A novel one-step method for oil-rich biomass production and harvesting by co-cultivating microalgae with filamentous fungi in molasses wastewater

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A novel one-step method for oil-rich biomass production and harvesting by co-cultivating microalgae with filamentous fungi in molasses wastewater Limin Yanga, Huankai Lib, Qin Wanga,b,

T

⁎

a State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center of Nanjing University, Nanjing 210061, China b Department of Food Science, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Chlorella sp. Aspergillus sp. Nutrients removal Lipid synthesis Biofuel

Aiming at simplifying the harvesting procedure, reducing the production cost, and improving the quality of microalgae-based biodiesel, herein, a novel one-step method for oil-rich biomass production and harvesting was proposed by growing Chlorella sp. with Aspergillus sp. in molasses wastewater. Lipid content and fatty acid profile were measured to assess the suitability of microalgal-fungal biomass for biodiesel production. The results showed that the highest biomass yield (4.215 g/L) was obtained when the inoculation ratio of fungi and microalgae was 100. Activities of fungi positive impacted the decolorization of wastewater and the removal of suspended solids. Thus, co-cultivation system had better performance than mono-system of microalgae in the removal of nutrients in wastewater. Analysis of biomass compositions showed that compared with mono-system of microalgae, co-cultivation system produced biomass with higher lipid content (35.2%) and yield microbial cell oil with lower unsaturation degree, potentially increasing the quality of microbial-cell-lipid based biodiesel.

1. Introduction In recent years, biodiesel production from microalgae, which have high biomass productivity and oil content, has been receiving considerable attention. However, the biodiesel production from microalgae still has not been widely commercialized because of high cost and low oil quality. As reported by Haas et al. (2006), the production cost of microalgae-based biodiesel ranged from 160 to 480 USD/bbl, which is much higher than the price of crude oil. Generally, the cost of freshwater, fertilizers, and harvesting process accounts for over 50% of the total cost (Acién et al., 2012; Zhang and Hu, 2012). In addition, low quality of biodiesel, partly caused by the very high percentages of polyunsaturated fatty acids (PUFAs) in microalgae oil, is another problem hindering the commercialization of microalgae-based biodiesel (Knothe, 2005; Kumar, 2017). The use of wastewater for low-cost microalgae biomass production has been widely documented in previous studies, but its application is rare in the industry. High energy consumption or high cost of biomass harvesting is one of the factors which limit the commercialization of microalgae-based biodiesel (de Boer et al., 2012). Also, in some cases, removal efficiency of nutrient in wastewater was not high since microalgae did not perform well in the removal of suspended solid

organics (Lu et al., 2015). Because of these technical barriers, wastewater-based microalgae cultivation for biodiesel production was rarely commercialized in a real-world application. Biofloculation, of which the energy consumption is low, is considered as a sustainable way to harvest microalgae biomass. Previous studies have used Moringa oleifera seed derivatives, auto-flocculating microorganisms, and filamentous fungi to harvest microalgae biomass by floculation (Chen et al., 2018; Hamid et al., 2014; Lananan et al., 2016). Low quality of microalgae-based biodiesel is another serious problem limiting the sustainable development of biodiesel industry (Knothe, 2005). Regardless of marine microalgae, many freshwater microalgae contain very high contents of PUFAs, which may change the oxidative stability and cetane number, resulting in low quality of biodiesel (Kumar, 2017). Previous study, which grew Chlorella zofingiensis for biodiesel production, discovered that the percentage of PUFAs is over 40% of total lipid in biomass (Liu et al., 2011). In some cases, percentage of PUFAs is even roughly 70% of total lipid in freshwater microalgae biomass (Lu et al., 2017). It is well known that the decrease of percentage of PUFAs in lipid is critical to the production of highquality biodiesel (Knothe, 2005). However, to our knowledge, until now, few studies have proposed simple and cost-saving methods to effectively reduce the percentage of PUFAs in microalgae lipid.

