Development of marine multi-algae cultures for biodiesel production

Algal Research 33 (2018) 462–469

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Algal Research journal homepage: www.elsevier.com/locate/algal

Development of marine multi-algae cultures for biodiesel production a

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Michalis Omirou , Ioannis Tzovenis , Panayiotis Charalampous , Panayiotis Tsaousis , Polycarpos Polycarpoua, Xanthi Chantzistrountsioub, Athena Economou-Amillib, ⁎ Ioannis M. Ioannidesa, a b

Αgricultural Research Institute, 1516 Nicosia, Cyprus Department of Ecology & Systematics, Faculty of Biology, National & Kapodistrian University of Athens, Athens 15784, Greece

A R T I C LE I N FO

A B S T R A C T

Keywords: Microalgae Multi-culture Biodiesel Wastewater

The concept of community ecology has become a central issue for sustainable biofuel production. However the development of appropriate multi-algae cultures for the industry remains challenging. Natural marine algae blooms collected from coastal areas of Cyprus were induced to form multi-algae cultures, and five (5) of those were developed and tested. Algae growth characteristics, biomass, and lipid productivity were assessed, and the dominant microalga was isolated in all cultures. Growth characteristics, lipid productivity, and FAME composition varied considerably among the different multi-algae cultures as well as their corresponding dominant species. Our results suggest that competitiveness and species complementarity could be crucial factors for biomass and lipid productivity. In material collected from the marina in Larnaca of Cyprus, the induced BL1_LCA bloom was the most promising culture, dominated by Nannochloropsis sp. and accompanied by cyanobacteria assemblages, exhibiting the highest biomass and lipid productivity compared to the other developed blooms. Biomass and lipid productivity of an axenic monoculture of Nannochloropsis sp. were 2.1 and 2.2 times lower compared to those measured in BL1_LCA. The growth parameters of BL1_LCA and its corresponding dominant species were further tested under culture media of different seawater and wastewater ratios. An increase of wastewater ratio in the culture media resulted in a significant reduction of lipid, FAME concentration as well as biomass productivity both in BL1_LCA and its dominant isolate. Overall, our findings suggest that the complex interactions within microalgae community might be crucial for biodiesel production; moreover, the general assumption that wastewater can be applied as an alternative nutrient source should be used cautiously since species-specific responses seem to take place.

1. Introduction Large-scale cultivation of microalgae is an energy-demanding process, which requires large amount of water resources as well as nutrients. A critical step for the development of a cost-effective biodiesel production is the isolation of appropriate strains of high growth rates that are capable of accumulating significant amounts of neutral lipids. Several studies showed that most of the selected strains that were tested under laboratory conditions do not exhibit high biomass productivity and do not accumulate high lipid concentrations when transferred under field conditions [1–4]. This is due to contamination, inadequate temperature control and light saturation and or limitation as well as water evaporation in open pond systems, and oxygen build-up in photobioreactors [1]. These findings suggest that the use of uni-algal cultures as feedstocks for biodiesel production is difficult and unprofitable [5]. Thus, to increase the potential of using microalgae as biofuel feedstocks

⁎

Corresponding author. E-mail address: [email protected] (I.M. Ioannides).

https://doi.org/10.1016/j.algal.2018.06.025 Received 14 October 2017; Received in revised form 30 June 2018; Accepted 30 June 2018 2211-9264/ © 2018 Elsevier B.V. All rights reserved.

it is necessary to develop simple and efficient ways to achieve proper cultivation systems with high lipid productivity. It has been suggested that the implementation of ecological principles during microalgae biomass production could lead to a more stable system in terms of productivity [6–8]. The proposed use of “multispecies communities” with carefully chosen co-habitants was expected to increase biomass productivity. Indeed, earlier studies showed that multi-algae communities generally exhibited higher productivity [9], and that the ecological advantages of microalgae consortia incorporated into cultivation systems might have the potential to improve biodiesel productivity [6,10]. However, very few studies further explored this possibility of developing and applying multi-algae cultures in biodiesel processes [11,12]. A primary concern during the development multi-algae systems is to include species that are functionally different and exhibit a differential response to environmental changes leading to enhanced ecosystem stability [7]. This could be

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Fig. 1. Coordinates of the two coastal sampling locations in Cyprus: the marina of Larnaca (A) and the fishing enclave of Liopetri (B).

established in blooms exhibit higher biomass and lipid productivity compared to their dominant species grown in monoculture conditions? and b) do tertiary-treated wastewater support high biomass and lipid production of the developed blooms and its dominant isolates? To answer these questions a two-step approach employed. In the first step, marine microalgae blooms were developed and evaluated for their biomass and lipid productivity. At the same time the dominant species from each bloom were isolated and evaluated for biomass, lipid productivity as well as for FAME composition. In the second step, different levels of tertiary-treated wastewater were used to evaluate the performance of the most promising bloom and its dominant isolate developed during the first stage of this study. Using this arrangement it was possible to underlie the importance of multi-algal diversity on the biomass and lipid productivity both in seawater and tertiary-treated wastewater.

particularly important when novel technologies are implemented for biofuel production. Recent studies demonstrated that hydrothermal liquefaction is an alternative process, providing a high biocrude yield that could be further upgraded to produce appropriate for the market, hydrocarbon fuels [13,14]. Indeed, the process can be applied to all types of algae without the restriction to high-lipid producing strains [15]. Recently, it has been shown that strain specific characteristics like lipid content and higher biomass positively affects biocrude yields [16]. Thus the development of high biomass yielding multi-algae cultures in the long term could be a valuable source of biomass for biofuel production [17]. Besides the search for the most appropriate strain or species, the use of wastewater might be a viable resource to enhance the environmental and economic sustainability of the microalgae-derived biodiesel [18]. Indeed, many microalgae species are capable of growing in wastewaters utilizing the available abundant organic carbon and other nutrients such as nitrogen and phosphorus [19,20]. Previous studies demonstrated that marine microalgae species are able to grow in wastewaters utilizing the available nutrients. For example, Nannochloropsis sp. (Eustigmatophycese) was able to grow in a medium containing low percentage of wastewater [21]. In another study, the marine haptophyte species Pleurochrysis carterae was able to grow in carpet-mill wastewater with low concentration of nitrogen and phosphorus [22]. These studies suggest that different types of wastewater might be used in microalgae cultivation. However, an optimization for high-lipid productivity is needed since the response of various microalgae species to different types and levels of wastewater is species-specific [23]. Many marine microalgae species are able to accumulate high quantities of lipids in response to unfavorable growth conditions; however, this is also associated with low biomass and lipid productivity [24,25]. Tertiary-treated wastewater is a valuable water resource in semiarid and arid regions like Eastern Mediterranean. Particularly in Cyprus even though the majority of the produced tertiary-treated wastewater is used in agriculture and for the enrichment of surface and ground water resources, substantial amounts are discarded annually to the sea. Tertiary-treated wastewater also contains low amounts of nutrients and it could be a cheap and environmental friendly water resource that can be used as a supplement for the culture of marine microalgae. For the biodiesel industry, it is imperative to optimize lipid and biomass productivity without increasing the operational cost and the environmental impact of the biomass production process. In the current study marine microalgae blooms were developed and tested for biodiesel production under laboratory conditions. The main questions addressed in this paper are: a) do microalgae communities

