement organic substrates by heterotrophy for energy and then produce biomass and useful products such as lipid, sugar, protein [69]. It was reported that for Scenedesmus sp. AARL G022, the highest biomass was obtained under 24:0 h and 16:8 h lightedark cycles [70]. Other reports showed that the growth of Isochrysis galbana and Chlorella sp. C2 was significantly affected by photoperiod, and the maximal dry weight was obtained at 12 h light and 12 h dark for both of strains [13,71]. Among the three pathways the light-dependent pathway contributes most of the hydrogen production in microalgae [21]. Since O2 production of PS II is light dependent, and microalgae hydrogenase HYDA1 only could catalyse hydrogen (H2) production under anaerobic conditions that oxygen (O2) is a strong inhibitor of both the enzyme's activity and expression [72], the light condition is important for hydrogen production [73,74]. Tsygankov et al. showed that controlling incident light flux could be applied in autotrophic conditions to induce anaerobiosis [75]. Laurinavichene and colleagues reported that average light intensity could be an important parameter to predict optimal conditions for biohydrogen production in photoheterotrophic conditions [76]. A recent study confirmed the combined effects of both light attenuation and acetate on the establishment of anoxic environment under mixotrophic conditions [77]. Not only the light intensity, but also the light quality, i.e. the different wavelength of the light could influence the hydrogen production of microalgae. The action spectrum of PS II is peaked at approximately 680 nm, while PS I is at 700 nm. Jennings et al. speculated that by applying PS I-light would thus suppress the PS II-coupled oxygen generation and create an anaerobic cellular environment and, with PS I remaining active, allowing hydrogen production to occur [78]. The theory was proved in C. reinhardtii cells [79], by applying 690 nm and 700 nm pulse light, the photosynthetic oxygen production rate dropped sharply to 40e50% and to less than 10% respectively, with the maximum hydrogen production rate occurred under 700 nm pulse light. Similarly, enhanced hydrogen production and longer sustainable hydrogen production term were observed under infrared light in C. reinhardtii [80] and Scenedesmus sp [81].

Carbon source Since carbon element is one of the essential elements for cell growth, the concentration and type of carbon source greatly affect the accumulation of biomass and lipid. Since both CO2 utilization and organic carbon utilization exist under mixotrophic condition, CO2 and organic compounds supply need to be finely optimized to achieve the best productivities in mixotrophic condition [52]. CO2 is found to be a major limiting factor for algal growth and its excess strongly enhances photosynthetic productivity at an appropriate range [54]. Under mixotrophic cultivation condition, microalgae cells can use inorganic carbon source for photosynthesis. The influence of exogenous CO2 on the mixotrophical cultivation of Auxenochlorella protothecoides was studied and it showed that pouring CO2 promoted the biomass accumulation after 4e5 days [82]. Organic carbon source also has important influence on microalgae growth under mixotrophic culture condition, and

the influence varies among organic carbon types, among organic carbon concentrations, and even among different microalgae species with the same carbon source. Among the organic carbon sources (such as glucose, xylose, rhamnose, fructose, sucrose and galactose), glucose may be the most effective carbon source for microalgae [83,84]. When comparing the effect of organic carbon sources such as glucose, glycerol and ethanol on Chlorella kessleri, it was found that glucose concentration of 300 mM was the most ideal for cell growth and fatty acid synthesis [85]. Compared with glucose, other carbon sources need more complicated interconversion metabolic process to provide energy for algal growth as well as bioenergy production. As for Chlorella vulgaris cultivated under mixotrophic condition, the maximum biomass was acquired when the glucose and glycerol was 4:1 [50]. Acetate is also one of the most used organic carbon in mixotrophy culture. It was found that sodium acetate could promote both growth and lipid content in many microalgae such as P. tricornutum [86], Brachiomonas submarina [87] and Chlorella vulgaris [50]. Liu, Chang [88] reported when cultivated Chlorella vulgaris ESP6 with dark hydrogen fermentation effluents, the acetate in the effluent was readily consumed by the microalga. Different initial concentration of glucose has different impact on the accumulation of biomass and fat synthesis. It was reported that the dry weight of Chlorella sp. and Nannochloropsis sp. increased continuously but the lipid content of the strains fell sharply with the increase of initial glucose concentration [89]. .

Temperature Temperature affects the growth and lipid accumulation of microalgae in mixotrophic culture. Cell density would increase below optimum temperature but decrease drastically on sudden change or increase in temperature [90]. Venkata Subhash, Rohit [91] cultured microalgae with domestic wastewater in mixotrophy and the maximum cell density and lipid productivity were observed with 30  C operation followed by 25  C and 35  C. Variation in the cell density concentrations might result from higher and lower temperatures causing adverse effects on growth of microalgae by ceasing growth protein synthesis and ultimately biomass. The mechanism of lipid accumulation associated with temperature may be referring to the instability of enzymes associated with carbon fixation (ribulose bisphosphate carboxylase/oxygenase: RuBisCO) and lipid biosynthesis (Acetyl CoA carboxylase) [92]. Overall, temperature is one of the key factors for bio-fuel production, which typically depends on the type of the biofuel-producing microorganism [21].