⁎ Corresponding author at: State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center of Nanjing University, Nanjing 210061, China. E-mail address: [email protected] (Q. Wang).

https://doi.org/10.1016/j.biortech.2018.12.036 Received 9 November 2018; Received in revised form 10 December 2018; Accepted 11 December 2018 Available online 12 December 2018 0960-8524/ © 2018 Elsevier Ltd. All rights reserved.

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measured. Fourthly, performance of two strategies, fungal pellets-assisted harvesting and co-cultivation assisted harvesting, in the wastewater remediation was assessed by measuring the nutrients removal and biomass yield. Fifthly, fatty acids profiles of microalgal and fungal biomass were measured and compared to identify the feasibility to use microbial fuel cells for biodiesel production. All the experiments and tests in this work were performed in triplicate. The results were expressed in average value ± standard deviation.

To overcome aforementioned problems, a novel concept of introducing Aspergillus sp., a strain of filamentous fungi, into the wastewater remediation process was proposed by our work. Theoretically, fungi could convert solid organics in wastewater to soluble nutrients, which may be easily assimilated by microalgae cells, by secreting extracellular enzymes (Li et al., 2015). As a result, in the wastewater, microalgae could uptake more nutrients, particularly organic carbon, for lipid synthesis. Also, Gultom and Hu (2013) reported that the construction of microalgal-fungal pellets could simplify the harvesting procedure of microalgae biomass and further reduce the cost of biodiesel production. The formation of fungal pellets begins with the aggregation of spores and then microalgal cells adhere to the fungal cell wall gradually (Chen et al., 2018). Zhang and Hu (2012) reported that the adherence can be of electronic nature due to the van Der Waals interactions. In our preliminary experiment, we observed that co-cultivated microalgae and fungi could form microalgal-fungal pellets and settle down under gravity. Therefore, it was expected that with the assistance of filamentous fungi, the whole process of wastewater remediation and biodiesel production will be upgraded (Gultom and Hu, 2013). The innovative points of this study include: (1) Actual wastewater, which could be obtained at no cost in a real-world application, was used for the co-cultivation of microalgae and filamentous fungi while most of previous studies used artificial culture medium. To our knowledge, it is the first time to co-cultivate microalgae with fungi for molasses wastewater treatment. (2) Based on the performances of fungal-pellets assisted harvesting and co-cultivation assisted harvesting, a novel concept of co-cultivating filamentous fungi with microalgae in actual wastewter for simultaneous production and harvesting of biomass was proposed. (3) Advantages of using microalgal-fungal lipid over conventional microalgal lipid for biodiesel production were fully studied.

2.3. Parameters measurement and analysis 2.3.1. Biomass yield and chlorophyll analysis Yield of microbial biomass (g/L) and density of suspended solids in wastewater were measured according to the methods described by Liu et al. (2017). In the co-cultivation system, to identify the ratio of microalgal biomass and fungal biomass, harvested biomass was dehydrated by vacuum dryer. Dehydrated biomass was collected for chlorophyll content (mg/g) measurement by measuring its absorbance at wavelengths of 645 nm and 663 nm (Chan et al., 2015). Ratio of microalgae in microbial biomass was calculated according to the linear relation between chlorophyll content and biomass concentration (Eq. (1) and Eq. (2)).

M = (a × C )/ Bt × 100%

(1)

F = (Bt − a × C )/ Bt × 100%

(2)

where M and F refer to the percentages of microalgal and fungal biomass in total biomass; Bt is the dry weight of total microbial biomass; C refers to the chlorophyll content in microalgae biomass; and a is the conversion factor of chlorophyll content to microalgal biomass concentration.

2. Materials and methods 2.3.2. Analysis of protein and lipid Protein content of dehydrated algal biomass was measured by using elemental analyzer to quantify the nitrogen element and the nitrogento-protein conversion factor was set as 6.25 (Lu et al., 2015). Lipids of dehydrated biomass were extracted by mixed solvent of chloroform and methanol according to the extraction method (Lee et al., 2010). Lipid content of microbial biomass was calculated according to Eq. (3).