2. Materials and methods 2.1. Sample preparation, marine bloom development, isolation of dominant species, and growth characteristic measurements Seawater column samples were collected from coastal areas in Cyprus, i.e. the marina of Larnaca (LCA) and the fishing enclave of Liopetri (LP), at 50 cm depth, and filtered through a 50 μm-mesh plankton net (Fig. 1). Blooms were established after enrichment of one (1) liter of the filtered seawater with 1 ml of sterilized Conway enrichment medium (commonly Walne, after Walne [26]). The enriched seawater samples were then stored in a growth room chamber under controlled conditions (25 °C, light intensity of 80 μmol photon m−2 s−1 m−2 and a 14:10 h L:D) in sterilized 2 L Duran bottles and with continuous mild aeration for 7 days until growth was observed. During the bloom development phase, algal growth was monitored using optical microscopy and optical density of the culture at 680 nm (OD 680). In addition, biomass dry weight (BDW, g L−1) was determined daily. Duplicate 10 mL samples were filtered through predried and pre-weighed fiber filters that were dried for 12 h at 100 °C, cooled in a desiccator and weighed. Bloom productivity (BP) was measured for 7 days using the following equation

BP (g BDW L−1 d−1) =

BDWt2 − BDWt1 t2 − t1

where t1 and t2 are the time intervals between early exponential and late exponential time, respectively. Once the initial blooming stage was stable, samples were collected 463

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‘Anthoupoli’ treatment plant (Nicosia-Cyprus) with the following physicochemical profile: pH 8.2, EC 1.71 mS cm−1, total N 7.38 mg L−1, and total P 2.3 mg L−1. The wastewater was sterilized twice in autoclave before use. A completely randomize design was implemented to test the ability of the most promising strain isolates as well as the bloom from which they were derived, for biodiesel production. Both strain isolates and blooms were grown on four different one-liter (1 L) volume media: 100% seawater (SW) medium enriched with Walne stock solution (100% SW), 70% seawater (without Walne addition) and 30% wastewater (70% SW), 50% seawater and 50% wastewater (50% SW), and 30% seawater and 70% wastewater (30% SW). Each treatment was repeated three times and the entire experiment was conducted twice. The growth characteristics were monitored, while the lipid content and FAME composition were determined at the stationary phase of growth.

to a) to isolate the dominant microalgal species and b) maintain the microalgal population and to achieve physiological adjustment of the microalgal population. To isolate the dominant microalgal species from each bloom, 100 μL of the final bloom solution was transferred onto Walne solidified medium under the same growth conditions. The purity of the cultures was accomplished by repeatedly streaking of the developed colonies on petri dishes, and the identification was performed using light microscopy. The dominant microalgal isolates were maintained in agar medium under the same conditions implemented during the blooming and isolation process. To maintain and achieve the physiological adjustment of the microalgal population after bloom development, we implemented serial transfers before the initiation of the experiments following a semicontinuous batch culture approach. In detail, 200 mL of the culture was transferred to a 1800 mL of UV sterilized seawater supplemented with Walne medium. The growing conditions of the developed blooms were the same as those implemented during the blooming phase. This process was repeated three times and the biomass production was similar for each bloom (Supplementary Fig. 1) and we maintained the multi-algal cultures over time. We also compared the micro-algal community of the cultures after the serial transfer with the initial bloom using light microscope and no differences were noticed. Before the experiment, a loop from the dominant isolates maintained in the agar plate was transferred in a 60 mL Walne liquid medium and grown until stationary phase. Two successive cultures were performed to achieve a physiological adjustment of the isolates in the same volume of liquid medium (Supplementary Fig. 2). To compare the growth characteristics, total lipids and FAME content between the dominant isolates and the multi-algal cultures, 20 mL of the physiological adjusted cultures were transferred in 180 mL Walne medium and grown under the conditions described during the bloom development.

2.4. Statistical analysis Determination of the different factors was performed in triplicate and the mean values were recorded. Differences were evaluated using the General Linear Model. Different letters were used to denote statistically significant differences between means of the different parameters (biomass productivity, total lipids and FAME concentration) using the Tukey's HSD post-hoc test with significance level set at p < 0.05. Similarly, the statistical significance of the wastewater use on microalgal growth and the biochemical characteristics were evaluated using one-way Analysis of Variance (ANOVA) with significance level set at p < 0.05. Principle Component Analysis was used to reveal the chemotaxonomic relationship between the isolates and the developed blooms based on FAMEs composition and concentration. Data analysis was performed using R statistics software [28]. 3. Results and discussion

2.2. Total lipids and FAME analysis 3.1. Characterization of multi-algae cultures and dominant species promising biodiesel production

Total lipids were determined using standard methodology and samples were taken before cultures got into the stationary phase [27]. Briefly, 10 mL culture aliquots were centrifuged and washed in triplicate using ammonium formate, and then immediately frozen in liquid nitrogen. The frozen biomass was freeze-dried and lipids were extracted from 200 mg dried biomass with a mixture of chloroform:methanol (2:1, v/v). After vigorous agitation, the biomass–solvent mixture was centrifuged for 20 min and the supernatant was collected and washed with KCl (0.9%) via centrifugation. Subsequently, the mixture was vigorously shaken and allowed to settle. The lower phase was collected and evaporated to dryness using N2, and the total lipids content was determined gravimetrically. Fatty acid composition was determined by methyl esters analysis (FAME). The fatty acid methyl esters were prepared using the oil extract as described above. In detail, a mixture of MeOH:H2SO4:CHCl3 (3.4:0.6:4 mL) was added in 0.2 g of oil. The mixture was heated for 40 min at 90 °C and samples were periodically manually agitated. After cooling, 2 mL of double distilled H2O was added and phases were let to separate. The lower chloroform (CHCl3) phase was collected and evaporated to dryness using N2. Total FAMEs were determined gravimetrically and then the residual oil was dissolved in chloroform. The dissolved FAMEs were separated and analyzed via gas chromatography. FAME's profile of each sample was evaluated using a 37 FAME mixture standard (Supelco) while each peak was confirmed with the National Institute of Standard and Technology (NIST) library. The quantitation of the samples was performed using n-heptadecanoic acid (C17:0) as internal standard, and calibration curves were constructed using the C4–C24 commercial standard (Supelco).

