Enhancing the productivity of microalgae cultivated in wastewater toward biofuel production: A critical review

Applied Energy 137 (2015) 282–291

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Applied Energy journal homepage: www.elsevier.com/locate/apenergy

Enhancing the productivity of microalgae cultivated in wastewater toward biofuel production: A critical review Guanyi Chen ⇑, Liu Zhao, Yun Qi ⇑ School of Environment Science and Engineering, State Key Lab of Engines, Tianjin University, Tianjin 300072, China

h i g h l i g h t s  Wastewater is a promising resource for microalgae cultivation toward biofuel production.  Biofuel productivity is restricted by the stability and productivity of culture.  The mixed native algae species and two-stage cultivation system are possible solutions.

a r t i c l e

i n f o

Article history: Received 6 March 2014 Received in revised form 6 October 2014 Accepted 7 October 2014

Keywords: Microalgae Lipid Wastewater Mixed native algae Culture Two stage cultivation strategy

a b s t r a c t Micro-algae have been recognized as a promising feedstock for biofuel production. Effective combining microalgae cultivation with wastewater treatment can reduce CO2 emissions and the cost of microalgae biofuel production, making it more feasible. However, the biomass and lipid productivity must be improved prior to large-scale production. The paper is therefore giving a critical review on microalgae productivity towards biofuel production focusing on the influencing factors in terms of strains and cultivation condition. Based on this review, we recommend the mixed native algae species and employment of two-stage cultivation strategy as potential breakthrough toward sustainable and economic microalgae biofuel production using wastewater as a medium for cultivation. Ó 2014 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3. 4.

5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microalgae-based biofuel and waste resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Microalgae and wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Municipal wastewater. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Agricultural wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Industrial wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Combining microalgae cultivation with wastewater treatment and CO2 capture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The influencing factors ivovlving microalgae-based biofuel production with wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potential breakthrough toward sustainable and economic microalgae biofuel production using wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Strains and diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Cultivation conditions control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⇑ Corresponding authors. E-mail addresses: [email protected] (G. Chen), [email protected] (Y. Qi). http://dx.doi.org/10.1016/j.apenergy.2014.10.032 0306-2619/Ó 2014 Elsevier Ltd. All rights reserved.

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1. Introduction Microalgae have emerged as a potential sustainable biomass resource because of their neutrality towards natural environment [1,2] and flexible cultivation. Compared to terrestrial plants, lowlipid microalgae exhibit faster growth rate [3], and the photosynthetic efficiency of microalgae can potentially exceed 10%, which is 10–50 times greater than that of terrestrial plants [4,5]. Under unfavorable environmental conditions, microalgae are able to accumulate large amounts of lipid [6–8], which is suitable for biodiesel production through transesterification [9–15]. It has been shown that the adoption of biofuel could reduce carbon emissions, and may help to increase energy security [16,17]. However, high lipid concentration is usually inversely correlated with biomass productivity and consequently lipid productivity. If the high growth rate and lipid content are combined, microalgae would become a promising feedstock for biofuel production, especially for biodiesel. Compared to plant-based biofuel crops, microalgae can also be adapt to a wider variety of water sources (fresh, brackish, saline and wastewater) [18–21], and potentially recycle other nutrient waste streams [22]. The application of non-potable water contributes to lower water footprint (WF) of microalgae based biofuel production. Because microalgae are capable of fixing ambient CO2 [5,23,24] and utilizing it as carbon source to grow and reproduce [25], the production of fuel from microalgae provides a promising alternative to conventional carbon capture and storage technologies (CCS). The fixed carbon is incorporated into carbohydrates and lipids and therefore stores energy, produces chemicals, and foods [26,27].

