Recycling of food waste as nutrients in Chlorella vulgaris cultivation Kin Yan Lau 1, Daniel Pleissner 1, Carol Sze Ki Lin ⇑ School of Energy and Environment, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong
h i g h l i g h t s Food waste hydrolysate used as medium in cultures of Chlorella vulgaris. Hydrolysate concentration affects microalgal growth. Better growth in hydrolysate than in deﬁned medium. Algal biomass rich in carbohydrates, proteins, lipids and fatty acids.
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Article history: Received 11 June 2014 Received in revised form 22 July 2014 Accepted 23 July 2014 Available online 1 August 2014 Keywords: Lipids Fatty acids Waste utilization Nutrient recovery Food waste hydrolysate
a b s t r a c t Heterotrophic cultivation of Chlorella vulgaris was investigated in food waste hydrolysate. The highest exponential growth rate in terms of biomass of 0.8 day1 was obtained in a hydrolysate consisting of 17.9 g L1 glucose, 0.1 g L1 free amino nitrogen, 0.3 g L1 phosphate and 4.8 mg L1 nitrate, while the growth rate was reduced in higher concentrated hydrolysates. C. vulgaris utilized the nutrients recovered from food waste for the formation of biomass and 0.9 g biomass was produced per gram glucose consumed. The microalgal biomass produced in nutrient sufﬁcient batch cultures consisted of around 400 mg g1 carbohydrates, 200 mg g1 proteins and 200 mg g1 lipids. The conversion of nutrients derived from food waste and the balanced biomass composition make C. vulgaris a promising strain for the recycling of food waste in food, feed and fuel productions. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The production of food waste is one of the most severe environmental problems in many countries. Every year, over one billion tonnes of food waste is produced, which needs to be treated or disposed in landﬁll sites (Gustavsson et al., 2011). Among developed countries in East Asia, Hong Kong has the highest food waste generation rate of 0.45 kg day1 per capita, which results in the daily production of around 3600 tonnes of food waste. This accounts for 40% of the total municipal solid waste that needs to be treated (Hong Kong SAR Environmental Protection Department, 2011; Hong Kong SAR Environment Bureau, 2014). At the moment, 52% of the municipal solid waste in Hong Kong is disposed in landﬁll sites, but limited capacity and possible water and soil contaminations by carbon and nitrogen-rich leachates create concern about future municipal solid waste treatment strategies (Cossua et al., 2003; Hong Kong SAR Environment Bureau, 2013).
http://dx.doi.org/10.1016/j.biortech.2014.07.096 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Food waste is deﬁned as any food that is wasted, degraded or lost during production or at consumer level (Gustavsson et al., 2011). However, food waste consists of around 300–600 mg g1 starch, 60–100 mg g1 proteins and 70–300 mg g1 lipids (Leung et al., 2012; Pleissner et al., 2014), which make it a promising source of nutrients in fermentation processes after hydrolysis. Recent studies have shown that hydrolysis of carbohydrates, proteins and polyphosphates using fungal enzymes can help to reduce the overall amount of food waste that needs to be disposed in landﬁll sites or incinerated. Furthermore, by hydrolysis of carbohydrates, proteins and polyphosphates a nutrient-rich hydrolysate containing glucose, free amino nitrogen (FAN), phosphate and nitrate can be prepared to be used for the fermentative production of high-value added products such as polyunsaturated fatty acids and succinic acid (Leung et al., 2012; Pleissner et al., 2013, 2014). Therefore, utilization of food waste as nutrient source in fermentative processes not only contributes to new waste treatment strategies, but also offers the opportunity to recycle waste organic matters by production of microbial biomass and subsequent formation of metabolites as value-added products. Due to diminishing fossil fuel reserves, production of fuel using sustainable sources has been studied intensively in the past