In the present study, samples were collected from 2 different eutrophic coastal areas of Cyprus (marina of Larnaca and fishing enclave of Liopetri), and 32 blooms were developed. Characterization of the microalgae found in blooms was based on the morphological characteristics and cellular appearance of several isolates. Three taxa of microalgae [i.e. Nannochloris sp. (Trebouxiophyceae), Nannochloropsis sp. (Eustigmatophyceae) and Tetraselmis sp. (Chlorodendrophyceae)] were the most dominant unicellular algae found, while diatoms [mostly Nitzschia sp. (probably N. longissima var. reversa) and Cylindrotheca sp.], and cyanobacteria (Pseudoanabaena sp.) were also detected. Unidentified microalgae with unicellular and/or colonial organization were present too, but their occurrence in the blooms was always below 10% of the total bio-volume (Supplementary Table 1). Five (5) of the induced blooms exhibited the highest potential for biodiesel production according to their lipid content and biomass productivity (Table 1). Doan et al. [58] screened and isolated 27 potential marine microalgae species for biodiesel feedstock from Singapore coastal areas and they found Nannochloropsis sp. to be the most promising isolate. Fatty acid profiling is known from the literature as a promising tool for studying chemotaxonomic features in microalgae [29]. In the current study, fatty acid methyl esters profile was also used to supplement microscopic identification of the developed multi-algae cultures and their dominant isolates. Indeed, FAME composition of the three main microalgae species isolated from the developed multi-algae cultures was markedly varied and it is in line with results published previously [30–32]. Principle Component Analysis clearly differentiated the developed multi-algae cultures and their dominant isolates (Fig. 3). For example, BL1_LCA formed a separate cluster from all other blooms mainly due to high proportions of C16:1, C20:5ω3 and C20:4ω6 in the total FAME. This multi-algae culture was also closely grouped with

2.3. Wastewater experiment Tertiary-treated wastewater effluent was collected from the 464

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previously recorded [36]. However, further research is needed to better understand the principles that govern the seasonal function and the structure of micro-algal communities and test whether multi-algae cultures perform better during HTL processing. In non-axenic cultures competitiveness and species complementarities could be crucial factors for biomass accumulation and productivity [37,38]. In our case, the lowest biomass found in BL19_LP could be the result of competition for resources between Nannochloropsis sp. and Nannochloris sp. Indeed, biomass production measured in axenic monocultures of these species was higher compared to that noticed in BL19_LP. These results are consistent with those of other studies and suggest that strain composition is a critical component of the system and that multi-algae cultures do not necessarily out-perform uni-algae cultures [12,39]. The lower biomass production in multialgae cultures has been attributed to the number of competitors in the culture [12,17]. Another possible explanation for this is allelopathy, where one species produce toxic compounds for the other species present in the culture [40]. On the other hand, the presence of cyanobacteria in BL1_LCA enhanced the growth of Nannochloropsis sp., i.e. the dominant species in this bloom, suggesting a positive interaction between them. This is supported by the lower biomass productivity measured in monospecific Nannochloropsis sp. cultures isolated from BL1_LCA (Table 1). In detail, biomass productivity of BL1_LCA (202.61 mg/L/day) was 2.1 times higher compared to the monospecific Nannochloropsis sp. (93.29 mg/L/day) culture. It has been suggested that cyanobacteria might be able to release nutrients to the water column stimulating microalgae growth [41,42]. Other studies have shown that microalgae associated bacteria could provide chemical compounds and nutrients able to enhance microalgae biomass [43,44]. However, the role of these bacteria remains largely unknown [43], and this study suggests complex interactions occur between microalgae and their associated microbial community that might be crucial for biomass productivity. Previously, Stockenreiter et al. suggested that complementarities might be the major mechanism for the increased biomass productivity in multi-algal communities; in particular, they demonstrated that the increased lipid productivity of a multi-algae culture was not related with the dominance of a single, highly-productive species suggesting that other mechanisms might be the control factors [11]. Indeed, resource-use complementarities have been considered as an important explanation for the increased biomass productivity in mixed micro-algae cultures [36]. In a diverse culture, species competition is lower when the present species exhibit different growth requirements compared to intraspecific competition of dense uni-algal cultures. Previous studies have shown that carbon accrual in multi-algal cultures increased with the increasing algal diversity [45]. This response was attributed to the increasing light-use complementarity of the diverse multi-algae cultures.

Table 1 Biomass dry matter (BM, mg/L), total lipids (mg/g), biomass productivity (mg/ L/day) and lipid productivity (mg/L/day) in the five (5) blooms developed from coastal marine waters of Cyprus (Larnaca, LCA and Liopesi, LP), and in the dominant three microalgae species (Nannochloropsis sp., Tetraselmis sp., Nannochloris sp.). Bloom

BM (mg/L)

Total lipids (mg/g)

Biomass productivity (mg/L/day)

Lipid productivity (mg/L/day)

BL1_LCA BL7_LCA BL18_LP BL19_LP BL28_LCA Nannochloropsis sp. Tetraselmis sp. Nannochloris sp.