2. Microalgae-based biofuel and waste resources Large-scale commercial production of algae, however, is potentially more costly than traditional crop production [28]. Algae cultivation requires abundance of water and nutrients, such as carbon, nitrogen and phosphorus. The application of external nutrient sources result in direct competition for fertilizers with food growers [29]. A major cause of high algae-based biofuel production costs is the huge consumption of nutrients and water resources [2,30,31]. Most important, nutrient expenses make the major part of the cost of microalgae production [32,33]. With nitrate as the nitrogen resource, 6–8 tons ha 1 year 1 is required in estimation, 55–111 times the requirement for field crops [34]. In addition, the low net energy ratio (energy out/energy in, NER) have been another great problem to perplex the commercialization of algal biofuel. Ideally, an NER of at least 7 is wanted [35], which was much higher than the values achieved in many cases. Under the best conditions considering reflection, respiration, photosaturation and photoinhibition, but with no losses for photorespiration, a maximum photosynthetic efficiency (PE) of 5.4% could be expected on total impinging solar radiation by algal cultures [36]. Given an annual horizontal global solar radiation of 2000 MJ/m2, a maximum annual energy yield of 108 MJ/m2 could be obtained from the outdoor microalgae cultivation system. Assuming the mixing and recirculation of pond water with the power of 0.34 W/m2, the net maximum annual energy yield of 97.20 MJ/m2 was obtained from the cultivation stage. However, PE is independent of irradiation level but depending on photobioreactor type [37], and achieved the lowest value (1.5%) in open pond. Consequently, open pond yield an annual energy yield of 19.20 MJ/m2, which was equal to the NER of 1.78 in the cultivation step. Even worse, lower life cycle NER value could be obtained with the supplement of chemical fertilizers, as well as the application of energy-intensive biomass harvesting and subsequent biofuel conversion techniques [38,39]. Developing algae production approaches that can effectively use non-fresh water resources and minimize both water

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and nutrient requirements will help reduce resource constraints. In order to make microalgae-based biofuel cost-effective, wastewater and flue gas can be employed as cheap nutrients sources. Such a method would reduce the reliance on chemicals providing the nutrients, making it also ecologically favorable [32,40]. 2.1. Microalgae and wastewater Global annual freshwater consumption was estimated at 3,908.3 billion m3 [41] during 2009, and most of the consumed water turned into wastewater. Furthermore, total N and P concentrations in wastewater can be as high as 10–100 mg/L in municipal wastewater and even more than 1000 mg/L in agricultural effluent [42]. Taking the city of Tianjin of China (population of about 14 million in 2012) as an example, the annual N and P emission in wastewater in 2011 was 36,700 t and 4700 t, respectively, corresponding to 7.18 and 0.92 g/d per capita. Without proper treatment, the release of N and P would lead to eutrophication and ecosystem damage in downstream watersheds [43–48]. Benefiting from its abundance and enrichment of nutrients, wastewater can be used as a low cost nutrient source for microalgae cultivation. Microalgae have high potential to remove nutrients from wastewater and to accumulate biomass for biofuel production [49–54]. Algal treatment of wastewater offers a cheaper and more efficient means to remove nutrients and metals from wastewater than conventional tertiary treatment [51,55–57]. For example, most commercial approaches of phosphorus removal do not recycle it as a fully sustainable product, as it is retrieved along with various other waste products, some of which are toxic [58]. But these nutrients can be incorporated into algae biomass [18] and subsequently removed from wastewater by algal treatment [33,59]. In addition, the application of microalgae eliminates the sludge treatment, which is logistically challenging [60]. Culturing of microalgae in wastewater also substantially reduces the need of chemical fertilizers and their related burden on life cycle [61,62]. Through the utilization of wastewater, the zero-waste concept is further implemented, and thus stimulates a more sustainable practice for the microalgae biofuel industry. It has even been proposed that integrated phyco-remediation and biofuel technology appears to be the only source of sustainable production of biofuels [63]. Taking Tianjin as an example, P would act as the limiting nutrient for algal growth in wastewater, because the molar N:P ratio available for algal uptake (17.3) exceeds the Redfield ratio for N:P (16). The P load available from wastewater is 1.52  108 moles. According to the Redfield ratio, 2.43  109 moles of N (34,020 t) and 1.61  1010 moles of C (193,200 t) are fixed respectively. As microalgae typically comprise 50% C by weight, this quantity of wastewater can theoretically support the maximum annual yield of 386,400 t biomass. Now, we assumed that the biomass composition was accordance with Roberts et al.’s result, viz. 29.0% dry weight (dw) ash, 48.9% ash-free dry weight (afdw) carbon, 37.5% afdw oxygen, and 14.0% afdw lipid [64]. According to Roberts et al. [64], the production of hydrothermal liquefaction contains 44.5 ± 4.7% afdw biocrude and 45.0 ± 5.9% dw solid biochar, with the energy density 39 and 8– 10 MJ/kg respectively. Consequently, an annual energy yield of 6.15–6.50  109 MJ could be obtained by the wastewater based miroalgae cultivation in Tianjin. However, 8500 t hydrogen was demanded for the fully hydroprocessing of biocrude (122,000 t) toward biodiesel production [65]. Attributed to the low sensitivity to feedstock price and low levelized cost, biogas steam methane reforming (SMR) was a feasible resource of hydrogen [66,67]. In the long run, the NMT-x catalysts render future practical and cost-effective applications in hydrogen production under visible light [68]. Additionally, on-site hydrogen production would be a practical solution with the elimation of storage and transportation.

