Fruit and Vegetable Waste Hydrolysates as Growth Medium for Higher Biomass and Lipid Production in Chlorella vulgaris

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1 Journal of Environment and Waste Management Vol. 4(2), pp , July, ISSN: XXXX-XXXX JEWM Research Article Fruit and Vegetable Waste Hydrolysates as Growth Medium for Higher Biomass and Lipid Production in Chlorella vulgaris Abhimanyu Pratap 1, Mritunjay Kumar 2 and Sibi G 3* 1,2 Department of Microbiology, Bangalore City College, Bengaluru, India 3* Department of Biotechnology, Indian Academy Degree College-Autonomous, Bengaluru, India Fruit and vegetable wastes include peels, pulp and seeds that constitute about 40% of the total mass and constitute huge environmental problems. Cultivation of microalgae that utilizes fruit and vegetable wastes as feedstock to produce value added products such as biomass and lipids is a unique approach. Different concentrations of fruit waste hydrolysate (FWH) and vegetable waste hydrolysate (VWH) were used for heterotropic cultivation of Chlorella vulgaris thereby optimizing the suitable hydrolysate concentration for higher biomass and lipid production. FWH in the ratio of 8:2 has produced maximum specific growth rate of 1.92 µ d -1. Higher biomass was recorded in growth medium supplemented with FWH (0.16 mg L -1 ) than VWH medium. Highest chlorophyll content of 7.2 mg L -1 was observed in 8:2 ratio of FWH whereas it was 4.3 mg L -1 in VWH at the same concentration. Carotenoid content was highest in VWH than FWH media with a maximum content of 0.52 and 0.42 mg L -1 respectively. Fruit waste hydrolysates significantly increased the total lipid content than the vegetable waste hydrolysate medium. Highest lipid content of 6.63 mg L -1 was recorded in 8:2 ratio of FWH. This work demonstrates the feasibility of fruit waste hydrolysate as a nutrient source for algal cultivation and a cost reduction of growth medium in algal biomass and lipid production. Keywords: Microalgae, fruit wastes, vegetable wastes, hydrolysates, biomass, lipid INTRODUCTION Today s world is highly dependent on the energy derived from fossil fuels (Doll and Pachauri, 2010) and development of alternative energy sources from plants and microbial origin is necessary (Berg and Boland, 2014; Gaurav et al., 2017). Oleaginous microorganisms accumulate lipid in their cell more than 20% of a dry biomass and the lipid can be used as a potential feedstock for biodiesel production due to their chemical composition. Oil accumulated in most microalgae is mainly triglyceride that can be applied to form biodiesel and glycerol through transesterification. Microalgae are well-adapted to survive under a large spectrum of environmental stresses (Tandeau-de-Marsac et al., 1993). Algal biomass can be fractionated into both bio-energy and food products (Wijffels et al., 2010). The price of algal biofuel ultimately depends on the substrate cost, lipid yield, and the quality of the products formed by the downstream process (Yang et al., 2006). Commercialization of biofuels using microalgae is hindered by the fact that it is more expensive due to the higher costs of heterotrophic growth nutrients (Hong et al., 2012). In algal cultivation, the cost of carbon source represents 50% of the cost of growth medium (Cheng et al., 2009; Li et al., 2007). *Corresponding author: Sibi G, Department of Biotechnology, Indian Academy Degree College- Autonomous, Bengaluru, India. address: gsibii@gmail.com

2 Pratap et al. 205 Recent studies have found that the biomass and lipid content of algae can be increased through changing cultivation conditions especially nutrient content of growth medium (Chiu et al., 2009; Converti et al., 2009). Microalgae are being cultivated in various culture media (Pleissner et al., 2013; Wu et al., 2014; Sibi, 2015) and use of natural wastes as source of growth medium will not only fulfil the nutrient requirements of microalgae but also reduce the cost of growth medium. Interest in the recovery of waste or by-products has been increasing for both economic and ecological reasons. Waste generation through fruits and vegetables use higher due to increase in world population. Wastes emanating from fruits and vegetables include peels, pulp