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decades. The US Department of Energy regarded microalgal lipids as a potential source of biofuel and funded a program named ‘Aquatic Species Program’ to investigate the feasibility to use microalgal lipids as a feedstock in biodiesel production. In this program Chlorella was considered, among others, as a promising genre for algal lipid production. Chlorella is further one of the genera extensively studied under heterotrophic, mixotrophic and photoautotrophic conditions in order to develop feasible algal biomass production processes, but also biomass treatment and lipid extraction methods (Sheehan et al., 1988; Miao and Wu, 2006; Liang et al., 2009; Šoštaricˇ et al., 2012). Although, microalgae can grow photoautotrophically in presence of carbon dioxide and sun-light, heterotrophic cultivation on organic carbon sources gives higher biomass productivities and higher speciﬁc lipid contents (Miao and Wu, 2006). However, the price of organic carbon sources can contribute by 80% to the production costs and utilization of nutrients recovered from food waste could beneﬁt to the economic feasibility (Li et al., 2007). Diluted food waste hydrolysate was used as nutrient source in heterotrophic microalgae cultivations for the production of lipidrich biomass and high exponential growth rates were obtained (Pleissner et al., 2013). However, in recently performed cultivations it was noted that with increasing concentration of food waste hydrolysate the growth of Chlorella spp. was inhibited (unpublished results). Additionally, in order to obtain high growth rates the presence of an appropriate nitrogen source is essential. Nitrate has been found to be more favorable to the growth of Chlorella spp. than organic nitrogen sources such as yeast extract and FAN (Li et al., 2013; Pleissner et al., 2013). In order to strengthen the knowledge of ‘food waste-based’ Chlorella spp. cultivation processes and to obtain high exponential growth rates, the investigation of appropriate food waste hydrolysate concentrations and the ability of utilization of the recovered carbon, nitrogen and also phosphorous compounds for growth and formation of biomass constituents is essential. Therefore, the aim of this study was the investigation of heterotrophic cultivation of Chlorella vulgaris in different concentrated food waste hydrolysates and compared to the growth in deﬁned media in the presence of different nitrogen sources such as in different waste streams derived media. Furthermore, the biomass of C. vulgaris grown in food waste hydrolysate was investigated for its carbohydrate, protein, lipid and speciﬁc fatty acid contents with the goal to evaluate its suitability as a feedstock in food, feed and fuel productions.
2. Methods 2.1. Handling of microorganism 2.1.1. Aspergillus awamori ATCC 14331 was purchased from the American Type Culture Collection (Rockville, MD, USA). Aspergillus oryzae was isolated from a soy sauce starter provided by the Amoy Food Ltd., Hong Kong. Spore solutions of A. awamori and A. oryzae were prepared as described in Lam et al. (2013) and stored at 80 °C.
2.1.2. C. vulgaris UTEX 259 was purchased from the University of Texas Culture Collection and grown in darkness in 250 mL conical ﬂasks containing 100 mL of food waste hydrolysate consisting of 4.8 g L1 glucose, 0.08 g L1 FAN, 0.04 g L1 phosphate and 1.6 mg L1 nitrate at 22 °C and an initial pH value of 6.8. Cultures were stirred using a magnetic stirrer bar and sub-cultivated every 6 days.
2.2. Bakery and food wastes Bakery (cake and pastry) and food (noodles, rice, meat and vegetables) wastes were collected from Starbucks and a local fast food restaurant, respectively. After collection, bakery and food wastes were separately blended and stored at 80 °C until use. 2.3. Solid-state fermentation Solid-state fermentation for the production of fungal solid mashes to be used as enzyme sources in food waste hydrolysis was carried out as described in Pleissner et al. (2013). To 10 g (8.5 g dry weight) of bakery waste, 1 mL of A. awamori (4.6 105 spores mL1) or 1 mL of A. oryzae (6.3 105 spores mL1) spore solution was added, mixed and the inoculated bakery waste was incubated without any stirring for 7 days at 30 °C. 2.4. Nitrate quantiﬁcation Nitrate was quantiﬁed using a commercially available nitrate kit (Aquarium Pharmaceuticals, www.apiﬁshcare.com, USA). Firstly, 40 lL of Reagent 1 (contains hydrochloric acid) was added to 400 lL of sample, then 40 lL of Reagent 2 (mixture of 85% (w/w) polyethylene glycol and 0.99, Fig. 1B). However, a 25% lower sensitivity was found in 50% (v/v)
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Fig. 1. (A) Absorption spectra of reaction mixtures of different sodium nitrate concentrations. (B) Calibration curve of nitrate measured at 544 nm in water (closed circle and solid line, y = 0.0141x) and 50% (v/v) food waste hydrolysate (open circle and dashed line, y = 0.0105x).