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Also, the application of wasterwater could cut the WF of microalgae biofuel production. In fact, the supply of freshwater is insufficient to support any substantial scale production of algal fuels anywhere [38]. Without water recycle, the WF value can be up to 3726 kg/kg biodiesel, which could be reduced by as much as 90% with wastewater using [30]. On the contrary, the WFs of bioethanol from cassava and sweet sorghum were 3708 and 17,156 kg/kg respectively, whereas the values for biodiesel produced from Jatropha curcas L. and soybean were 5787 and 13,676 kg/kg respectively [30,69]. Thus, the WF of wastewater based algal biodiesel is much lower than that for biodiesel from other sources, as well as bioethanol. With wastewater supplement, the WF mainly originates from evaporative loss during cultivation [30,38]. Also, it has been revealed that lipid productivity have positive influence on WF [70]. Consequently, further reduction in WF could be achieved with the site selection for lower evaporation rate (and the replacement of open pond by photobioreactors), as well as the cultivation system with higher lipid productivity. Recently, a number of studies have reported reasonable lipid accumulation in wastewater-grown microalgae, ranging from low (<10% DW) to moderate (25–30% DW) lipid content, and in some studies this can translate to relatively high lipid productivity when coupled to high biomass. But it should be noted that all these experiments are conducted in laboratory rather than in large scale production. Some factors, including land occupation and harvesting cost, have been neglected. Consequently, these results may not be applicable to industrial large scale of biofuel production. Although wastewater contains valuable nutrients for algal growth [71,72], the presence of toxin, predators and competitors in wastewater may have negative impact on microalgae [73]. Furthermore, all these properties of a specific wastewater vary with sources. As a result, the ability of microalgae strains to valorize waste stream and accumulate lipid varies for each strain and each waste stream. Herewith we briefly review the studies involving microalgae cultivation in different types of wastewater including municipal wastewater, agricultural wastewater, and industrial wastewater. 2.2. Municipal wastewater During municipal sewage treatment, high levels of N and P are removed from wastewater in the tertiary advanced treatment phase, and these nutrients can be actually applied in growing microalgae. It has been confirmed that microalgae are able to remove N and P from municipal wastewater efficiently [51,74,75]. Some microalgae species can even grow in raw wastewater [76], and remove nutrients efficiently [77]. Li et al. found that Chlorella sp. removes ammonia, TN, TP by 93.9%, 89.1%, and 80.9% respectively, from raw concentrate [77]. Municipal wastewater can serve as the nutrient resource for microalgae cultivation toward biofuel production [58,78]. Currently, most studies on the combination of wastewater treatment with algae based biofuel production focus on municipal wastewater treatment [77,79–82]. Kong et al. cultivated Chlamydomonas reinhardtii in municipal wastewater. 55.8 mg/L/d N and 17.4 mg/ L/d P are effectively removed from wastewater. The maximum biomass productivity and lipid content are 2.0 g/L/day and 25.25% (w/ w) respectively [79]. But all the results come from laboratory-scale experiments, and require confirmation in long term and at relevant volume, as there is significant difference between laboratory-scale and large-scale production. 2.3. Agricultural wastewater A significant difference between agricultural wastewater and municipal wastewater lies in the higher nutrients (such as N and P) concentration in the former. The abundance of nutrients indi-