and seeds that constitute about 40% of the total mass. The majority of these waste materials are often improperly disposed, hence constitute huge environmental problems (Essien et al., 2005; Lim et al., 2010). Cultivation of microalgae that utilizes fruit and vegetable wastes to produce value added products such as biomass and lipids is a unique approach. Media formulation and optimization are key considerations in development of bioprocesses that can produce affordable by products. The study of growth medium components affecting significantly growth rate, biomass production, pigments content, biochemical composition of microorganisms is a step required to advance in the design of a low-cost culture medium for the efficient production of value added products. This study aimed primarily at utilization of fruit and vegetable waste hydrolysates as the growth medium for the cultivation of microalgae and secondly to determine the effect of hydrolysates growth and biochemical composition of microalgae thereby optimizing the suitable hydrolysate concentration for higher biomass and lipid production in microalgae. MATERIALS AND METHODS Collection of Fruit and Vegetable Wastes The fruit and vegetable wastes were collected from juice shops, supermarkets, local markets located in Bangalore urban areas. Fruit wastes are mixture of banana peels- 13.4%; sapota-8.3%, sweet lime-11.2%, orange-6.4%; apple-10.3%; mango-14.7%, pomegranate-8.6%, watermelon-9%, musk melon-5.8% and papaya-12.3%. Vegetable wastes are foliage-26.8%, corn-4.3%, pumpkin- 4.9%, carrots and beans-11%, tomatoes-19.2%, potatoes- 9.3%, cucumber-8.4%, capsicum-4.9%, cauliflower-8.5% and cabbage-3.7%. Pre-treatment of fruit and vegetable wastes Size-reduced and homogenised FVW was mixed with deionised water to achieve 250 g solids L -1 and then refrigerated at 4 C for 24 h. This process allows for passive leaching of soluble sugars and essential nutrients, whilst minimising microbial mediated leaching processes. After 24 h incubation, the fruit and vegetable waste slurry was pretreated by thermal hydrolysis. Hydrolysis via thermal hydrolysis employed autoclaving of the fruit and vegetable waste slurry under standardised heat and pressure conditions of 121 C for 15 min. Growth medium Fruit and vegetable waste hydrolysates prepared as described earlier was diluted into various concentrations (7:3, 8:2; 9:1, 10:0) with sterile distilled water and used as growth medium for the cultivation of C. vulgaris. Growth rate and Biomass concentration Specific growth rate (μ) of the microalgae was calculated according to the following formula (Levasseur et al., 1993). µ= ln (N t /N 0) T t -T 0 Where, Nt and N0 are the dry cell weight concentration (g L -1 ) at the end (Tt) and start (T0) of log phase respectively. Biomass (g L -1 ) of microalgae grown in the fruit and vegetable waste hydrolysate medium was determined by measuring the optical density of samples at 600 nm (OD600) using UV-Vis spectrophotometer. Biomass concentration was then calculated by multiplying OD600 values with 0.6, a predetermined conversion factor obtained by plotting OD600 versus dry cell weight (DCW). DCW was determined gravimetrically by centrifuging the algal cells (3,000 g, 10 min) and drying. Biomass concentration = OD Eq. (1) Chlorophyll Estimation Algal cells were centrifuged and extracted with acetone overnight. The extract was centrifuged at 3000 x g for 5 mins and the chlorophyll content in the supernatant were determined by measuring the optical densities at 645 and 663 nm in a spectrophotometer (Becker, 1994) and then calculated using the Eq. (2). Chl (mg/l) = 8.02 OD OD645.. Eq. (2) Carotenoids Estimation Algal cells were centrifuged and treated with centrifuging the algal cells and treated with KOH (60% w/w). The mixture was homogenized and warmed to 40 C for 40 mins and extracted using ethyl ether. The solvent was evaporated followed by resuspending in acetone and the optical density was measured at 444 nm (Whyte, 1987). Total carotenoids were calculated using the Eq. (3). Ct (mg/l) = 4.32 OD Eq. (3)