2.1. Microorganisms and wastewater Microalgal strain, isolated from local lake (Guangzhou, China), was identified as Chlorella vulgaris by using molecular technology. In this work, microalgae were preserved at 30 °C under continuous fluorescent light (120 µmol photons m−2 s−1) in artificial culture medium, of which the compositions include KH2PO4 (0.054 g/L), NH4Cl (0.375 g/ L), (HOCH2)3CNH2 (0.50 g/L), CaCl2·2H2O (0.050 g/L), MgSO4·7H2O (0.100 g/L), CH3COOH (1.0 mL/L), K2HPO4 (0.108 g/L), and trace elements stock solution (1.0 mL/L). Aspergillus sp. was preserved on Potato Dextrose Broth (PDB) medium at 30 °C. Pretreated molasses wastewater, which was obtained from local plant (Guangzhou, China), was centrifuged at 6000 rpm to accelerate the sedimentation of suspended solids with high density and supernatant was sterilized at 121 °C for 30 min before the inoculation of microalgae or fungi. In the experiment, microorganisms were grown in 250 mL Erlenmeyer flasks, filled with 100 mL actual wastewater, shaken at 80 rpm.

Q = L/ Bt × 100%

(3)

where Q refers to the percentage of lipid in microbial biomass by dry weight; L is the dry weight of total lipid extracted by aforementioned method; and Bt is the dry weight of total microbial biomass. To analyze the fatty acids profile, dehydrated biomass was sent for fatty acid methyl ester (FAME) preparation and GC–MS analysis. The initial oven temperature of GC–MS was set at 80 °C for 4 min and the temperature increased to 320 °C at a rate of 2 °C/min. At the end of temperature increase program, oven temperature was held at 320 °C for 3 min. Injector and detector temperatures were set at 250 and 230 °C, respectively. Flow rate of carrier gas (helium) was controlled at 1.2 mL/ min. The compounds were identified by database and quantified by measuring the peak area. Percentages of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) were calculated accordingly.

2.2. Experimental design This work, aiming at producing low-cost oil for biodiesel use by constructing pellets consisted of microalgae and filamentous fungi in molasses wastewater, was carried out in five steps. Firstly, parameters critical to the construction of microalgal-fungal pellets in molasses wastewater were studied. Secondly, effects of co-cultivation on wastewater properties, including oxidation-reduction potential (ORP), pH, color intensity, and density of suspended solid, were assess to explore the mechanisms of microalgal-fungal pellets formation. Thirdly, removal of nutrients, including chemical oxygen demand (COD), ammonia nitrogen (NH3-N), total nitrogen (TN), and total phosphorus (TP), by microalgal-fungal pellets grown in molasses wastewater was

2.3.3. Nutrients removal Concentrations of COD, TN, NH3-N, and TP, expressed as mg/L, were measured daily by using spectrophotometer according to the method described by Liu et al. (2017). Removal efficiencies of these nutrients were calculated according to Eq. (4).

R = (N0 − Nt )/ N0 × 100%

(4)

where R refers to the removal efficiency of certain nutrient in wastewater; No and Nt are concentrations of nutrients on Day 0 and Day t, 36

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respectively; and t refers to the cultivation period (day).

Table 1 Nutrients profile of wastewater and artificial medium.

2.3.4. Other parameters The values of ORP (mV) and pH were measured daily by using analyzers (Liu et al., 2017). Color intensity (A475) of wastewater was determined by measuring OD of supernatant at 475 nm and decolorization efficiency was calculated according to the published method (Gengec, 2015). 2.4. Microalgal-fungal pellets for nutrients removal Biomass yield and biomass composition of microalgal-fungal system grown at four different temperatures (25 °C, 30 °C, 35 °C, and 40 °C) were measured. In addition, effects of inoculation ratio on the construction of microalgal-fungal pellets were assessed. Inoculation ratio refers to the ratio of microalgal cells to fungal spores inoculated in the wastewater (Chen et al., 2018; Zhang and Hu, 2012). In this experiment, initial density of fungal spores was 1.5 × 104/mL and inoculation ratios of microalgal cells and fungal spores were set as 20:1, 60:1, 100:1, 300:1, and 500:1. Biomass yield and biomass composition of cocultivation system with different inoculation ratios were measured. The optimum temperature and inoculation ratio were identified according to the biomass yield. As the optimum temperature and inoculation ratio were identified, nutrients removal in molasses wastewater was measured and calculated. Based on the results of biomass production and nutrients removal, metabolic interaction between microalgae and fungi was discussed.