cates that agricultural wastewater could be an alternative nutrient source for microalgae production [83]. Microalgae are able to grow in, and efficiently remove nutrients from agricultural wastewater [84–89]. Agricultural wastewater can also be used as the nutrient source for algae based biofuel production. Chlorella sp. cultivated in dairy manure wastewater achieves a fatty acid productivity of 0.23 g/m2/d [90]. However, high concentration of nutrients and other compounds in agricultural wastewater might inhibit microalgae growth [91,92]. In addition, high turbidity of agricultural wastewater would reduce light penetration necessary for algal growth. As a result, wastewater should be diluted during the storage and before use [93,94]. It has been confirmed that dilution has an impact on biomass accumulation and nutrients removal from agricultural wastewater [88,95,96]. In an experiment by Johnson et al. [88], the highest algal production was achieved in original dairy wastewater and 75% diluted wastewater, with almost all soluble phosphorus removed. Dilution is also correlated to lipid production [87,97,98]. Zhu et al. cultivated Chlorella zofingiensis in piggery wastewater, and achieved the maximum lipid productivity of 110.56 mg/L/d when the initial COD was diluted to 1900 mg/L [98]. 2.4. Industrial wastewater Previously, the majority of the researches on microalgae cultivation in industrial wastewater emphasize on the removal of heavy metal pollutants and organic chemical toxins, rather than nitrogen and phosphorus [40,57,58,99].There have been only three studies involving microalgae cultivation in industrial wastewater for biofuel production, i.e. the production of microalgae with palm oil mill effluent [100], carpet mill wastewater [101–103] and olive mill wastewater (OMW) [91,104–106]. For Scenedesmus obliquus growth in OMW, the highest value of maximum specific growth rate is 0.044 h 1 [91] and the carbohydrates content can reach 65.8 wt% [104]. The total N and P concentration of OMW (532 mg/L and 182 mg/L, respectively) [107] corresponds to N:P ratio of 6.5, which means N would act as the limiting nutrient for microalgae growth. Taking into account large available amount of OMW (5.4  106 m3/year) [105,106] in the world, the maximum annual microalgae biomass (50% C by weight) and lipid (25% by weight) yield theoretically supported by OWM is 3.26  105 t and 8.2  104 t, respectively. However, the high metal concentration, existence of organic chemical toxins and relative low N and P concentration in industrial wastewater have negative effects on microalgae growth. Consequently, the application of industrial wastewater in microalgae based biofuel production is limited. 2.5. Combining microalgae cultivation with wastewater treatment and CO2 capture In order to make microalgae cultivation more cost-effective, efficient combination of waste exhaust CO2 and wastewater streams is recommended [33,108–110], which provides a pathway for removing nutrients from wastewater, capturing CO2, and producing feedstock for biofuel production, without using freshwater [111,112]. The potential of such approaches has been proven both theoretically and experimentally [102,112–116]. Acting as the carbon source, CO2 has a positive effect on biomass and lipid accumulation [79,108,117–120]. Hu et al. indicated that CO2 has a positive effect on biomass and lipid productivity of Auxenochlorella protothecoides cultivated in concentrated municipal wastewater [117]. CO2 has also been shown to affect the composition of fatty acids in microalgae [121–125]. Tang and Devi confirmed that CO2 supplementation has significant effect, both positive and negative, on the degree