3 J. Environ. Waste Manag Specific growth rate (µ) d Growth period (days) 7:3 8:2 9:1 10:0 Control Fig-1: Specific growth rate of Chlorella species grown in fruit waste hydrolysate Protein Assay The extraction of proteins from microalgae was performed using alkali method. Aliquots of algal sample were centrifuged and 0.5 N NaOH was added to the pellet followed by extraction at 80 C for 10 mins. The mixture was centrifuged and protein content of the supernatant was estimated using Bovine Serum Albumin (BSA) as standard (Lowry et al., 1951). Carbohydrate Assay Cellular carbohydrates were estimated using the anthrone method described by Gerhardt et al., (1994) after hot alkaline extraction (Levya et al., 2008). Briefly, microalgal pellets were resuspended in distilled water and then heated in 40% (w/v) KOH at 90 C for 1 h. After cooling down, ice cold ethanol was added and stored at -20 C overnight followed by centrifugation. The pellet was resuspended in distilled water and then reacted with anthrone reagent. D-glucose was used as standard and the colour development was read at 578 nm in a spectrophotometer. Determination of Total Lipids Algae cells were harvested by centrifugation and then dried for the analysis of lipid content. The lipids were extracted using a one step extraction method (Folch et al., 1956). Dried algal cells added with distilled water were ultrasonicated and mixed with chloroform: methanol (2:1). The mixture was left for 30 mins in a water bath (30 C) and filtered through a Whatman No.1 filter paper. The filtrate was transferred to another screw cap tube containing NaCl solution (0.9%) and the purified chloroform layer was evaporated to a constant weight in a fuming hood under vacuum at 60 C. The total lipid content of dry weight was calculated using the following Equation (4). Lipid content (%) = (m2-m0)/m Eq. (4) where m1 is the weight of the dried algal cells, m0 is the weight of the empty new screw cap tube and m2 is the weight of the new screw cap tube with the dried lipids. Lipid productivity (g L 1 d 1 ) was determined using the following Equation (5). Lipid productivity = Biomass productivity Lipid content. Eq. (5) Statistical Analysis All the experiments were carried out in triplicates and the results were expressed as mean values and the standard deviation (SD). The statistical differences were obtained through one-way analysis of variance (ANOVA) (p < 0.05). RESULTS AND DISCUSSION Specific growth rate of C. vulgaris grown in fruit waste hydrolysate (FWH) and vegetable waste hydrolysate (VWH) media was determined in alternate days for a period of 14 days. Control flasks contained Bristol media alone to compare the effect of hydrolysate on microalgal growth. It was found that fruit waste hydrolysate in the ratio of 8:2 has produced maximum specific growth rate until the end of cultivation period (Fig-1) which was significantly higher than the control. This was followed by 9:1 and 7:3 which recorded 1.68 and 1.52 µ d -1. Lower specific growth rate was observed when the ratio was 10:0.

4 Pratap et al Specific growth rate (µ) d Growth period (days) 7:3 8:2 9:1 10:0 Control Fig-2: Specific growth rate of Chlorella species grown in vegetable waste hydrolysate The effect of vegetable waste hydrolysate on the specific growth rate of C. vulgaris is represented in Fig-2. Hydrolysates in the ratio of 9:1 and 8:2 recorded highest growth rate of 1.82 and 1.42 µ d -1. It was found that vegetable waste hydrolysate in the ratio of 9:1 has produced maximum specific growth rate at the end of cultivation period (Fig-2). Lower specific growth rate was observed when the ratio was 10:0. The lowest growth rate of 1.08 µ d -1 was observed in control group at the end of 14 days cultivation period. The biomass concentration of C. vulgaris grown on FWH and VWH were represented in Fig-3 and 4. In general, higher biomass was recorded in growth medium supplemented with fruit waste hydrolysate (0.16 mg L -1 ). The biomass was increased with increasing cultivation period however the concentration started declining after 12 days. However, increase in biomass concentration was observed with 10:0 ratio of hydrolysate and control experiments. The hydrolysates compositions, such as the carbon, nitrogen, pigments, polyphenols, polypeptide, minerals, volatile substances, and even the ph of the hydrolysates, might be the factors that influence the biomass