Wastewater

Artificial medium

SS COD TN NH3-N TP ORP pH Color intensity

1.38 g/L 11230 mg/L 407.5 mg/L 170.3 mg/L 30.4 mg/L 113 mV 6.12 97.5

0 3894 mg/L 364.5 mg/L 132.0 mg/L 28.6 mg/L / 7.0 /

which may not be assimilated by microalgae cells in the treatment process, would cause serious environmental pollution. Second, as the values of ORP and pH were 113 mV and 6.12, respectively, the wastewater provided a neutral and aerobic environment which is suitable for the growth of microalgae and fungi (Liu et al., 2017). Third, high value of color intensity (97.5), which may inhibit the photosynthesis of microalgae cells, is partly attributed to the suspended solids in wastewater. Hence, in terms of color intensity, artificial culture medium created a better environment for microalgae growth. Generally, the molasses wastewater rich in various nutrients is a good medium for biomass production although it has some limiting factors. Conventional microalgae-based technology did not perform well in the remediation of molasses wastewater. Firstly, microalgae growth entered stationary stage on Day 5, yielding very low density (1.465 g/L) of biomass. Secondly, due to the low density of microalgae biomass in wastewater, harvesting by some conventional technologies will be an expensive process. For example, under the optimal conditions of centrifugation, the cost of microalgae oil could reach $0.864/L (Dassey and Theegala, 2013). Previous studies showed that the cost of harvesting even accounted for 35% of total cost of biomass production (Acién et al., 2012; Chen et al., 2018). Thus, it may not be an economically feasible way to produce biomass in molasses wastewater for biodiesel industry. Thirdly, after 5-day treatment, removal efficiencies of COD, TN, NH3-N, and TP only reached 25.50%, 45.46%, 79.64%, and 33.88%, respectively. Suspended-solid density was reduced by 13.77%, suggesting that mono-system of microalgae did not perform well in the removal of suspended solids in molasses wastewater. This result is in accordance with previous studies which used microalgae for food processing wastewater, manure, municipal effluent and other wastewater with high density of suspended solids (Hongyang et al., 2011; Lu et al., 2015; Zhou et al., 2012). Due to the high concentrations of residual nutrients, wastewater is not dischargeable after microalgae cultivation.

2.5. Comparison of two strategies 2.5.1. Fungal pellets assisted harvesting Microalgae were inoculated in molasses wastewater for 5 days to remove nutrients. At the end of microalgae cultivation, 2 g/L (dry weight basis) fungal pellets, which were obtained by growing fungi in culture medium according to the optimized conditions, were added into wastewater for microalgae harvesting. Harvesting efficiency was calculated according to Eq. (5).

H = (W − S )/ W × 100%

Parameter

(5)

where H refers to the harvesting efficiency (%); W is the total microbial biomass (g/L); and S refers to the microbial biomass density (g/L) in supernatant after harvesting. The correlation between harvesting time (h) and harvesting efficiency (%) was established to evaluate the performance of fungal pellets assisted harvesting.

3.2. Construction of microalgal-fungal pellets

2.5.2. Co-cultivation assisted harvesting Co-cultivation assisted harvesting was carried out by inoculating microalgae and fungi in wastewater under the optimized conditions. After 5-day cultivation, harvesting of microalgal-fungal biomass was performed by gravity-driven sedimentation. To evaluate the performance of co-cultivation assisted harvesting, changes of harvesting efficiency (%) with the sedimentation time (h) were recorded and analyzed.