Metal ion The synthesis of pigments and fatty acids are complex enzyme catalytic reaction processes which need some metal ions such as Fe2þ, Mg2þ and Mn2þ as cofactors in higher plants and some algae [93,94]. These metal ions affect the photosynthetic pigment content and ratio, even the accumulation of fatty acids. It was reported that in the cultivation process of Chromochloris zofingiensis under mixotrophic condition,

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addition of Mn2þ and Mg2þ did not significantly affect the biomass and production of astaxanthin and total fatty acids in C. zofingiensis, while addition of Fe2þ decreased growth but increased production of astaxanthin and total fatty acids [95].

pH pH is also importantly affects enzyme activity [96,97], and it may also influence the cell growth and metabolism of microalgae cultivated under mixotrophic condition. pH was reported as one of the most important factors for green alga Chlorella sp. C2 mixotrophic cultivation for biological DeNOx [13]. It was also reported that pH could influence the absorption of different kinds of carbon source by Chlorella [56], which may take the effect on the cell growth and metabolism of microalgae as well. The pH of the system is also an integral expression of the redox conditions for any anaerobic process [21]. Furthermore, Proton supply to the hydrogenase is key for the hydrogen production [98] and the changes in external pH values also affect several physiological parameters in cells [99]. Thus, pH also has a key role in the regulation of metabolic pathways and hydrogen production using microalgae.

Other factors special for bio-hydrogen production Besides the aforementioned factors, there exists some special factors for bio-hydrogen production with microalgae under mixotrophy. Hydrogen production of microalgae is sensitive to O2. This is because O2 inhibits hydrogenase enzyme function [100]. The establishment of anoxic conditions in the microalgae by sulphur (S) starvation was first reported in year 2000 by Melis and co-workers [61]. S starvation in green algae results in lower activity of PS II but balance the capacities of photosynthesis and respiration in the cell, thus allowing the formation of anaerobic microenvironment [61]. A two stage photosynthesis and H2 production in green algae were constructed with a first growing algae aerobically for biomass accumulation and a followed sulphur (S) starvation for H2 generation, during which the stored carbohydrates were used. C. reinhardtii culture was reported to become anaerobic in the light after ~22 h of S deprivation and started to produce H2 [101]. A different approach used to avoid O2 inhibition is to control the O2 concentration of the culture. It was reported that continuous purging of C. reinhardtii cell suspensions with Ar could result in higher rates of H2 photoproduction [76]. The biological pathways of H2 production are also highly sensitive to the partial pressure of H2 (HPP), because hydrogenase activity intend to decrease because of feedback inhibition [102]. Many studies reported previously focused on the effect of the H2 partial pressure in the photo bioreactor headspace on the H2 photoproduction activity in green algal [21,103]. As reported in C. reinhardtii the maximum rate and yield of H2 photoproduction could be observed if the H2 is effectively removed from the medium [104]. Immobilization of microbial cultures also significantly affects the hydrogen production [105,106]. Immobilization processes can occur as in the form of biofilms, or by the attachment of the cells on solid surfaces or entrapment of the cells within matrices. It was reported that immobilized cyanobacteria can produce H2 at much higher volumetric rates than suspension cultures

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[107]. The improved and longer-term H2 photoproduction by immobilized was also demonstrated in C. reinhardtii [108].