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of saturation, which determines the properties of biodiesel [124,125]. In addition, CO2 can be used to regulate pH value of the cultivation system, and therefore avoids the use of acid, alkaline or buffer for the same purpose, which is difficult to realize and also unsafe for the environment. [126]. Because of its high concentration of CO2, flue gases are optimal CO2 source for the microalgae based biofuel production [127,128]. Although NOx and SOx components in flue gas may be toxic for microalgae, their actual effect is highly dependent on the type of strains [109]. Some microalgae strains are able to eliminate both CO2 and NO from flue gas [129–134]. It has even been reported that flue gas derived from a cement plant has no significant adverse effects on microalgae cultivation compared to pure CO2 [135]. However, the low mass transfer efficiency of CO2 from gaseous to liquid phase is the major limiting step in microalgae cultivation [136]. The direct injection of flue gases into microalgae culture, which always accompanies the release of CO2 to the atmosphere, may be less effective [137]. In addition, the additional cost of carbon capture and transportation must be considered when flue gas is used as the carbon source for microalgae cultivation. Some works proposed separating CO2 from flue gases before utilization by techniques such as chemical absorption [138]. However, the energy consumption for CO2 capture and regeneration would again increase the cost of microalgae production. Some researchers are now trying to capture CO2 in the form of bicarbonate [127,139,140]. Chang et al. applied alkaline wastewater as the scrubbing liquid for capturing CO2 from exhaust such as that from natural gas boilers, and achieved commercially competitive substantial biomass productivity (0.036 g/L/d) [140]. However, the treated wastewater, from which pollutants and nutrients are partially removed, should be reused rather than discharged directly into the environment. Longer hydraulic detention time arising from wastewater reuse and the cost of alkalis (NaOH) would add to the difficulty of commercial microalgae-based fuel production, especially in large scale. 3. The influencing factors ivovlving microalgae-based biofuel production with wastewater Effective combination of microalgae cultivation with wastewater treatment (and CO2 mitigation) can reduce the cost of microalgae-based biofuel production. However, the low biomass and lipid productivity is its major impediment [141]. Low productivity increases the consumptions of resource and energy involved in biomass cultivation and processing and consequently makes microalgae biofuel unsustainable. The low productivity also increases the land occupation for microalgae cultivation. In large scale production, an important issue is the number of suitable sites for algae cultivation, which must be close to both sources of CO2 and wastewater [142]. But it is nearly impossible to find a suitable site to cultivate microalgae in large scale that is close to the wastewater treatment plants (as well as flue gas resources). The low productivity means that a vast area of land would be used for cultivation system and ancillary facilities, and extend the distances between cultivation position and resources. Furthermore, the collection and transportation cost of the waste resources affected the final profits of biofuel adversely [143]. Consequently, the benefit from using waste resources is still not sufficient in compensating for high cost of waste resources capture and transportation.

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To realize an economically viable algal biofuel industry, it is indispensable to optimize the productivity [144]. Fortunatelly, the huge metabolic plasticity of microalgae means that production of lipid can easily be triggered [145–148]. Nutrient starvation under phototrophic condition is the most common approach to enhance lipid accumulation. A number of nutrient stresses, such as nitrogen [149], phosphate [150], iron [151], silicon [152], and sulfur [153] can trigger the change of metabolic flux from carbonhydrate to lipid biosynthesis and consequently promote lipid accumulation. Higher lipid content can even be achieved by prolonging the exposure of the culture to nutrient-depleted conditions [154,155]. But the response to nutrient stress differs from one algae specie to another [156]. Most importantly, higher lipid content always accompanies lower biomass productivity [157–160] and consequently decrease in overall lipid productivity [148,161,162]. For example, under nitrogen limitation, the lipid content of Scenedesmus sp. LX1 reaches as high as 30%, but the lipid productivity decreases from 20.3 mg/L/d to 8.3 mg/L/d [162]. One possible solution to this dilemma is optimizing nutrient limitation conditions rather than complete starvation [163–165]. For example, although increasing nitrogen concentration in the medium (0.04-3.66 mM) leads to a decrease of lipid content, the maximum lipid productivity of 0.019 g/L/d has been achieved by Botryococcus braunii cultivated with an initial 0.37 mM nitrate [164]. However, this approach is only applicable to those species that show significantly different lipid productivity in low-nutrient medium than in an intermediate-nutrient medium. In addition, microalgal photosynthesis will be significantly impeded if colored wastewater is applied as cultivation medium. Even dilution can induce such negativity, the requirement of larger cultivation volume would counteract the application of wastewater. Consequently, such a strategy is not applicable to microalgae biofuel production, especially in industrial large scale. An alternative is heterotrophic/mixotrophic growth with external organic carbon source. Organic carbon source can increase both cell density and lipid productivity of microalgae, especially under mixotrophic conditions [77,166–169]. Compared to heterotrophic conditions, the lipid yield of Chlorella protothecoides under mixotrophic conditions is 69% higher [170]. Heterotrophic and mixotrophic operations are also superior because they do not rely on light. On the contrary, autotrophic microalgae productivity is strongly dependent on the availability of solar resource [171]. Most importantly, organic carbon from wastewater stimulates a more sustainable practice for the combination of algae based biofuel production and wastewater treatment. Such organic carbon sources avoid the cost of external organic carbon sources [158,172,173]. In fact, carbon sources from wastewater have been reported to offer great promises for cultivation of heterotrophic and mixotrophic algae [80,174–177]. In addition, heterotrophic/mixotrophic strains have very good potential in wastewater treatment [24]. However, additional organic carbon source leads to the potential of contamination and competition of cultures with other microorganisms, and results in the culture instability, especially with wastewater as a carbon resource (see Table 1). Such a condition may deteriorate for the case of applying limited microalgal strain monoculture. In fact, the natural biodiversity of microalgae is estimated to be 350,000 species [178], and about 3000 strains have been screened during the Aquatic Species Program [8]. But much research work and commercial production