1230.00a 630.00b 357.33f 482.67e 528.00d 576.21c 432.76ef 529.89d

264.00e 504.33b 430.33c 567.50a 314.00d 245.87e 416.71c 253.67e

202.61a 91.00b 56.81d 77.70c 85.26 93.29b 70.08c 85.75b

46.33a 43.59a 21.68cd 38.79b 23.42c 20.24cd 25.89c 18.98d

Nannochloropsis sp., which was its dominant species (Fig. 3). Similarly, BL7_LCA and BL18_LP were clustered together with their dominant isolate, Tetraselmis sp. (Fig. 3). Surprisingly, the orientation of BL19_LP and BL28_LCA did not match with their corresponding dominant species. Particularly, BL19_LP was clearly separated from all other groups in the positive values of PC1 and this was due to the higher values of C14:0, C18:1ω9 and C20:1. Probably, the presence of both microalgae would create resource-deprived conditions, which in turn could induce lipid accumulation as response to nutrient and/or light stress. Indeed, microalgae are highly responsive to stresses and can accumulate saturated fatty acids to overcome adverse conditions [33]. 3.2. Multi- and uni-algal biodiesel potential 3.2.1. Biomass and lipid productivity Total lipid content in the five most promising multi-algae cultures ranged from 264.0 to 567.50 mg/g dw, and the highest content was noticed in the bloom developed from water samples collected from Liopetri (LP) (Table 1). Particularly, BL19_LP was the bloom with the highest total lipid content (567.5 mg/g dw), and the dominant species identified were Nannochloris sp. and Nannochloropsis sp. with a relative abundance equal to 40 and 55%, respectively (Supplementary Table 1). On the contrary, the lowest lipid content was measured in BL1_LCA (264.0 mg/g dw), a bloom developed in water samples from Larnaca (LCA). Even though BL1_LCA exhibited the lowest lipid content, it retained the highest biomass and lipid productivity in comparison to the other blooms (Table 1). Griffiths and Harrison [1] proposed that high lipid productivity is a desirable key characteristic for biodiesel production. This is particularly important for scale-up purposes since high biomass density increases yield per harvest and improves the economic feasibility of biodiesel production [31]. Additionally, recent findings suggest that implementing novel procedures like HTL could increase biofuel productivity [15]. For HTL, the overall biomass productivity is the critical factor since this technology processes the entire micro-algae biomass. Several studies reported that biocrude oil yield using HTL ranged between 15 and 60% depending on the conditions prevailing during the process as well as the inherent characteristics of the microalgal strains [34]. A recent study showed that multi-algae cultures exhibited more balanced performance and maintained biofuel production at higher levels that un-algae cultures [35]. Our findings clearly show that BL1_LCA could be a promising source for biofuel production through the HTL process due to its high biomass productivity (Fig. 2). Thereby, even if the lipid productivity is low a multi or uni-algae culture could be still appropriate for biofuel production if HTL is the processing pathway. In terms of micro-algae production systems, biomass yield, availability and its quality have to be the main selection traits to further develop HTL processing [16]. Implementing ecological principles to construct high yielding micro-algal communities has been

3.2.2. Impact of microalgae diversity on FAME content and biodiesel quality The fatty acid methyl ester (FAME) concentration of the developed blooms and their dominant isolates are presented in Table 2. The highest FAME content was noticed in the BL19_LP bloom and accounted to 349.3 mg g−1 dw, while the lowest was observed in the BL1_LCA bloom and accounted to 106.3 mg g−1 dw. Intermediate total FAME content was observed in the blooms BL7_LCA, BL18_LP and BL28_LCA (Table 2). Saturated and monounsaturated FAME represented more than the 60% of the total FAME measured. In this study, the major type of FAME detected was palmitic (C16:0), palmitoleic (C16:1) and oleic acid (C18:1ω6). Palmitic acid was the major fatty acid detected in blooms where Tetraselmis sp. was the dominant species and its concentration ranked between 53.3 and 104.5 mg g−1 dw. Palmitoleic acid was the dominant fatty acid detected in the BL1_LCA bloom and in Nannochloropsis sp. strains. In addition, elevated amounts of C16:3 were detected in Nannochloris sp. isolates and accounted for 28.4% of the total FAME detected (Table 2). Interestingly, all blooms exhibited 465

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Fig. 2. Mean biomass production (mg/L) of the 5 developed blooms and the corresponding dominant isolates during time. Error bars represent standard error of the mean (n = 3).

Fig. 3. Bi-plot representation of the 5 developed blooms and the corresponding dominant isolates, derived from Principle Component Analysis of FAME data matrix with the first two principle components.

derived from this bloom (p < 0.001). On the contrary, total FAME found in the BL7_LCA bloom that was dominated by a Tetraselmis sp. isolate was lower compared to that found in monospecific Tetraselmis sp. cultures (p < 0.001). Finally, in Nannochloropsis sp. monospecific cultures derived from the BL1_LCA bloom, the concentration of total FAME was similar to that found in the bloom (p > 0.05). It was also found that the concentration of specific FAME in blooms was lower compared to uni-algal cultures (Table 2). For example, in the BL1_LCA bloom, eicosapentaenoic acid concentration (19 mg g−1 dw) was 10.4% lower compared to that found in the isolated Nannochloropsis strain, further supporting the hypothesis that species-specific interaction could determine lipid concentration in microalgae. It has been suggested that

increased concentration of polyunsaturated fatty acids (PUFA). In particular, PUFA content varied markedly between blooms and the most common fatty acid detected was linoleic (C18:2ω6), linolenic (C18:3ω3), arachidonic (C20:4ω6), and eicosapentaenoic (C20:5ω3) acid. The highest amount of linolenic acid was measured in the BL19_LP bloom (65.8 mg g−1 dw) and accounted for 18.8% of the total FAME determined. Post-hoc comparisons showed that total FAME content in blooms was not always different with those measured in the dominant isolates (Table 2). For example, the Nannochloropsis and Nannochloris sp. dominated BL19_LP bloom, and exhibited substantially higher total FAME content compared to monospecific cultures of both species 466

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Table 2 Fatty acid (FA) profile and individual concentration (mg/g dw) as measured in selected blooms of Larnaca (BL1_LCA, BL28_LCA, BL7_LCA) and Liopetri (BL18_LP BL19_LP) and the corresponding dominant microalgae strains (Nannochloris sp., Nannochloropsis sp., Tetraselmis sp.). FA (mg/g dw)

C14:0 C16:0 C16:1 C16:3 C18:0 C18:1 C18:2ω6 C18:3ω6 C18:3ω3 C18:4ω3 C20:1 C20:4ω6 C20:5ω3 Otherb SFA MUFA PUFA Total FAME a b c d e

Bloom/isolate BL1_LCAa

BL18_LP

BL19_LP

BL28_LCA

BL7_LCA

Nchlor.c

Nanno.d

Tetra.e

5.93 c 7.94 e 49.15 a 0 2.82 c 8.36 e 1.39 d 0.5 cd 2.03 d 0 1.87 e 6.45 a 18.97 b 0.85 cd 16.69 e 59.37 b 29.34 f 106.25 f