The application of mixotrophic microalgae cultivation in bioremediation It was reported that the ability of CO2 fixation by microalgae photosynthesis was 10e50 times of that by plant [109]. Studies had reported that the autotrophic cultivation of microalgae could be used to deal with flue gas to decrease the environment pollution without harm to microalgae growth. Doucha, Straka [110] cultivated Chlorella in outdoor open thin-layer photo bioreactor bubbled in flue gas generated by combustion of natural gas in a boiler, and the CO2 in the flue gas decreased by 10%e50% and the CO2 removal rate increased with the addition of flue gas into the bioreactor, with little harm to microalgae growth. Most of the applications of microalgae for CO2 fixation are focused on photoautotrophic cultivation because of the higher fixation efficiency [111]. Although reports on mixotrophic CO2 biofixation are limited, it is believed that the mixotrophic cultivated microalgae can be used to deal with flue gas accompanied by the accumulation of biomass and biolipid. The cultivation of Botyrococcus braunii mixotrophically with CO2 and glucose achieved a 75% CO2 reduction [112]. By growth with excess CO2 during the day and heterotrophic growth in glycerol without added CO2 during the night, another study reported that a nighteday cycle regime could reach maximal biomass and lipid production with the cultivation of Chlorella protothecoides and N. salina under mixotrophic condition [52]. Some studies had reported the cultivating of microalgae with swage under mixotrophic or photoheterotrophic conditions to process sewage and produce biomass and lipid, suggesting the application of mixotrophic cultivated microalgae in wastewater treatment. Chlamydomonas globosa, Chlorella minutissima and Scenedesmus bijuga have been cultivated with glucose, sucrose, acetic acid salt and sewage respectively as carbon source [113], and the biomass of algae cultivated in sewage was found 3 to 10 times larger than those cultivated in other media. Another study found that mixotrophic microalgaebacteria system significantly promoted algal growth and nutrients removal efficiency with the maximal biomass and lipid productivity when used Desmodesmus sp. CHX1 to treat piggery wastewater [8]. Moreover, the co-culture of microalgae and bacteria with wastewater was reported to get a 50e60% and a 68e81% of DOC (Dissolved Organic Carbon) removal efficiency from municipal wastewater and industrial wastewater mixtures respectively [114]. So microalgae mixotrophic cultivation suggests a convenient way to reduce the high organic content of wastewater along with the production of algal lipids. Carbohydrate rich, nitrogen deficient solid wastes and some food industry wastewaters such as olive mill wastewaters can be used for hydrogen production [115,116]. It was reported that photosynthetic H2 evolution from C. reinhardtii grown in Advanced Solid State Fermentation wastewater was increased by more than 700% compared to the cells grown in TAP medium [3]. Similarly, the C. reinhardtii cells grown on a mixture of pretreated olive mill wastewaters and TAP (tris-

Please cite this article in press as: Zhan J, et al., Mixotrophic cultivation, a preferable microalgae cultivation mode for biomass/bioenergy production, and bioremediation, advances and prospect, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/ j.ijhydene.2016.12.021

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acetate-phosphate) (50% dilution) were found to exhibit a greater production of hydrogen, compared to the control cells grown in TAP only [117]. Chlorella vulgaris MSU 01, isolated from a pond sediment was reported to be capable of generating H2 from corn stalk via anaerobic fermentation, and also decreasing the butyrate concentrations of effluent [118]. By using acetate and butyrate-rich wastewater effluent as substrate, Micractinium reisseri YSW05 got a maximum hydrogen production of 191.2 ± 14.7 mL/L [119]. According to the above discussion, microalgal mixotrophy is useful for flue gas CO2 fixation and wastewater treatment in the process of bioenergy production. Microalgae cultivation as a promising methods for bioenergy production is not a new idea, however the life cycle analysis (LCA) [120] showed an urgent need for new technologies to make algae biofuels more sustainable and commercially reality. The cultivation, dewatering and harvesting of microalgae are all energy intensive and are the major bottlenecks in the algae-to-fuel production chain. For microalgae cultivation, the need of carbon source, nutrient substance, light, water and even the emission of CO2 from the bioreactor are major problems. The LCA concluded that these upstream obstacle and environment burden may be reduced by the associated microalgae biodiesel production with wastewater treatment and industrial CO2 capture [120,121]. The combination of CO2 fixation from flue gas and nutrient removal from wastewater may provide a very promising alternative to current CO2 capture strategies. A simplified flow chart of the combination of CO2 fixation from flue gas and nutrient removal from wastewater is summarized in Fig. 1. The combinations improve the economics and efficiency and minimize the environmental and resource implications since wastewater readily contains available water source and major substrates required by microalgae proliferation and the CO2 from the flue gas act as inorganic carbon. For example, a study explored the ability of microalgae to utilize higher concentration of CO2 by bubbling CO2 with different sparging period into wastewater to enhance microalgae lipid accumulation under mixotrophy [122]. Yun et al. [123] cultivated Chlorella vulgaris in wastewater discharged by steel-making plant to develop an economically feasible system to remove ammonia

from wastewater and CO2 from flue gas, simultaneously. The CO2 fixation and ammonia removal could reach to 26.0 g m3 h1 and 0.92 g m3 h1, respectively. Algal strains including Chlorella globosa, Chlorella minutissima and S. bijuga were cultivated in untreated wastewater of carpet industry supplied with a gas stream containing 5e6% of CO2 [124]. During the process biomass productivity could reach 5.9e21.1 g m2 d1. Cyanobacteria Aphanothece microscopica €geli cultivated in refinery wastewater was found to assimNa ilate the organic carbon presented in the absence of light but fix CO2 under light, the CO2 fixation efficiency was strongly impacted by light conditions [125].