Table 1 The advantages and disadvantages of different microalgae culture modes. Culture mode

Advantages

Disadvantages

Phototrophic Heterotrophic & Mixotrophic

Low cost High productivity

Low lipid productivity High cost, contamination and competition of cultures with other microorganisms

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focus only on the monoculture of a small number of species with high growth rate and lipid content. Genetic engineering has also been restricted to improve well documented strains to enhance lipid production [179–181]. However, interspecies differences in lipid production under different nutrient conditions or cultivation modes may result in differences in the cultivation efficiency. Most importantly, the application of sterilized bioreactor in microalgal biofuel production is too energy-intensive and costly to be economically viable, whilst open systems impose risk in culture contamination and instability. Lots of factors, such as competition and predation, have negative impacts on microalgae cultivation, and may contribute to the instability of the culture. Monocultures are susceptible to contamination [182], and strains with high lipid productivity are likely to be outperformed by other faster growing species [183]. Additionally, the stability of microalgae productivity in monoculture is doubtful when supplied with wastewater [184]. It is difficult to maintain a pure culture during the operation because of constant airborne contamination in the open system and impacts from the wastewater [185]. In fact, only mixed culture of algae can sustain themselves in wastewater treatment systems [33]. 4. Potential breakthrough toward sustainable and economic microalgae biofuel production using wastewater Commercial production of microalgae biofuel with wastewater is possible only if three main parameters can be fulfilled, i.e. high biomass productivity, high lipid content and productivity, and high tolerance to wastewater. High biomass and lipid productivity helps reduce the resource and energy consumption involved in biomass cultivation and processing. High tolerance to wastewater contributes to the health and stability of cultivation system. Microalgae strains and diversity determine the potential productivity of a cultivation system, whilst the actual production depends greatly on culture condition. 4.1. Strains and diversity As a critical factor [186,187], selection of proper microalgae species is crucial to the success of effective combination of wastewater treatment and algae-based biofuel production [162]. Taking all factors into account, cultivation of mixed native algae species may be the only resolution that meets all the requirements. Native microalgae species perform better than most other species in commercial scale cultivation with wastewaters [80,188]. Acclimation in accordance with the local environment can facilitate the growth of native species [51,97,103,189,190], and even physiological acclimation of commercial cells in wastewater prior to the utilization improves their nutrient removal efficiency [191]. Cho et al. found that the maximum biomass production of the five native microalgal isolates showed increases of 1.3–1.7 times compared with that of Chlorella sp. 227 cultured with diluted anaerobic digestion tank wastewater and the growths were active from the initial stage without any lag period [192]. Moreover, native species showed higher removal rates of nutrients from local environment [188,193,194]. Pérez et al. reported that species isolated from wastewater achieve remarkably higher nutrients removal rates than those achieved in experiments with commercial species [188]. Hence, it is possible to maximize the efficiency of nutrient removal by endogenous microalgae [188,195]. The native species cultivated in wastewater also have higher growth rate, biomass productivity, and even lipid content [80,82,96,101]. Compared to 11 species of high-lipid content microalgae reported by other researchers, Scenedesmus sp. LX1 isolated from stored tap water [173,196] achieves the highest biomass (0.11 g/L DW) and lipid content (31–33%) when grown in secondary effluent [82].