food waste hydrolysate compared to water. Reagent 2 contains polyethylene glycol and sulfanilamide. Sulfanilamide is known to react with thiol groups and thus, thiol group containing compounds like cysteine, most likely to be present in food waste hydrolysate, may interfere with the formation of diazo-compounds and lower the sensitivity (Nims et al., 1995). Nevertheless, the method showed an acceptable performance and therefore, it was used to quantify nitrate in food waste hydrolysate and supernatant samples of C. vulgaris cultures. Furthermore, the method allows a prompt quantiﬁcation of nitrate, which is particularly for interesting for the control of bioprocesses.
3.2. C. vulgaris 3.2.1. Cultivation in deﬁned and waste streams derived media Growth experiments in different concentrated food waste hydrolysates, but also in modiﬁed basal medium with addition of either yeast extract or nitrate as nitrogen source were performed. Table 1 shows the average initial glucose, FAN, phosphate and nitrate concentrations. Fig. 2 shows the growth and nutrient consumption in C. vulgaris growth experiments. C. vulgaris grew well in all tested food waste hydrolysate concentrations ranged
Fig. 2. Growth and nutrient consumption of C. vulgaris in 2.5% (A), 5% (B), 10% (C), 20% (D), 30% (E), 40% (F) and 50% (v/v) (G) food waste hydrolysates, in 20% (v/v) food waste hydrolysate supplemented with 1 g L1 nitrate (H) and in modiﬁed basal medium supplemented with 2.5 g L1 yeast extract (I) or 1 g L1 of nitrate (J).
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Fig. 3. Exponential growth rate of C. vulgaris as a function of food waste hydrolysate concentration.
from 2.5% to 50% (v/v) and utilized the nutrients supplied (Fig. 2A–G). The exponential growth rate, however, was dependent on the food waste hydrolysate concentration (Fig. 3). From 2.5% to 20% (v/v), the average exponential growth rate of C. vulgaris increased with increasing food waste hydrolysate concentration and the highest rate of around 0.8 day1 was found in 20% (v/v). This is comparable to the resultant growth rates (0.7–1.1 day1, Table 2) in mixo- and heterotrophic cultures of C. vulgaris in deﬁned medium and wastewater (Orús et al., 1991; Liang et al., 2009; Ji et al., 2014). The resultant exponential growth rate (0.8 day1) is also twice as high as the growth rates obtained using dairy waste hydrolysate by Abreu et al. (2012) and using deﬁned medium in this study (Table 2). In more concentrated food waste hydrolysates (30%, 40% and 50%, v/v), the growth rate declined and leveled off at 0.4 day1. Previous studies carried out in deﬁned medium revealed that a glucose concentration beyond 20 g L1 exhibited a negative impact on the exponential growth rate of Chlorella vulgaris, Chlorella protothecoides and Chlorella zoﬁngiensis (Shi et al., 1999; Ip and Chen, 2005; Liang et al., 2009). Furthermore, exponential growth rate of Chlorella pyrenoidosa was reduced in presence of 30 g L1 glucose and beyond (unpublished results). In this study, the 20% and 30% (v/v) food waste hydrolysates contained 17.9 and 27.2 g L1 glucose, respectively, and thus it is concluded that a glucose concentration beyond 20 g L1 also inhibits the growth of C. vulgaris in food waste hydrolysate. This fact is of importance for the development of a ‘food waste-based’ Chlorella spp. cultivation process.