3.2.1. Effects of temperature on biomass production As shown in Fig. 1(a), biomass yield increased from 0.996 g/L to 3.342 g/L with the increase of temperature. When the temperature reached 40 °C, yield of microbial biomass dropped to 2.756 g/L. Hence, in terms of biomass productivity, 35 °C is the optimum temperature for the microorganism growth. Fig. 1(b) showed that the percentage of microalgal biomass dropped from 90.20% to 26.30% while the percentage of fungal biomass increased to 73.70%. When the temperature increased from 35 °C to 40 °C, biomass yield of microalgae dropped from 1.641 g/L to 0.725 g/L, causing the decrease of microbial biomass yield. The main reason for this phenomenon is that high temperature (over 35 °C) was favorable to fungi while limited the growth of microalgae. This result is in accordance with previous studies which reported that the optimum temperature for Aspergillus sp. ranged between 40 °C and 60 °C, but high temperature would seriously disturb the metabolisms of Chlorella sp. and even cause the failure of microalgae cultivation (Contesini et al., 2010; Converti et al., 2009).

3. Results and discussion 3.1. Properties of wastewater Properties of pretreated molasses wastewater and a commonly used artificial mixotrophic medium are shown in Table 1. First, wastewater provided sufficient nutrients for microorganisms’ growth. Interestingly, compared with artificial mixotrophic medium, wastewater has much higher concentration of COD (11230 mg/L). Due to the high content of suspended solids (1.38 g/L), some wastewater-borne solid organics, 37

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Fig. 1. Growth of microalgae and fungi at different temperatures or inoculation ratios: (a) Effects of temperature on biomass yield; (b) Effects of temperature on biomass composition; (c) Effects of inoculation ratio on biomass yield; (d) Effects of inoculation ratio on biomass composition.

issue, effects of microorganisms on a couple of wastewater properties, including ORP, pH, color intensity, and density of suspended solids, were studied.

3.2.2. Effects of inoculation ratio on biomass production Inoculation ratio is another parameter influencing the growth of microalgae and fungi. Fig. 1(c) indicated that the highest biomass yield (4.215 g/L) was obtained when the inoculation ratio was 100. In terms of biomass yield, 100 is the optimum inoculation ratio for the co-cultivation of microalgae and fungi. According to previous studies, the inoculation ratio could partly determine the biomass yield by impacting the growth of microalgae and fungi (Chen et al., 2018; Zhang and Hu, 2012). Similar phenomenon was observed in this work. For example, the biomass yields were only 3.465 g/L and 1.851 g/L when the inoculation ratios were 20 and 500, respectively. Besides biomass yield, biomass composition was also impacted by the inoculation ratio. As shown in Fig. 1(d), with the decrease of inoculation ratio, percentage of fungal biomass dropped from 75.10% to 4.80%. In a real-world application, for the purpose of producing high percentage of microalgae or fungi in the mixed biomass, inoculation ratio could be adjusted accordingly. Based on the results above, fungi and microalgae were cocultivated at 35 °C with an inoculation ratio of 100 for the following experiment.

3.3.1. Values of OPR and pH Fig. 2(a) showed that fungi and microalgae have totally different effects on ORP value of wastewater. ORP value decreased gradually with the growth of fungi while increased slightly with the growth of microalgae. The main reason is that the growth of fungi is a heterotrophic process, which consumes O2 and produces CO2, resulting in the drop of ORP value in wastewater while the photosynthesis of microalgae increases ORP value by generating O2. In the mono-system of fungi, with the consumption of O2 and the decrease of ORP value, wastewater environment will be changed from aerobic condition to anaerobic condition, which may prohibit the growth of fungi (Muniraj et al., 2013; Sankaran et al., 2010). In a real-world application, selfinhibition caused by O2 accumulation was also observed in the monosystem of microalgae (Wang et al., 2012). As shown in Fig. 2(a), in the co-cultivation system, values of ORP ranged between 92.6 mV and 116.2 mV, creating a favorable environment for the growth of microalgae and fungi. Similar phenomenon was observed in the changes of pH value (Fig. 2(b)). In the mono-system of fungi, pH value decreased slightly while microalgae growth increased the pH value of wastewater (Li et al., 2011). Fluctuation of pH value might negatively impact the

3.3. Effects of microorganisms on wastewater properties Although the optimization of temperature and inoculation ratio improved the biomass yield, relationship between microalgae and fungi in molasses wastewater still remained uncertain. To further explain this 38