High-diversity native algae system may result in more robust operation and high productivity. Compared to monocultures, microalgae communities of higher diversity are considered to be more stable [197–200] and lower invasion risk [201]. Bhatnagar et al. confirmed that the diversity of microalgae cultivation system has positive effect on the stability of the cultivation system as well as the types of wastewater applicable in microalgae cultivation [103]. Species diversity also has a positive effect on bioremediation of wastewater. Combining species with different metabolic abilities enhances the retention of nutrients [202–205], as well as the overall remediation capacity [184,206,207]. Compared to the monocultures, mixed algal culture of C. reinhardtii, S. rubescens, and C. vulgaris achieved higher nutrient removal rates from wastewater [208]. Additionally, mixed cultivation is cheap and easy to operate and maintain, making the wastewater treatment and algae cultivation more cost-effective and efficient [208]. Furthermore, high diversity results in higher biomass [209,210] and lipid productivity [211]. Communities of higher diversity have higher efficiency of resources usage [212], which ultimately leads to better nutrients depletion and consequently better lipid accumulation [213]. However, high diversity does not necessarily mean a potential increase in lipid content. According to a study by Chinnasamy et al. on a consortium of 15 algaes, its lipid content is not higher than that of any monoculture in wastewater [101]. Thus, more work should be conducted at the biochemical and physiological levels to understand the inhibitory factors of lipid accumulation during mixed microalgae cultivation in wastewater. The dominance of oleaginous species in mixed microalgae cultivation should be the most pragmatic goal. 4.2. Cultivation conditions control Cultivation conditions play an important role in the biomass and lipid productivity of microalgae strain or consortium. Taking into consideration the merits and drawbacks of different growth modes of microalgae, two-stage cultivation strategy is recommended, i.e. a first stage for cell reproduction and a second for lipid production [179]. Within such a system, cell proliferation and lipid production are conducted and optimized during different phases, and the first phase can be separated or succeeded with the second one. However, harvesting (especially filtration or centrifugation) and the consequential cost from biomass transfer between the two stages means that the process without the two separated phases is more practical, especially on a large scale. One alternative is the combination of nutrient-sufficient and nutrient-limited conditions. With an initial nutrient-sufficient condition followed by nutrient deprivation, such a strategy could balance the rapid and efficient biomass generation with high lipid content to maximize the lipid productivity [214]. Taking into account the productivity of different growth modes, it is feasible to apply mixotrophic/heterotrophic rather than phototrophic mode in the two-stage nitrogen starvation process. Zheng et al. cultivated Chlorella sorokiniana in such a process and yielded high lipid productivity (4.2 g/L/d) [215]. Another alternative is the combination of phototrophic stage with mixotrophic/heterotrophic stage in the microalgae cultivation process. Such a system takes advantage of both the high efficiency of mixotrophic/heterotrophic cultures and low production costs of phototrophic culture. One approach is the phototrophic–heterotrophic culture mode [216]. According to Xiong et al.’s research, such a strategy enhances carbon conversion ratio of sugar to oil and provides an efficient approach for the production of algal lipid [217]. The heterotrophic/mixotrophic -phototrophic culture strategy is also an alternative system [50,117]. In fact, heterotrophic culture provides an efficient way for seed cells production, which can be used as inoculums in the subsequent cultivation for biomass and

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Light Flue gas

CO2 Capture

Phototrophic Culture

A

Water Wastewater

Mixotrophic Culture

B

Heterotrophic Culture

C

Biomass Pretreatment

Lipid

Waste organic carbon resource

Biodiesel Fig. 1. The schedule of two stage microalgae cultivation system toward biodiesel production with waste resources. A – the combination of heterotrophic/mixotrophic with phototrophic cultivation system; B – two stage mixotrophic cultivation system; C – two stage heterotrophic cultivation system.