In general, the ﬁnal optical density increased with increasing food waste hydrolysate concentration (Fig. 2). The pH value decreased during cultivation only slightly from 6.8 to 6.2–6.6 (not shown). However, the growth of C. vulgaris reached a plateau even though the carbon, nitrogen and phosphorous sources were still present in excess. This effect has not been observed in cultures of C. pyrenoidosa where all food waste obtained nutrients were consumed and around 20 g L1 biomass was formed (Pleissner et al., 2013). In order to utilize all nutrients, the investigation of the possibility to reuse the remaining nutrients in cultures of C. vulgaris is considered. Food waste hydrolysate is an abundant source of amino acids and the FAN concentration in the hydrolysates used for the growth studies ranged from 0.03 to 0.74 g L1 (Fig. 2A–G). Even when C. vulgaris grew well in food waste hydrolysate, only a small fraction of FAN was consumed and used for the production of biomass and intracellular proteins. Since nitrate is commonly used as nitrate source in Chlorella spp. cultivations (Li et al., 2013), it was hypothesized that FAN may not be an appropriate nitrogen source. Therefore, 20% (v/v) food waste hydrolysate supplemented with 1 g L1 nitrate was tested as culture medium. However, results from this study revealed that the addition of nitrate neither promotes the growth nor the consumption of other nutrients, and the growth stagnated before carbon, nitrogen and phosphorous sources were depleted (Fig. 2H). A stagnation of growth before nutrient depletion was also observed in the modiﬁed basal medium supplemented with yeast extract or nitrate (Fig. 2I and J). This could be due to the depletion of certain essential amino acids, which are needed in order to maintain the growth of C. vulgaris. Therefore, further experiments are being planned to demonstrate the validity of this assumption. Interestingly, C. vulgaris did grow twice as fast (0.8 day1) in 20% (v/v) food waste hydrolysate compared to modiﬁed basal medium supplemented with yeast extract or nitrate (0.5 day1, Fig. 2I and J). Food waste has been found to be rich in trace elements (Zhang et al., 2011). Thus, the complexity of food waste and consequently its hydrolysate seemingly beneﬁts to the growth of C. vulgaris and makes an additional supply of trace elements such as Co2+, Fe2+ and Mn2+ unnecessary. 3.2.2. Bench-top scale fermentation in food waste hydrolysate In order to produce sufﬁcient C. vulgaris biomass for the investigation of speciﬁc contents of biomass constituents, two bench-top scale fermentations were conducted at near identical conditions (Fig. 4). Both fermentations were carried out in 20% (v/v) food waste hydrolysate with an average initial concentration of 18.6 g L1 glucose, 0.23 g L1 FAN, 0.16 g L1 phosphate and around 5 mg L1 nitrate. The exponential growth rate of cells was between 0.7 and 0.8 day1, which is comparable to the growth rate
Table 2 Exponential growth rates (l) and lipid contents of C. vulgaris obtained under mixo- and heterotrophic conditions in different cultivation media using different carbon (CS) and nitrogen (NS) sources.
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Fig. 4. Two bench-top scale fermentations of C. vulgaris in 20% (v/v) food waste hydrolysate (Batch 1: A–D and Batch 2: E–H). Increase in biomass concentration and consumption of glucose (A and D), phosphate and FAN (B and F), changes in weight speciﬁc carbohydrate, protein, lipid (C and G) and weight speciﬁc fatty acid contents (D and H).
obtained in ﬂask cultures. After 7 days of fermentation, 3.8 g L1 of biomass was formed after the consumption of 4.3 g L1 glucose and negligible amounts of phosphate and FAN (Fig. 4A, B, E and F). C. vulgaris efﬁciently converted glucose into biomass at a yield of 0.9 g g1 and used the food waste derived nutrients for the formation of biomass constituents. At Day 4, during exponential growth phase, the biomass consisted of around 200 mg g1 carbohydrates, 300 mg g1 lipids and 300 mg g1 proteins (Fig. 4C and G). Between Days 4 and 6, the carbohydrate content increased, probably due to a stagnation of growth, to about 400 mg g1, while the lipid and protein contents decreased to about 200 mg g1. At Day 7 the lipid content increased to 300 mg g1. Changes in carbohydrate, protein and lipid contents are mainly correlated to depletion of carbon,
nitrogen and phosphorous sources (Pleissner and Eriksen, 2012). However, in this study nutrients were still present in excess. An inhibition of the cell cycle caused by depletion in essential growth factors may additionally affect the biomass composition (Kwok and Wong, 2005). In this case, cells would still consume nutrients, and synthesize and accumulate carbohydrates and lipids initially designated for the formation of daughter cells. The average lipid content was comparable to the contents in C. vulgaris biomass produced under mixotrophic conditions in dairy waste hydrolysate, while a lower content of 10–20% (w/w) was found in cells grown in deﬁned media (Table 2, Orús et al., 1991; Liang et al., 2009; Abreu et al., 2012; Ji et al., 2014). The lipids of C. vulgaris biomass contained considerable amounts of alpha-linolenic, linoleic, oleic, stearic and palmitic acids (Fig. 4D and H). Oleic acid was the most
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dominant fatty acid with a speciﬁc content of 90–100 mg g1, which is two to ten times higher than the contents of the other detected fatty acids.
crude algal lipids into biodiesel and makes CN and CV values less an issue when it comes to the evaluation of potential biodiesel sources.