Prospect Mixotrophic microalgae can not only absorb CO2 and release O2 by photosynthesis, but also grow with organic carbon, which give it a promising application in environmental treatment of flue gases and wastewater. Compared to the high cost of heterotrophic cultivation and lower biomass yield of autotrophic cultivation, the mixotrophic cultivation may be one optimal culture method for microalgae large-scale culture and application. It also provides a new insight into the economically viable application of microalgae in the synergistic combination of environmental bioremediation and biofuel production. Even though the mixotrophy cultivation of microalgae has great promise for biodiesel production, there are many problems ahead for industrial application of microalgae for biofuel production. The most important is the high cost of microalgae cultivation, harvesting, dewatering, drying, cell disruption, extraction and product purification. To achieve satisfactory productivity and lipid accumulation for industry application, large quantities of inorganic/organic carbon substrates, nutrients, and water and photo bioreactor are required. All of those contribute to the high cost of microalgal biodiesel production. The combination of wastewater and flue gas treatment will made microalgae biodiesel production more environmentally sustainable and cost-effective, as fresh

Fig. 1 e Simplified flow chart of the combination of CO2 fixation from flue gas and nutrient removal from wastewater. Please cite this article in press as: Zhan J, et al., Mixotrophic cultivation, a preferable microalgae cultivation mode for biomass/bioenergy production, and bioremediation, advances and prospect, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/ j.ijhydene.2016.12.021

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 6 ) 1 e1 3

water were replaced with wastewater and flue gases were used as a carbon and inorganic nutrient source for culturing microalgae. In addition the microalgae cultivating system should be located near the wastewater treatment/CO2 emission plant to shorten the rout and cost for wastewater/gas treatment [126]. Lipids extraction and transesterification is one of most important processes in overall biodiesel production. The most used methods were chemical solvent extraction [127] and organic solvent (supercritical lipids) [128]. With the high toxicity of chemical solvent and high cost of organic solvent, new methods of lipid extraction are required for industrial scale, typically in terms of energy efficiency and costeffectiveness. Another approach might be to test other microscopic algae (green, blueegreen, brown and red) or improve the existed algae by genetic engineering for bio-fuel production. Although getting a higher biofuel production rates, the nutrient deprivation method such as N starvation for lipid production [129e131] or S starvation for hydrogen production [61,101] is not a good choice for industrial biofuel production. S deprivation or N starvation impairs cellular protein biosynthesis and always inhibit the growth of microalgae and finally inhibit the biofuel production [5]. The selection and engineering of high-efficiency microalgae cell lines under normal growth condition is central to overcome all the problems. As special for biohydrogen, H2 has distinct advantages over alternative carriers of energy, which shows a notably energy density and produces H2O as the only byproduct. The fact that microalgae could metabolize hydrogen was initially reported a century ago, however development and application of biological hydrogen production to commercialization is still a long-term objective [132]. Nowadays the cost for microalgae hydrogen production is unacceptably high, and the only approach to solve the problem is to increase the productivity. Dedicated investigators have tried to innovate development ranging from photo bioreactor design, metabolic alterations via nutrient-deprivation to genetic modifications to increase the production efficiency [5]. The biggest contradiction exists between a higher photosynthetic efficiency and the O2 sensitivity of hydrogenase enzyme. The selection and engineering of high-efficiency microalgae cell lines also quite important for the bio-hydrogen production. Recently, two algae strains were reported to produce low levels of H2 and show substantial hydrogenase activity at almost atmospheric partial pressure of oxygen (19%), thus the S starvation is not essential [119]. Other approaches aim to develop O2-tolerant hydrogenases or balance O2 production with metabolic O2 utilization using C. reinhardtii [133]. With the mixotrophic cultivation mode, a higher growth rate, higher biomass production, and higher hydrogen production were obtained. However, under such cultivation mode, research areas including integrated biosystems for H2 production in mixed-cultures and direct utilization of residual algal biomass as a feedstock for H2 production by a consecutive anaerobic digestion stage should be paid more attention to increase the hydrogen production rate further. To increase the photochemically-useful range of irradiance provided with the bioreactor and to lowering the total energy demands of the biofuel production, integration of the different microorganisms within a comprehensive biosystem

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is supposed [5]. A good example is that the integration of C. reinhardtii (green microalgae) with Rhodospirillum rubrum (purple anoxygenic photosynthetic bacteria) cultures to produce bio-hydrogen was tested to expand the absorption range of solar irradiance from 400e700 nm to 400e900 nm [134,135]. Rather than remaining as a waste product, algae biomass used for bio-hydrogen photolysis production could be good substrates for biogas production from dark fermentation by using anaerobic fermentation as final step [136e138]. It is believed that the application of these integrated bioprocesses would improve the economics and practicality of biohydrogen production in the near future.

Acknowledgements This work was supported jointly by the National Program on Key Basic Research Project (2012CB224803), the National Natural Science Foundation of China (31300030, 31270094), Sinopec (S213049), and the State Key Laboratory of Freshwater Ecology and Biotechnology (2016FB11).

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