12.61 b 72.58 b 4.42 d 0 2.28 cd 49.1 b 26.44 b 0.61 c 23.47 c 1.27 c 3.15 a 2.2 f 4.79 ef 0.95 bc 87.47 b 56.67 bc 58.78 d 203.87 c

21.64 a 104.5 a 7.18 c 0 8.79 a 67.72 a 54.61 a 2.66 a 65.84 a 0 5.4 c 3.45 d 6.15 d 1.37 a 134.93 a 80.31 a 132.71 a 349.31 a

2.53 d 72.47 b 22.76 b 0 3.39 b 34.44 c 11.93 c 0.93 b 32.04 b 1.95 b 2.52 d 5.72 b 13.18 c 0.51 e 78.39 c 59.72 b 65.70 c 204.33 c

11.81 b 67.43 c 3.99 0 1.98 d 46.69 b 25.82 b 0.56 c 22.67 c 1.34 c 2.84 b 1.83 f 4.68 f 0.97 bc 81.22 c 53.52 c 56.88 d 192.59 d

2.63 d 53.25 5.65 d 22.69 1.54 e 14.07 d 26.35 b 0.8 b 19.4 c 0 0.23 f 1.38 g 2.52 g 1b 57.42 d 19.95 e 73.15 b 151.52 e

5.09 c 9.02 d 44.67 a 0 1.27 e 2.28 f 0.91 d 0.39 d 0.08 e 0 0.08 g 5.13 c 27.52 a 0.86 cd 15.39 e 47.03 d 34.03 e 97.31 g

12.83 b 69.72 bc 4.9 d 0 2.59 c 49.08 b 25.6 b 0.62 c 29.46 b 3.72 a 3.13 a 2.44 e 5.06 e 0.77 d 85.14 b 57.11 b 66.90 c 209.92 b

The content of each fatty acid represents the mean of 3 biological replicates. Minor fatty acids (below 1% of total FAME detected in the chromatogram) are designated as other. Nchlor. = Nannochloris sp. Nanno. = Nannochloropsis sp. Tetra. = Tetraselmis sp.

concentration was higher than the maximum levels established in biodiesel standards, suggesting that the biodiesel derived from these blooms could be included in blends with petroleum diesel to increase the oxidation stability of the fuel [53]. However, further research is needed to evaluate possible interactions between species in multi-algae cultivation systems that are reflected to FAME composition and to reveal the responsible mechanisms of this response.

diverse communities can utilize available resources more efficiently than monocultures when the consortium is comprised by complementary species in their use of resources [46]. Indeed, previous studies showed that cultures with high microalgal diversity exhibited higher lipid and biomass production compared to monocultures [11]. Shurin et al. suggested that multi-algal cultures might be a useful approach to increase biodiesel production; however, in this study, it has been postulated that cultures with increased diversity varied greatly in biofuel production compared to the component monocultures [17]. In this study, the developed blooms varied considerably regarding their FAME concentration and the response was species-specific. The highest FAME concentration found in the BL19_LP bloom might be attributed to the competition of the two dominant microalgae species Nannochloropsis sp. and Nannochloris sp. for nutrients. In corroboration, previous studies showed that nutrient stress might enhance lipid accumulation in many microalgae species and suppress biomass production [47–49]. On the contrary, the BL1_LCA bloom displayed low FAME concentration but exhibited the highest biomass and lipid productivity. This bloom was dominated by a Nannochloropsis sp. and cyanobacteria, suggesting a positive association between them. Cyanobacteria are microorganisms suitable for bioenergy production; it has been suggested that they could be used for fuel and high-added value co-products [50]. The interaction between cyanobacteria and microalgae for biodiesel production is unknown and further research is needed to reveal the mechanisms and the ecological perspectives. The profile of fatty acids in microalgae is particularly important as it affects the quality of the biodiesel [51]. Particularly, triglycerides are the desired component within microalgae that are potentially appropriate for biodiesel production. The double bonds found in PUFA are susceptible to autoxidation thereby resulting in poor oxidative stability of the fuel [52], which is the main problem noticed in microalgae-derived biodiesel. The present results indicate that the selected marine species had increased amounts of PUFA, a non-desirable qualitative characteristic for biodiesel, further supporting previous findings [32]. Our results are also suggesting that multi-algae cultivation systems could improve the biodiesel qualitative characteristics, i.e. by reducing the unsaturation levels of FAME. Although PUFA content in the selected blooms was substantially lower compared to uni-algal cultures, their

3.3. Use of treated wastewater in blooms and uni-algae cultures The impact of treated domestic wastewater and seawater mixtures on the growth and lipid characteristics of the most promising developed bloom (BL1_LCA) and its most dominant isolate (Nannochloropsis sp.) were tested. Increase of wastewater percentage in the culture medium resulted in a substantial reduction of biomass productivity both in BL1_LCA and Nannochloropsis sp. (Table 3). Growth of BL1_LCA and Nannochloropsis sp. in culture medium with 70% wastewater was critically suppressed and biomass productivity was reduced by 93 and 89%, respectively (Table 3). This response could be attributed to the low salinity and nitrogen level of the growing media. However, increasing the adaptation period beyond the time implemented in this study might result in an increase of biomass productivity. It has been shown that the growth of Nannochloropsis sp. was completely inhibited Table 3 Biomass productivity (mg/L/day) and total lipids (mg/g dw) measured in the BL1_LCA bloom and in an axenic culture of the dominant microalga species (Nannochloropsis sp.) grown in different seawater levels (SW). Growth medium

100% SW 70 % SW 50 % SW 30 % SW

467

BL1_LCA

Nannochloropsis sp.

Biomass productivity (mg/ L/day)

Total lipids (mg/ g)

Biomass productivity (mg/ L/day)

Total lipids (mg/g)

186.97 a 123.51 b 56.61 c 13.19 d

257.77 b 335.63 a 201.51 c 83.93 d

94.53 72.38 23.43 11.29

241.72 b 273.15 a 165.02 c 78.99 d

a b c d

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findings and contributes additional evidence that the role of minor microalgae players within a bloom can be crucial for biodiesel production and the resilience of multi-algae cultures. This response was found to be species–specific; hence, carefully designed experiments are needed to reveal the role of the different microalgae species within a bloom community. Random species selection was found to be ineffective and prone to unnecessary complexity in cultures with low performance in terms of biomass and biodiesel production. The present findings support the idea of applying community ecology to algal cultivation for biodiesel production. However technologies like HTL where biomass productivity and availability are more critical than lipid content might be more effective in terms of enhanced biofuel production. Thus, the current study could serve as a base for future studies answering the question whether multi-algal cultures are more suitable for biofuel production using HTL processing. Concerning the inclusion of tertiary-treated wastewater in culture media, this study showed that the most promising developed bloom, BL1_LCA, exhibited higher biomass productivity compared to Nannochloropsis sp. monoculture in moderately saline levels. This finding suggests that multi-algae cultures are more resilient under stress conditions supporting previous findings. In addition, we found that low levels of tertiary-treated wastewater caused a significant increase of saturated and monounsaturated FAME concentration possibly due to low nitrogen levels in the culture medium.