3.3. Evaluation of C. vulgaris as feedstock in food, feed and fuel productions
Milk and soybean contain 260 and 370 mg g1 proteins, 380 and 300 mg g1 carbohydrates and 280 and 370 mg g1 lipids, respectively (Becker, 2007). The composition of both foods and feeds is similar to the biomass composition of C. vulgaris (Fig. 4C and G) and this illustrates the feasible applicability of C. vulgaris biomass as a source of food and feed. Furthermore, it has been that its amino acid content is comparable to the amino acids found in eggs and to the amino acid pattern for human diet suggested by the World Health Organization (WHO), and the Food and Agriculture Organization of the United Nations (Becker, 2007). C. vulgaris is not only a valuable source of amino acids, but also a source of essential and healthy fatty acids. In this study, oleic acid was found to be the most abundant fatty acid. This fatty acid could slow down the progression of atherosclerosis and lower low density lipoprotein cholesterol concentration in blood (Parthasarathy et al., 1990). Furthermore, the two essential fatty acids linoleic and alpha-linolenic acids make up of around 30% (w/w) of the total fatty acids found in C. vulgaris biomass (Fig. 4D and H). Both polyunsaturated fatty acids could beneﬁt to the development of infants if supplied in the right ratio by diet (Jensen et al., 1997). The biomass constituents with their beneﬁts to human health make C. vulgaris to a promising source of food, but also animals could beneﬁt from this nutritional diet. Halle et al. (2009) found that hens laid a higher number of eggs with a better quality when their diet was supplemented with C. vulgaris compared to a control group. Therefore, the cultivation of C. vulgaris in this study opens an innovative opportunity to recycle nutrients from food waste for the sustainable production of novel food and feed. Microalgal lipids are not only a source of nutritional fatty acids, but also a source of fatty acids convertible into biodiesel. The requirements for biodiesel from microalgal lipids are regulated by International Standards such as European standard EN 14213 and EN 14214. Both standards deﬁne twenty biodiesel properties such as cetane number (CN), iodine value (IV) and saponiﬁcation value (SV) (Knothe, 2006). Rather than testing a biodiesel feedstock for its properties, Krisnangkura (1986) and Kalayasiri et al. (1996) proposed an empirical formula for the estimation of CN, IV and SV based on the fatty acids proﬁle. CN, IV and SV are calculated by the following three equations:
CN ¼ 46:3 þ ð5458=SVÞ ð0:225 IVÞ; SV ¼ IV ¼
ð560 NÞ=M; and
X ð254 D NÞ=M:
ð1Þ ð2Þ ð3Þ
Where D is the number of double bonds, N is the percentage of each fatty acid in the feedstock and M its molecular mass. According to the equations, the CN and IV of the fatty acids from C. vulgaris biomass are 52 and 102, respectively. The European standard EN 14214 allows a minimum CN of 51 and maximum IV of 120. Therefore, the obtained lipid extract from this study would fall within the requirements for biodiesel (Knothe, 2006). However, the presence of 17% (mol mol1) alpha-linolenic acid exceeds the European limit of 12% (mol mol1) and a partial catalytic hydrogenation of double bonds is required. Alternatively, microalgal lipids containing unsaturated fatty acids can be converted by hydrodeoxygenation into diesel-range alkanes (Kandel et al., 2014). This technology supports the conversion of
