Biotechnological production of lipids by Rhodotorula mucilaginosa using cassava wastewater

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1 Biotechnological production of lipids by Rhodotorula mucilaginosa using cassava wastewater J. Silva 1, F. L. H. Silva 2, J. E. S. Ribeiro 3, M. S. Madruga 4, L. L. Medeiros 5, A. L. O. Ferreira 6 1,3,5 Program of Pos-graduation in Food Science and Technology Federal University of Paraíba Zip Code: João Pessoa PB Brasil Phone: Fax: (jo_sivansilva@hotmail.com) 2 - Department of Chemistry Engineering - Federal University of Paraíba Zip Code: João Pessoa PB Brasil Phone: Fax (flavioluizh@yahoo.com.br) 4 - Department of Food Engineering - Federal University of Paraíba Zip Code: João Pessoa PB Brasil Phone: Fax (msmadruga@uol.com.br) 6 Federal Institute of Bahia Zip Code: Salvador BA- Brasil Phone: Fax: e- mail:adreaferreira456@gmail.com RESUMO - Essa pesquisa teve como objetivo avaliar os efeitos dos componentes do meio de cultivo sobre a biomassa e lipídios produzidos utilizando a levedura Rhodotorula mucilaginosa e a manipueira. As células de R. mucilaginosa foram inoculadas em um meio com 20% de manipueira, 10 g.l -1 glicose, 1,0 g.l -1 sulfato de magnésio e 0,8 g.l -1 de extrato de levedura. Os cultivos foram submetidos à temperatura de 30 C sob agitação constante de 200 rpm por 120 h. A manipueira apresentou 92,38% de umidade, 30,2 g.l -1 de açúcares redutores, 51 g.l -1 de açúcares totais, 1,17% de proteínas e 0,53% de cinzas, acidez titulável de 3,20 (meq NaOH.100 ml -1 ), sólidos solúveis totais de 7,0 ºBrix, e ph 5,26. A biomassa máxima foi 7,49 g.l -1, 96 h após o inóculo, sendo produzidos 30% de lipídios em biomassa seca. A manipueira mostrou-se um bom substrato para a produção de lipídios utilizando R. mucilaginosa. PALAVRAS-CHAVE: resíduo, levedura, mandioca, biomassa. ABSTRACT - This research aimed to evaluate the effects of the components of the culture medium on biomass and lipids production by yeast Rhodotorula mucilaginosa cultured in a medium containing cassava wastewater. R. mucilaginosa cells were inoculated in a medium with 20% cassava wastewater, 10 g.l -1 of glucose, 1.0 g.l -1 magnesium sulphate and 0.8 g.l -1 yeast extract. Cultivation process was performed at 30 C under constant agitation of 200 rpm for 120 h. Cassava wastewater showed 92.38% moisture, 30.2 g L -1 of reducing sugars, 51 g L-1 of total sugars, 1.17% protein and 0.53% ash, titratable acidity of 3.20 (meq NaOH.100 ml -1 ), total soluble solids of 7,0 ºBrix and ph The maximum biomass was 7.49 g.l -1, 96 h after inoculation, and were produced 30% lipid in dry biomass. Cassava wastewater was observed as a suitable substrate to grow R. mucilaginosa for the production of lipids. KEYWORDS: waste, yeast, cassava, biomass. 1. INTRODUCTION