Table 4 Concentration (mg/g dw) of saturated (SFA), mono-unsaturated (MUFA) and poly-unsaturated (PUFA) fatty acid methyl esters measured in the BL1_LCA bloom and in an axenic culture of the dominant microalga species (Nannochloropsis sp.) grown in different seawater levels (SW). Isolate/bloom

Nannochloropsis sp.

BL1_LCA

Growth medium

100% SW 70% SW 50% SW 30% SW 100% SW 70% SW 50% SW 30% SW

Lipids concentration (mg/g dw) SFA

MUFA

PUFA

Total FAME

25.4 b 44.7 a 22.6 c 7.5 d 27.9 b 56.1 a 25.1 b 8.2 c

44.5 61.3 35.1 19.2 48.9 74.1 39.7 23.9

23.2 a 18.8 b 18.9 b 5.1 c 25.5 a 20.9 b 21.4 b 5.6 c

93.1 b 124.8 a 76.6 c 31.8 d 102.3 b 151.1 a 86.2 c 37.7 d

b a c d a b c d

when grown in a culture medium with 80 and 100% wastewater [21]. Interestingly, a differential response between BL1_LCA and Nannochloropsis sp. was noticed at lower levels of wastewater. In particular, BL1_LCA biomass productivity was 2.4 and 1.7 times higher compared to Nannochloropsis sp. in 30 and 50% wastewater treatments respectively. The present results show that the presence of more than one species in the BL1_LCA enhances biomass productivity under moderate salinity. This finding corroborates with early ideas of the positive correlation between diversity and ecological communities and particularly the ability of a consortium to withstand perturbance [54]. A recent study showed that species richness had a positive effect on growth rate at different temperature levels and this was associated with a more efficient nutrient utilization due to the different response of the phytoplankton community to temperature [55]. It has been suggested that diverse multi-algal cultures containing species with complimentary traits maintain the resilience of the culture to environmental changes [7]. Wastewater level also affected significantly FAME's concentration. FAME content increased in cultures containing 30% WW in both BL1_LCA and Nannochloropsis sp. by 44 and 38%, respectively (Table 4). However, any further increase of wastewater resulted in a substantial reduction of FAME content in both BL1_LCA and Nannochloropsis sp. In detail, the lowest lipid content was measured in cultures grown in medium with 70% wastewater. Under these conditions, the reduction of total FAME in BL1_LCA and Nannochloropsis sp. was 63 and 65% compared to cultures grown only in seawater medium, respectively. This finding was associated with the critically low biomass production. These results suggest that the inclusion of certain levels of wastewater in the culture medium might possibly enhance the potential of biodiesel production from microalgae due to low nitrogen levels. In the current study, the increase of wastewater by 30% reduced nitrogen concentration in the culture medium from 17.61 mg/L to 12.11 mg/L. Previous studies demonstrated that nitrogen limitation is the most critical factor affecting lipid accumulation as well as biomass accumulation in several microalgae species [56]. These results are in line with previous studies, which reported an increase of saturated and unsaturated FAME under low nitrogen levels [57]. Interestingly, the developed bloom exhibited considerable higher increase of saturated and unsaturated FAME content compared to monospecific Nannochloropsis sp. cultures (Table 3). To our knowledge, lipid accumulation and the response of specific FAME types in mixed microalgae grown under nitrogen-depleted conditions are unknown.

Acknowledgements This research was financially supported by the European Neighborhood and Partnership Instrument (ENPI) (I-B/2.2/099), Mediterranean Sea Basin Joint Operational Program within the framework of the project entitled: Production of biodiesel from Algae in selected Mediterranean countries, Med-Algae (www.medalgae.com). We express our thanks to Louisa Konstandinou and Evdokia Neophytou for their lab assistance. Declarations of authors' contribution All authors had made a substantial contribution to the completion of this study. M.O., I.M.I. and I.T. designed, directed and coordinated this study. P.T., P.C. and X.C. collected and assembled the data from the experiments. M.O. and I.T. performed the statistical analysis and interpretation of the data. M.O. and I.M.I. drafted the article and I.T., P.P. and A.E.-A. critically revised the article for important intellectual content. Conflict of interest statement The authors whose names are listed in the manuscript entitle “Development of marine multi-algal cultures for biodiesel production” confirm that they have no financial or other interest that could be perceived to influence the outcomes of the research. Statement of informed consent, human/animal rights No conflicts, informed consent, human or animal rights applicable. Authors' agreement to authorship and submission of the manuscript for peer review

4. Conclusion

This statement is to certify that all Authors have seen and approved the manuscript being submitted and entitled “Development of marine multi-algal cultures for biodiesel production”. We warrant that the article is the Authors' original work. We confirm that the article has not received prior publication and is not under consideration for publication elsewhere.

The present findings corroborate the view that the implementation of ecological principle strategies could be a feasible and effective process for the development of induced microalgae blooms with enhanced biomass and lipid productivity. The present study confirms previous 468

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Appendix A. Supplementary data [31]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.algal.2018.06.025.