In this study, the recycling of nutrients from food waste was investigated by cultivation of C. vulgaris in different composed food waste hydrolysates and nutrient media. The highest exponential growth rate in terms of biomass of 0.8 day1 and a balanced biomass composition were obtained in a hydrolysate consisting of 17.9 g L1 glucose, 0.1 g L1 FAN, 0.3 g L1 phosphate and 4.8 mg L1 nitrate. This study demonstrated the feasibility of C. vulgaris cultivation in food waste hydrolysate for the sustainable production of microalgal biomass. This exciting opportunity to produce food, feed and fuel from C. vulgaris biomass contributes to the development of ‘food waste-based’ bioreﬁnery concept, which enables the successful transition to the biobased economy. Acknowledgements The authors acknowledge the Innovation and Technology Funding (ITS/353/12) from the Innovation and Technology Commission in Hong Kong. References Abreu, A.P., Fernandes, B., Vicente, A.A., Teixeira, J., Dragone, G., 2012. Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source. Bioresour. Technol. 118, 61–66. Becker, W., 2007. Microalgae in human and animal nutrition. In: Richmond, A. (Ed.), Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Publishing Ltd., Oxford, pp. 312–351. Cossua, R., Ragaa, R., Rossettib, D., 2003. The PAF model: an integrated approach for landﬁll sustainability. Waste Manag. 23, 37–44. Gustavsson, J., Cederberg, C., Sonesson, U., van Otterdijk, R., Meybeck, A., 2011. Global food losses and food waste – extent, causes and prevention. Food and Agriculture Organization of the United Nations, Italy, Rome. (17.04.14). Halle, I., Janczyk, P., Freyer, G., Souffrant, W.B., 2009. Effect of microalgae Chlorella vulgaris on laying hen performance. Arch. Zootec. 12, 5–13. Hong Kong SAR Environment Bureau, 2013. Hong Kong blueprint for sustainable use of resources 2013–2022. (17.04.14). Hong Kong SAR Environment Bureau, 2014. A food waste and yard waste plan for Hong Kong 2014–2022. (17.04.14). Hong Kong SAR Environmental Protection Department, 2011. Monitoring of solid waste in Hong Kong – waste statistics for 2011. (17.04.14). Ip, P.F., Chen, F., 2005. Production of astaxanthin by the green microalga Chlorella zoﬁngiensis in the dark. Process Biochem. 40, 733–738. Jensen, C.L., Prager, T.C., Fraley, J.K., Chen, H., Anderson, R.E., Heird, W.C., 1997. Effect of dietary linoleic/alpha-linolenic acid ratio on growth and visual function of terms infants. J. Pediatr. 131, 200–209. Ji, Y., Hu, W., Li, X., Ma, G., Song, M., Pei, H., 2014. Mixotrophic growth and biochemical analysis of Chlorella vulgaris cultivated with diluted monosodium glutamate wastewater. Bioresour. Technol. 152, 471–476. Kalayasiri, P., Jeyashoke, N., Krisnangkura, K., 1996. Survey of seed oils for use as diesel fuels. J. Am. Oil Chem. Soc. 73, 471–474. Kandel, K., Anderegg, J.W., Nelson, N.C., Chaudhary, U., Slowing, I.I., 2014. Supported iron nanoparticles for the hydrodeoxygenation of microalgal oil to green diesel. J. Catal. 314, 142–148. Knothe, G., 2006. Analyzing biodiesel: standards and other methods. J. Am. Oil Chem. Soc. 83, 823–833. Krisnangkura, K., 1986. A simple method for estimation of cetane index of vegetable oil methyl esters. J. Am. Oil Chem. Soc. 63, 552–553. Kwok, A.C.M., Wong, J.T.Y., 2005. Lipid biosynthesis and its coordination with cell cycle progression. Plant Cell Physiol. 46, 1973–1986. Lam, W.C., Pleissner, D., Lin, C.S.K., 2013. Production of fungal glucoamylase for glucose production from food waste. Biomolecules 3, 651–661. Leung, C.C.J., Cheung, A.S.Y., Zhang, A.Y.Z., Lam, K.F., Lin, C.S.K., 2012. Utilisation of waste bread for fermentative succinic acid production. Biochem. Eng. J. 65, 10– 15. Li, T., Zheng, Y., Yu, L., Chen, Z., 2013. High productivity cultivation of a heatresistant microalga Chlorella sorokiniana for biofuel production. Bioresour. Technol. 131, 60–67.
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Recycling of food waste as nutrients in Chlorella vulgaris cultivation.
Heterotrophic cultivation of Chlorella vulgaris was investigated in food waste hydrolysate. The highest exponential growth rate in terms of biomass of...