2 The increased of the exploration of natural resources, due to development of industrialization, result in a large accumulation of waste in the environment. An alternative related to the use of these industrial wastes, is their utilization as substrates in biotechnological process for the production of antioxidants, antimicrobial agents, vitamins, lipids, proteins and pigments, great interest in the food industry substances (Santos et al., 2013). One of these waste, cassava wastewater, originates from cassava (Manihot esculenta Crantz), a plant whose roots are used for human and animal nutrition serving as raw material for the production of several products, among them the most important are the flour, starch and cassava powder (Avancini et al., 2009). Most of these subproducts or substances that would normally be discarded, can be used for producing microbial lipids. In most cases, it is possible to produce lipids in high concentrations. Thus, this study aimed to evaluate the influence of some nutrients of the culture medium on biomass production and accumulation of lipids by the yeast R. mucilaginosa, using cassava wastewater as substrate. 2. MATERIAL AND METHODS 2.1 Cassava Wastewater Cassava wastewater was gently provided by a cassava flour industry in the municipality of Sobrado, in the state of Paraiba, Brazil and stored at -18 C soon after collection. For removal of excess solids and subsequent clarification, cassava wastewater was centrifuged (RB7 Refrigerated Centrifuge) at 3200 rpm for 10 minutes, then the precipitate was removed by filtration and the supernatant was used for the fermentation process. Physicochemical analysis of the substrate: analyses of total and reducing sugars were performed using modified DNS method originally proposed by Miller (1959). The determination of protein, moisture, ash, total acidity, soluble solids ( Brix) and ph followed AOAC (2000). 2.2 Culture in synthetic medium R. mucilaginosa cells were diluted in sterile distilled water and inoculated into 1000 ml Erlenmeyer flasks containing 400 ml of synthetic medium composed of 40 g.l -1 de glucose, 8,0 g.l -1 KH 2 PO 4, 0,5 g.l -1 MgSO 4.7H 2 O and 3,0 g.l -1 yeast extract (Frengova et al. 1994). The inoculum was kept in a "Shaker" type incubator (Logen - LS THZ), at 30 C and 200 rpm. During the incubation period, aliquots were periodically collected during time intervals of 0, 4, 8, 12, 24, 48, 72, 96 and 120 hours to determine yeast growth (biomass). 2.3 Culture Conditions The experiments were performed in 1000 ml Erlenmeyer flasks containing 400 ml of culture medium resulting from the addition of the culture medium, yeast suspension (inoculum) and sterile distilled water and placed on a rotary shaker and incubated for 120 hours. The culture conditions were determined according to Spier (2014) and are described in Table 1. Preliminary tests defined the concentration of cassava wastewater and the nutrients used to suplepplement. Table 1 Culture conditions for the production of lipids by R. mucilaginosa yeast.

3 Component Concentration Cassava wastewater (% v/v) 20 Glucose (g.l -1 ) 10 MgSO 4. 7H 2 O (g.l -1 ) 1.0 Yeast extract (g.l -1 ) Analytical Methods Biomass: aliquots of 2 ml were removed during time intervals of 0, 4, 8, 12, 24, 48, 72, 96 and 120 hours for analysis of biomass concentration (g.l -1 ) using a spectrophotometer (Bioespectro SP 220) at wavelength of 600 nm. The absorbance value was converted into concentration through a biomass standard curve. Lipids: for quantification of total lipids, biomasses produced after 96 and 120 hours of culture were used. The lipid content was determined by the method of Bligh e Dyer (1959), using a mixture of chloroform, methanol and water. 3. RESULTS AND DISCUSSION 3.1 Chemical And Physicochemical Analysis Of The Substrate The results of chemical and physicochemical composition of cassava wastewater are apresented in the Table 2. Table 2 - Physicochemical composition of cassava wastewater Determination Quantification Moisture (%) Proteins (%) 1.17 Reducing sugars (g.l -1 ) 30.20* Non- reducing sugars (g.l -1 ) Total sugars (g.l -1 ) Ash (%) 0.54 Total soluble solids (ºBrix) 7.0 ph 5.26 Titratable acidity (meq NaOH.100 ml -1 ) 3.20 * Reducing sugars are included in total sugars. Avancini et al. (2007) performing characterization and toxicological evaluation of cassava starch wastewater found protein content equal to 1.35%, which is very close to value found in this study. The ph and the ash content found were far lower compared to the current study, 3.20% and 0.02, respectively. Nitschke and Pastore (2006) evaluated the production and properties of a biosurfactant obtained from Bacillus subtilis using cassava wastewater as substrate and found residue with 12.8 g.l - 1 of reducing sugar content, far below value found in this study. The content of non-reducing sugars, equivalent to 22.2 g.l -1, was very similar to that of this study. The total nitrogen found by the authors was 2.5%, slightly above the value found, and ph was equivalent to 5.9, slightly more basic than ph observed in this work.