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11 10

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60 50 40 30 20 10 0 Microalgae

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(c) Fig. 2. Effects of co-cultivation on wastewater properties: (a) ORP; (b) pH value; (c) Decolorization and suspended solids removal.

filamentous fungi, which could secrete various extracellular enzymes to digest organics for heterotrophic metabolisms, might be another possible mechanism for the removal of suspended solids and decolorization in wastewater (Novotný et al., 2004; Romaní et al., 2006).

growth of fungi or microalgae. However, in the co-cultivation system, the pH value of wastewater remained stable (Fig. 2(b)), creating a better environment for the growth of fungi and microalgae. According to the results above, in the industrial application of this technology, pH value and ORP value of the wastewater should be monitored and controlled.

3.4. Nutrients removal by microalgal-fungal pellets 3.4.1. Removal of COD and TP Fig. 3(a) showed that removal efficiency of COD by microalgae, fungi, and co-cultivation system reached 25.96%, 59.00%, and 70.68%, respectively, at the end of cultivation period. Fungi, which used the organic carbon as the only carbon source and major energy source, efficiently reduced the concentration of COD. On the contrary, in the wastewater with mono-system of microalgae, residual concentration of COD was higher than 8300 mg/L. As reported by previous studies, since the diameter of transportation channel on plasma membrane is small, large-sized suspended solids could not be directly absorbed by microalgae cells (Lu et al., 2015; Zemke-White et al., 2000). As a result, removal of COD, which is partly attributed to suspended solids in molasses wastewater, by microalgae was limited. As fungi had much better performance in COD removal than microalgae, with the construction of co-cultivation system, removal efficiency of COD reached 70.68%. Not only the removal of COD, but also the removal of TP was enhanced by growing fungi, which also perform well in phosphorus

3.3.2. Color intensity and density of suspended solid As shown in Fig. 2(c), after 5-day cultivation, color intensity of wastewater with microalgae, fungi, and co-cultivation systems decreased by 66.32%, 15.14%, and 69.98%, respectively, suggesting that fungi had much better performance in decolorization than microalgae. Similar trend was observed in the removal of suspended solids (Fig. 2(c)). Therefore, in terms of decolorization and suspended solid removal, compared with mono-system of microalgae, it is a more promising way to co-cultivate microalgae with fungi for wastewater remediation. Previous study which explored the surface physical properties of filamentous fungi confirmed that driven by electrostatic force, some suspended solids in wastewater could be captured by fungi and attached on the cell surface (Michniewicz et al., 2006). Undeniably, the removal of suspended solids in molasses wastewater was partly attributed to the physical interaction between microbial cells and suspended solids. In addition to this mechanism, nutrients degradation by 39

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(e) Fig. 3. Nutrients removal by microalgae and fungi: (a) Removal of COD; (b) Removal of TP; (c) Removal of TN; (d) Removal of NH3-N; (e) Contribution of microalgalfungal system to nutrients removal.

assimilation, with microalgae in molasses wastewater. In the 5-day cultivation, co-cultivation system removed 88.39% of TP in the wastewater, reducing the concentration of TP to 3.6 mg/L (Fig. 3(b)).

3.4.2. Removal of TN and NH3-N As shown in Fig. 3(c), removal efficiency of TN by co-cultivation system reached 67.09% while mono-system of microalgae and fungi only removed 44.39% and 18.20% of TN in molasses wastewater. Interestingly, although the removal efficiencies of TN and NH3-N by fungi 40

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were not high (< 20%), removal efficiencies of TN and NH3-N were improved when fungi were introduced into the wastewater remediation for co-cultivation. Similar trend was observed in the removal of NH3-N as well. Fig. 3(d) showed that the introduction of fungi into microalgae system increased the removal efficiency of NH3-N to 94.72%, reducing the concentration of NH3-N to 8.9 mg/L. Hence, it is a promising strategy to co-cultivate microalgae with filamentous fungi in molasses wastewater for nutrients removal.

Table 2 Composition of microbial biomass.