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References [33] [1] M.J. Griffiths, S.T.L. Harrison, Lipid productivity as a key characteristic for choosing algal species for biodiesel production, J. Appl. Phycol. 21 (2009) 493–507. [2] K.M. Weyer, D.R. Bush, A. Darzins, B.D. Willson, Theoretical maximum algal oil production, BioEnergy Research 3 (2010) 204–213. [3] J.W. Richardson, M.D. Johnson, X. Zhang, P. Zemke, W. Chen, Q. Hu, A financial assessment of two alternative cultivation systems and their contributions to algae biofuel economic viability, Algal Res. 4 (2014) 96–104. [4] E. Stephens, I.L. Ross, J.H. Mussgnug, L.D. Wagner, M.A. Borowitzka, C. Posten, O. Kruse, B. Hankamer, Future prospects of microalgal biofuel production systems, Trends Plant Sci., 15, 554-564. [5] J. Benemann, Microalgae for biofuels and animal feeds, Energies 6 (2013) 5869. [6] E. Kazamia, D.C. Aldridge, A.G. Smith, Synthetic ecology — a way forward for sustainable algal biofuel production? J. Biotechnol. 162 (2012) 163–169. [7] D.T. Newby, T.J. Mathews, R.C. Pate, M.H. Huesemann, T.W. Lane, B.D. Wahlen, S. Mandal, R.K. Engler, K.P. Feris, J.B. Shurin, Assessing the potential of polyculture to accelerate algal biofuel production, Algal Res. 19 (2016) 264–277. [8] J.K. Fredrickson, Ecological communities by design, Science 348 (2015) 1425–1427. [9] J.J. Weis, D.S. Madrigal, B.J. Cardinale, Effects of algal diversity on the production of biomass in homogeneous and heterogeneous nutrient environments: a microcosm experiment, PLoS One 3 (2008) e2825. [10] V.H. Smith, T. Crews, Applying ecological principles of crop cultivation in largescale algal biomass production, Algal Res. 4 (2014) 23–34. [11] M. Stockenreiter, A.-K. Graber, F. Haupt, H. Stibor, The effect of species diversity on lipid production by micro-algal communities, J. Appl. Phycol. 24 (2012) 45–54. [12] J.B. Shurin, R.L. Abbott, M.S. Deal, G.T. Kwan, E. Litchman, R.C. McBride, S. Mandal, V.H. Smith, Industrial-strength ecology: trade-offs and opportunities in algal biofuel production, Ecol. Lett. 16 (2013) 1393–1404. [13] P.J. Valdez, M.C. Nelson, H.Y. Wang, X.N. Lin, P.E. Savage, Hydrothermal liquefaction of Nannochloropsis sp.: systematic study of process variables and analysis of the product fractions, Biomass Bioenergy 46 (2012) 317–331. [14] P. Biller, A.B. Ross, Potential yields and properties of oil from the hydrothermal liquefaction of microalgae with different biochemical content, Bioresour. Technol. 102 (2011) 215–225. [15] D.C. Elliott, P. Biller, A.B. Ross, A.J. Schmidt, S.B. Jones, Hydrothermal liquefaction of biomass: developments from batch to continuous process, Bioresour. Technol. 178 (2015) 147–156. [16] S. Jones, Y. Zhu, D. Anderson, R. Hallen, D. Elliot, A. Schmidt, K. Albrecht, T. Hart, M. Butcher, C. Drennan, L. Snowden-Swan, R. Davis, C. Kinchin, Process Design and Economics for the Conversion of Algal Biomass to Hydrocarbons: Whole Algae Hydrothermal Liquefaction and Upgrading, PNNL-23227, Pacific Northwest National Laboratory, 2014. [17] J.B. Shurin, S. Mandal, R.L. Abbott, Trait diversity enhances yield in algal biofuel assemblages, J. Appl. Ecol. 51 (2014) 603–611. [18] J.K. Pittman, A.P. Dean, O. Osundeko, The potential of sustainable algal biofuel production using wastewater resources, Bioresour. Technol. 102 (2011) 17–25. [19] W.Y. Cheah, T.C. Ling, P.L. Show, J.C. Juan, J.-S. Chang, D.-J. Lee, Cultivation in wastewaters for energy: a microalgae platform, Appl. Energy 179 (2016) 609–625. [20] E.-S. Salama, M.B. Kurade, R.A.I. Abou-Shanab, M.M. El-Dalatony, I.-S. Yang, B. Min, B.-H. Jeon, Recent progress in microalgal biomass production coupled with wastewater treatment for biofuel generation, Renew. Sust. Energ. Rev. 79 (2017) 1189–1211. [21] L. Jiang, S. Luo, X. Fan, Z. Yang, R. Guo, Biomass and lipid production of marine microalgae using municipal wastewater and high concentration of CO2, Appl. Energy 88 (2011) 3336–3341. [22] S. Chinnasamy, A. Bhatnagar, R.W. Hunt, K.C. Das, Microalgae cultivation in a wastewater dominated by carpet mill effluents for biofuel applications, Bioresour. Technol. 101 (2010) 3097–3105. [23] C.S. Vairappan, A.M. Yen, Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture, J. Appl. Phycol. 20 (2008) 603–608. [24] A.J. Klok, D.E. Martens, R.H. Wijffels, P.P. Lamers, Simultaneous growth and neutral lipid accumulation in microalgae, Bioresour. Technol. 134 (2013) 233–243. [25] S.A. Scott, M.P. Davey, J.S. Dennis, I. Horst, C.J. Howe, D.J. Lea-Smith, A.G. Smith, Biodiesel from algae: challenges and prospects, Curr. Opin. Biotechnol. 21 (2010) 277–286. [26] P.R. Walne, Studies on food value of nineteen genera of algae to juvenile bivalves of the genera Ostrea, Crassostrea, Mercenaria and Mytilus, Fish. Invest. Lond. Ser. 2 (26) (1970) 1–62. [27] J. Folch, M. Lees, G.H. Sloane Stanley, A simple method for the isolation and purification of total lipides from animal tissues, J. Biol. Chem. 226 (1957) 497–509. [28] R Development Core Team, R: A Language and Environment for Statistical Computing R Foundation for Statistical Computing. Retrieved from http://www.Rproject.org, Vienna, Austria, 2014. [29] A. Sahu, I. Pancha, D. Jain, C. Paliwal, T. Ghosh, S. Patidar, S. Bhattacharya, S. Mishra, Fatty acids as biomarkers of microalgae, Phytochemistry 89 (2013) 53–58. [30] I. Tzovenis, E. Fountoulaki, N. Dolapsakis, I. Kotzamanis, I. Nengas, I. Bitis, Y. Cladas, A. Economou-Amilli, Screening for marine nanoplanktic microalgae from

[34]

[35]

[36] [37]

[38]

[39] [40] [41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52] [53]

[54] [55]

[56]

[57]

[58]