growth of Chlorella vulgaris. Both chlorophyll and carotenoid contents of C. vulgaris grown on hydrolysates medium was shown in Fig-5 and Fig 6. Highest chlorophyll content of 7.2 mg L -1 was observed in 8:2 ratio of FWH whereas it was 4.3 mg L -1 in VWH at the same concentration. Carotenoid content was highest in VWH than FWH media with a maximum content of 0.52 and 0.42 mg L -1 respectively. Both protein and carbohydrate contents were determined photometrically and higher levels of protein were found in cells grown in FWH. In the case of carbohydrates, highest content of 18.5 mg L -1 was found in VWH at the ratio of 10:0. Gas chromatography analysis revealed that the lipid extract from C. vulgaris after 14 days of cultivation in fruit waste hydrolysate medium composed of three main types of fatty acids that could be present in a triglyceride: saturated (Cn:0), monounsaturated (Cn:1) and polyunsaturated with two to four double bonds (Cn:2,3,4) (Table-1). The fatty acid composition of the microalgal lipid is similar to that of vegetable oils, and so the lipid with this fatty acid composition is a promising feedstock for biodiesel production through transesterification (Christophe et al., 2012). Considering the amount of monounsaturated and polyunsaturated fatty acids present in the sample, the degree of unsaturation (DU) was calculated in accordance with the empirical equation [DU=(monounsaturated Cn: 1; wt:%)+2 (polyunsaturated Cn: 2;3;4; wt:%)] described by Ramos et al. (2009). The DU, which was calculated as 61.39, might have influenced the cetane number of the biodiesel to be synthesized with the extracted lipid. Higher the cetane number, the better the ignition properties of the biodiesel (Meher et al., 2006). The higher cetane number is associated with lower DU and vice versa (Knothe et al., 2003), and higher DU than 137 makes lipids unsuitable to meet the European Standard for the cetane number. Again, the abundance of lignoceric acid, the longer saturated fatty acid including other saturated fatty acids might be associated with higher cetane number. Furthermore, the lower DU indicated the higher oxidation stability of the biodiesel to be synthesized with the microbial lipid produced from fruit waste hydrolysate by C. vulgaris. However, further research is necessary to synthesize biodiesel with this microalgal lipid and to study its physico-chemical properties.

5 J. Environ. Waste Manag. 208 Table-1: Fatty acid profiles of C. vulgaris grown on FWH media Length Fatty acid Percentage C13:1 Tridecanoic acid 0.92 C16:0 Palmitic acid 3.57 C18:0 Stearic acid 2.74 C18:1 Oleic acid C18:3 Linolenic acid 6.59 C20:4 Arachidonic acid 4.37 C24:0 Lignoceric acid Fig-3: Biomass concentration of Chlorella species grown in fruit waste hydrolysate Fig-4: Biomass concentration of Chlorella species grown in vegetable waste hydrolysate Fig-5: Chlorophyll content of C. vulgaris on FWH and VWH media Fig-6: Carotenoid content of C. vulgaris on FWH and VWH media Production of lipids by algal species is very much importance in terms of their use in biodiesel, as lipid is the essential source of biofuel production. The production and content of lipids from microalgae can be manipulated by introducing the algae with various carbon sources. Enzymatic hydrolysates of carbohydrates from various agroindustrial wastes were used for heterotrophic cultivation of microalgae as alternative carbon feed stocks (Xu et al., 2006; Gao et al., 2010; Lu et al., 2010; Wei et al., 2009; Li et al., 2011; Cheng et al., 2009; Yan et al., 2011; Liu et al., 2012). In this study, fruit waste hydrolysates produced highest specific growth rate and biomass productivity than vegetable waste hydrolysates. Similar results were obtained in cellular pigment levels, carbohydrate and protein content. The hydrolysates compositions, such as the ph, carbon, nitrogen, pigments, polyphenols, polypeptide, minerals, volatile substances might be the factors that influence the biomass growth of Chlorella vulgaris. This work demonstrates the feasibility of fruit waste hydrolysate as a nutrient source for algal cultivation and a cost reduction of growth medium in algal lipid production may be expected.

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