Microalgae Fungi Co-cultivation system

Protein

Lipid

Other*

61.5% 15.4% 38.6%

22.2% 37.7% 35.2%

16.3% 46.9% 26.2%

* “Other” refers to carbohydrate, nucleic acid, and other compositions not included in the categories of protein or lipid.

3.4.3. Discussion on the mechanisms of nutrients removal According to aforementioned results and previous publications, three mechanisms contributed to the distinguished performance of microalgal-fungal system in the molasses wastewater remediation (Fig. 3(e)). First, microalgae and fungi, which grew well in the cocultivation system, removed more nutrients in wastewater. Second, in the co-cultivation system, carbon dioxide released by fungal cells through heterotrophic metabolisms could be assimilated by microalgal cells through photosynthesis (Abinandan et al., 2018). Thus, the utilization efficiency of carbon in wastewater would be improved. Third, as some nutrients, particularly nitrogen and carbon, were embedded into suspended solids, with the conversion, driven by fungal enzymes, of solids organics to soluble low-molecular-weight nutrients, microalgae could uptake more nutrients (Zhao et al., 2016). Because of this mechanism, in the co-cultivation system, microalgae were prone to assimilate more nitrogen elements with the assistance of fungi. Thus, nutrients removal was enhanced by the co-cultivation system even if mono-system of fungi did not perform well in the removal of TN and NH3-N. Similar results were reported by previous studies which explored the interaction between microalgae and bacteria in wastewater treatment.

previous studies (Prajapati et al., 2014; Wrede et al., 2014), but its application in microalgae industry is rare due to some technical barriers. First, the cost of producing fungal pellets is high since artificial medium is needed to cultivate fungi. For example, in the study of Chen et al. (2018), about 10 g/L glucose, of which the cost is very high, was used for the production of fungal pellets. Second, special equipment and intensive labor might be necessary for the collection of fungal pellets in medium. According to the results and discussion above, advantages of cocultivation are listed as follows: First, since fungi and microalgae are co-cultivated in wastewater, no culture medium is needed to produce fungal pellets. Thus, the cost of co-cultivation assisted harvesting should be lower than that of fungi-pellets assisted harvesting. Second, as fungi contribute to the degradation of solid organics in wastewater, concentrations of residual nutrients were reduced to a low level. Third, co-cultivation assisted harvesting does not have much requirement on the equipment and labor work. Due to the low capital cost and operation cost, this technology is more likely to be applied in the industry. Considering these advantages, in a real-world application, co-cultivation of microalgae with fungi might be a promising way for wastewater remediation and microbial biomass production and harvesting.

3.5. Comparison of two strategies 3.6. Composition and fatty acids profile of harvested biomass As shown in Fig. 4(a), harvesting efficiencies of two strategies were higher than 97% in 4 h, suggesting that both of them could be used for efficient biomass harvesting. However, in terms of nutrients removal, co-cultivation assisted harvesting is much better than fungi-pellets assisted harvesting. For example, Fig. 4(b) showed that at the end of cocultivation, concentrations of COD, TN, TP, and NH3-N in wastewater were reduced to 3328 mg/L, 130.5 mg/L, 3.1 mg/L, and 9.2 mg/L, much lower than the concentrations of residual nutrients in wastewater after fungi- pellets assisted harvesting. Fungi-pellets assisted harvesting has been widely reported by

As shown in Table 2, lipid content of microbial biomass reached 35.2% while protein content was about 38.6% by co-cultivating fungi with microalgae in molasses wastewater. Compared with mono-system of microalgae, co-cultivation system contained more lipids, making the biomass more valuable for biodiesel industry. Fig. 5(a) showed that Aspergillus sp. contained higher percentages of SFA (33.04%) and MUFA (26.79%) but lower percentage of PUFA (40.17%) than microalgae. According to previous study, at the molecular level, deletion of odeA (ΔodeA), encoding a Δ-12 desaturase 8000

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Fig. 4. Comparison of two strategies: (a) Harvesting efficiency; (b) Nutrients removal. 41

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100 Other C20:4 C20:3 C18:3 C18:2 C18:1 C18:0 C16:3 C16:2 C16:1 C16:0

90

Percentages of fatty acids (%)