469

Greek coastal lagoons (Ionian Sea) for use in mariculture, J. Appl. Phycol. 21 (2009) 457–469. R. Huerlimann, R. de Nys, K. Heimann, Growth, lipid content, productivity, and fatty acid composition of tropical microalgae for scale-up production, Biotechnol. Bioeng. 107 (2010) 245–257. M.J. Griffiths, R.P. van Hille, S.T.L. Harrison, Lipid productivity, settling potential and fatty acid profile of 11 microalgal species grown under nitrogen replete and limited conditions, J. Appl. Phycol. 24 (2012) 989–1001. N. Lu, D. Wei, X.-L. Jiang, F. Chen, S.-T. Yang, Fatty acids profiling and biomarker identification in snow alga Chlamydomonas nivalis by NaCl stress using GC/MS and multivariate statistical analysis, Anal. Lett. 45 (2012) 1172–1183. H.K. Reddy, T. Muppaneni, S. Ponnusamy, N. Sudasinghe, A. Pegallapati, T. Selvaratnam, M. Seger, B. Dungan, N. Nirmalakhandan, T. Schaub, F.O. Holguin, P. Lammers, W. Voorhies, S. Deng, Temperature effect on hydrothermal liquefaction of Nannochloropsis gaditana and Chlorella sp. Appl. Energy 165 (2016) 943–951. C.M. Godwin, D.C. Hietala, A.R. Lashaway, A. Narwani, P.E. Savage, B.J. Cardinale, Ecological stoichiometry meets ecological engineering: using polycultures to enhance the multifunctionality of algal biocrude systems, Environ. Sci. Technol. 51 (2017) 11450–11458. E. Kazamia, A.S. Riseley, C.J. Howe, A.G. Smith, An engineered community approach for industrial cultivation of microalgae, Ind. Biotechnol. 10 (2014) 184–190. J.N. Griffin, V. Méndez, A.F. Johnson, S.R. Jenkins, A. Foggo, Functional diversity predicts overyielding effect of species combination on primary productivity, Oikos 118 (2009) 37–44. J.-H. Yun, V.H. Smith, H.-J. La, Y. Keun Chang, Towards managing food-web structure and algal crop diversity in industrial-scale algal biomass production, Curr. Biotechnol. 5 (2016) 118–129. A. Schmidtke, U. Gaedke, G. Weithoff, A mechanistic basis for underyielding in phytoplankton communities, Ecology 91 (2010) 212–221. A. Ianora, A. Miralto, Toxigenic effects of diatoms on grazers, phytoplankton and other microbes: a review, Ecotoxicology 19 (2010) 493–511. C.C. Carey, K.L. Cottingham, K.C. Weathers, J.A. Brentrup, N.M. Ruppertsberger, H.A. Ewing, J.N.G. Hairston, Experimental blooms of the cyanobacterium Gloeotrichia echinulata increase phytoplankton biomass, richness and diversity in an oligotrophic lake, J. Plankton Res. 36 (2014) 364–377. N.S.R. Agawin, S. Rabouille, M.J.W. Veldhuis, L. Servatius, S. Hol, H.M.J. van Overzee, J. Huisman, Competition and facilitation between unicellular nitrogenfixing cyanobacteria and non-nitrogen-fixing phytoplankton species, Limnol. Oceanogr. 52 (2007) 2233–2248. R. Ramanan, B.-H. Kim, D.-H. Cho, H.-M. Oh, H.-S. Kim, Algae–bacteria interactions: evolution, ecology and emerging applications, Biotechnol. Adv. 34 (2016) 14–29. M.T. Croft, A.D. Lawrence, E. Raux-Deery, M.J. Warren, A.G. Smith, Algae acquire vitamin B12 through a symbiotic relationship with bacteria, Nature 438 (2005) 90–93. S. Behl, A. Donval, H. Stibor, The relative importance of species diversity and functional group diversity on carbon uptake in phytoplankton communities, Limnol. Oceanogr. 56 (2011) 683–694. R. Ptacnik, A.G. Solimini, T. Andersen, T. Tamminen, P. Brettum, L. Lepistö, E. Willén, S. Rekolainen, Diversity predicts stability and resource use efficiency in natural phytoplankton communities, Proc. Natl. Acad. Sci. 105 (2008) 5134–5138. L. Rodolfi, G. Chini Zittelli, N. Bassi, G. Padovani, N. Biondi, G. Bonini, M.R. Tredici, Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor, Biotechnol. Bioeng. 102 (2009) 100–112. A.P. Dean, D.C. Sigee, B. Estrada, J.K. Pittman, Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae, Bioresour. Technol. 101 (2010) 4499–4507. Y. Li, M. Horsman, B. Wang, N. Wu, C.Q. Lan, Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans, Appl. Microbiol. Biotechnol. 81 (2008) 629–636. F.S. Steinhoff, M. Karlberg, M. Graeve, A. Wulff, Cyanobacteria in Scandinavian coastal waters — a potential source for biofuels and fatty acids? Algal Res. 5 (2014) 42–51. S.K. Hoekman, A. Broch, C. Robbins, E. Ceniceros, M. Natarajan, Review of biodiesel composition, properties, and specifications, Renew. Sust. Energ. Rev. 16 (2012) 143–169. G.R. Stansell, V.M. Gray, S.D. Sym, Microalgal fatty acid composition: implications for biodiesel quality, J. Appl. Phycol. 24 (2012) 791–801. Y.-H. Chen, B.-Y. Huang, T.-H. Chiang, T.-C. Tang, Fuel properties of microalgae (Chlorella protothecoides) oil biodiesel and its blends with petroleum diesel, Fuel 94 (2012) 270–273. D. Tilman, C.L. Lehman, K.T. Thomson, Plant diversity and ecosystem productivity: theoretical considerations, Proc. Natl. Acad. Sci. 94 (1997) 1857–1861. S. Schabhüttl, P. Hingsamer, G. Weigelhofer, T. Hein, A. Weigert, M. Striebel, Temperature and species richness effects in phytoplankton communities, Oecologia 171 (2013) 527–536. Q. Hu, M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert, A. Darzins, Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances, Plant J. 54 (2008) 621–639. S.-H. Ho, A. Nakanishi, X. Ye, J.-S. Chang, K. Hara, T. Hasunuma, A. Kondo, Optimizing biodiesel production in marine Chlamydomonas sp. JSC4 through metabolic profiling and an innovative salinity-gradient strategy, Biotechnol. Biofuels 7 (2014) 97. T.T.Y. Doan, B. Sivaloganathan, J.P. Obbard, Screening of marine microalgae for biodiesel feedstock, Biomass Bioenergy 35 (2011) 2534–2544.