lipid production [218]. High inoculation rate of heterotrophic algal seed can be utilized as an effective method for contamination control [218]. Zhou et al. applied such a strategy and achieved high biomass concentration (1.16 g/L), high lipid content (33.22%), as well as high nutrient removal efficiency from municipal wastewater [50]. However, the O2 produced in photosynthesis would go against microalgae growth. High O2 level could reduce biomass and lipid production [36,219]. Consequently, O2 concentration should be maintained at a relative low level for phototrophic culture, especially with the application of mixed algae species which lead to higher O2 production [210]. Physical and biological process could be applied as O2 scavenger. As a common physical method, proper gas exchange for algae growth could attribute to sufficient removal of O2 [33,220]. However, fluctuating O2 evolution capacity of algae cultivation would lead to the variance of the optimal air-flow rate value, and negative impact on life-cycle NER ultimately. For biological process, addition of the O2 scavenger might permit the algae to avoid poisoning themselves [221] but will introduce a competitor for the nutrients. Such a drawback could be overcomed by the application of nitrogen-fixing growth-promoting bacteria. Consumption of O2 and extracellular matter production (such as exopolysaccharydes) by algae can enhance bacterial growth rate, as well as CO2 and growth promoter substances production by bacteria can enhance microalgal growth [222]. Compared with monoculture, higher biomass and lipid yield of C. vulgaris was obtained from the jointly cultivation with Azospirillum brasilense in wastewater [223,224]. As shown in Fig. 1, waste resource can be integrated into the two stage system as cultivation medium. Wastewater with organic carbon resource can be used as medium for heterotrophic/mixotrophic growth, whilst that without organic carbon can be applied in the phototrophic stage. The combination of different wastewaters can also be used as microalgae cultivation medium. Benefiting from their abundance and relatively low cost, waste organic carbon resource and flue gas can also be used as carbon resources. Hu et al. confirmed that CO2 aeration can be conducted in the whole process of the two-stage cultivation strategy [117]. Most important, the diversification of nutrients could attribute to a flexible biofuel supply, which would foster the supply security involving biofuel [225]. However, the two-stage cultivation strategy requires further assessment when wastewater is used as medium, especially when waste resources are applied in large scale continuous cultures.

5. Conclusion Although considered as a promising feedstock of biofuel production, the algae towards biofuel production is limited by its huge consumption of resources and the high cost. To make microalgae cultivation more cost-effective, wastewater treatment is combined with the process. On the other hand, microalgae-based biofuel is restricted by stability and productivity of the culture. Low stability of limited microalgal strain monoculture has a negative effect on the health of cultivation system, and may lead to collapse. Low biomass and lipid productivity adds to the cost of biomass cultivation and processing. Here we recommended mixed native algae species and two-stage cultivation strategy as possible solutions. The cultivation of mixed native algae species improves the stability of cultivation system in wastewater, and promotes the biomass and lipid productivity. The two-stage strategy, with cell proliferation and lipid production conducted in separate phases, combines high biomass productivity with high lipid content, and accordingly results in high lipid productivity. Waste resource can be applied in the two stage cultivation strategy of mixed native microalgal species. Wastewater, flue gas, and waste organic carbon resource supply microalgae with nutrients, inorganic and organic carbon resources. The combination of wastewater and different carbon resource can be used in different growth mode, and achieve higher biomass and lipid for biofuel production. However, such a combination solution requires more further research as there is big difference between laboratory and large-scale production. meanwhile the total process should be simplified by further investigation prior to large scale application. Acknowledgements Financial support from National Key Basic Research Program of China (Grant No. 2012CB215303), National Natural Science Foundation of China (Grant Nos. 51076158, 51108310) and Project for Developing Marine Economy by Science and Technology in Tianjin (KX2010-0005) are highly appreciated. References [1] Karemore A, Pal R, Sen R. Strategic enhancement of algal biomass and lipid in Chlorococcum infusionum as bioenergy feedstock. Algal Res 2013;2:113–21. [2] Amaro HM, Guedes AC, Malcata FX. Advances and perspectives in using microalgae to produce biodiesel. Appl Energy 2011;88:3402–10.

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