80 70 60 50 40 30 20 10 0 Microalgae

Fungi

Co-cultivation

Biomass category Fig. 5. Fatty acids profiles of microalgae and fungi.

this work mainly confirmed that it is a feasible way to obtain the most appropriate fatty acids profile of biomass for biodiesel production by co-cultivating microalgae with fungi. In a real-world application, to use fungal-microalgal lipid for biodiesel production, more parameters should be measured. For example, effects of external conditions, such as temperature and light intensity, on lipid yield and fatty acid profile of fungal-microalgal biomass should be assessed. With further improvement of cultivation technologies, in the coming future, co-cultivation of fungi and microalgae may become a promising way to produce highquality lipid for biodiesel industry.

which could convert oleic acid to linoleic acid, is one of the main reasons for the low percentages of PUFA (C18:2 and C18:3) and high percentages of MUFA and SFA in Aspergillus sp. (Calvo et al., 2001). As shown in Fig. 5(b), percentages of SFA, MUFA, and PUFA in pellets reached 31.97%, 23.23%, and 44.80%, respectively, suggesting that it is an effective way to reduce the unsaturation degree of microalgal cell lipid by co-cultivating microalgae with Aspergillus sp. In a real-world application, to commercialize the microbial biodiesel, not only the production cost, but also the biodiesel quality should be comprehensively considered. Previous studies which used microalgae biomass for biodiesel production discovered that high percentages of PUFA in lipid may reduce the cetane number and oxidative stability, limiting the commercial use of biodiesel (Knothe, 2005; Kumar, 2017; Ramírez-Verduzco et al., 2012). For example, as cetane number is an inverse function of fuel’s ignition delay, biodiesel with low cetane number will have longer ignition delay periods (Canakci and Sanli, 2008). In addition, since PUFA with much lower oxidative stability than SFA and MUFA when exposed to air conditions, high levels of unsaturation in lipid and presence of double bonds in fatty acid chain make the biodiesel more prone to be oxidized (Fattah et al., 2014). In traditional biodiesel industry, peanut, corn, and sunflower with low contents of PUFA are preferred for biodiesel production. To survive in some harsh conditions, most microalgal strains developed the synthesis pathway of PUFA which could reduce the freezing point of plasma membrane and increase the survival efficiency of cells (Roleda et al., 2013). However, as mentioned above, biodiesel made from microalgae lipid with very high contents of PUFA may not be qualified for industrial or commercial use (Kumar, 2017; RamírezVerduzco et al., 2012). In this work, by the co-cultivation strategy, percentage of PUFA in biomass was reduced to 44.80%, alleviating potential problems caused by low cetane number or low oxidative stability. Since the criteria to assess biodiesel quality are complex, it is not reasonable to claim that fungal-microalgal lipid with lower percentage of PUFA is better than microalgal lipid for biodiesel production. If the percentages of SFA and MUFA are too high, quality of biodiesel might be negatively impacted as well. For example, high percentages of SFA and MUFA may reduce the low temperature operability of biodiesel, limiting its use in cold environment (Smith et al., 2010). The results of

4. Conclusions It is concluded that (1) Organics-rich molasses wastewater with high content of suspended solids could not be efficiently treated by monosystem of microalgae; (2) Co-cultivation of microalgae with fungi not only increased the biomass yield, but also promoted the docolorization and the nutrients removal; (3) Compared with conventional fungi-pellets assisted harvesting, co-cultivation assisted harvesting had similar harvesting efficiency but removed more nutrients from wastewater; and (4) Microalgal-fungal biomass with low percentages of PUFA might be a good feedstock for biodiesel production. Acknowledgement This study was funded by the Guangzhou Science and Technology Program of China (Project No. 201704030084), and the Guangdong Science and Technology Program of China (Project No. 2016A040402045). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2018.12.036. References Abinandan, S., Subashchandrabose, S.R., Venkateswarlu, K., Megharaj, M., 2018. Microalgae–bacteria biofilms: a sustainable synergistic approach in remediation of acid mine drainage. Appl. Microbiol. Biotechnol. 102 (3), 1131–1144.

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