4 Biomass (g.l -1 ) ln de X Biomass (g.l -1 ) ln de X 3.2 Culture Held In Synthetic Medium The Figure 1 shows the growth of R. mucilaginosa yeast in synthetic medium with the specific growth rate profile. Figure 1 - (a) Growth of R. mucilaginosa yeast in synthetic medium. (b) Specific growth rate profile of the microorganism in synthetic medium. 20,00 18,00 16,00 14,00 12,00 10,00 8,00 6,00 4,00 2,00 0, (a) 3,00 2,50 2,00 1,50 1,00 0,50 y = 0,0551x - 0,1692 R² = 0,9781 0, (b) With respect to the culture of the microorganism in synthetic medium, Figure 1 (a) shows yeast growth in synthetic medium up to 96 hours after the start of fermentation, which is the point of maximum biomass produced, equivalent to g.l -1. Through Figure 1 (a), it was observed that there was no growth in the first 12 hours of cultivation, characterizing the lag phase of the microorganism, reaching its peak 96 hours after the inoculation of the microorganism, emphasizing the short stationary phase, lasting a few hours, followed by the decline phase or cell death. The Figure 1 (b) shows the profile of the maximum specific cell growth rate (μ max), obtaining value of h Culture Using Cassava Wastewater as substrate The Figure 2 shows the growth curve of R. mucilaginosa yeast with the specific growth rate profile in medium containing cassava wastewater. Figure 2 - (a) Growth of R. mucilaginosa yeast in medium containing cassava wastewater. (b) Specific growth rate profile of the microorganism in medium containing cassava wastewater (a) 2,50 2,00 1,50 1,00 0,50 y = 0,0242x - 0,1304 R² = 0,9146 0, (b) In culture using medium based on cassava wastewater supplemented with glucose, yeast extract and magnesium sulfate, maximum biomass produced was 7.49 g.l hours after start of fermentation. The maximum specific cell growth rate (μ max) was equivalent to h -1 (only half the value with synthetic medium).

5 Comparing the biomass produced at times of 96 hours and 120 hours, it could be concluded that there was a considerable reduction in this time interval, with a decrease in biomass production, close to the time of 80 hours, characterizing decline phase or cell death, similarly to what was observed for the synthetic culture (Figure 1 (a)). Aksu and Eren (2007) found that the biomass in dried cells increased with increase in the initial glucose concentration up to 20 gl -1, reaching maximum value at this point. The authors also highlight the ability of R. mucilaginosa yeast to grow in a variety of carbon sources such as glucose, sucrose and lactose, being a great advantage. With maximum biomass (7.49 g.l -1 in 96 hours), the percentage of equivalent lipids was analyzed, yielding 30 %, value still not optimized, i.e., further studies will seek to optimize the process, verifying the effect of variables % cassava wastewater, % glucose, MgSO 4 7H 2 O concentration and yeast extract on the medium. Castanha et al. (2013) compared two methods for the extraction of lipids produced by oleaginous yeast in cheese whey, Rhodotorula graminis CBS 2826 yeast produced 9.71 g.l -1 of biomass and 0.09 g L -1 of total lipids, and comparing these values to those of the present study, it appears that the production of lipids by R. mucilaginosa was higher. Cazetta et al. (2005) used sugarcane vinasse and molasses for the synthesis of lipidic biomass by yeast and bacteria. Using raw vinasse as a culture medium and R. mucilaginosa yeast, the authors obtained the largest biomass production, 7.05 g L -1, very similar to value that found in this study. In sugarcane molasses, Candida lipolytica produced 6.28 g.l -1 in biomass and the largest amount of lipids, % in the vinasse and % in the molasses. R. mucilaginosa produced 18.38% in the vinasse and % in the molasses after 48 hours of culture. Xue et al. (2008) investigated the influence of addition of glucose in the medium on the biomass and lipids produced using Rhodotorula glutinis yeast and monosodium glutamate wastewater as substrate. The addition of glucose was shown to be favorable not only for cell growth but also for the synthesis of lipids. Among the methods tested, the addition of glucose during back feeding proved to be effective, achieving the production of 25 g.l -1 of biomass and 20 % of lipids. The biomass produced in this study was higher than that obtained by Reyna-Martinez et al. (2014) in the synthesis of lipids and by yeasts and by mixed cultures of yeast and microalgae produced 6.27 g.l -1 of dry biomass using R. mucilaginosa yeast, and the lipid content produced by the yeast was equivalent to % by dry biomass, which is lower than values found in the present study. 4. CONCLUSION The use of culture medium based on cassava wastewater, supplemented with some nutrients, enabled good biomass and lipid production, as well as a considerable yield of lipids in the dry biomass. Cassava wastewater proved to be a good alternative for producing microbial lipids, and can be used in fermentation processes, adding value to waste and minimizing possible environmental impacts by improper disposal. 5. ACKNOWLEDGMENTS

6 The authors would like to thank the financial support from the National Council for Scientific and Technological Development (CNPq) and the André Tosello Foundation for the microorganism donation. 6. REFERENCES Aksu, Z.; Eren, A. T. (2007). Production of carotenoids by the isolated yeast of Rhodotorula glutinis. Biochemical Engineering Journal, 35 (2), Aoac. Association of Official Analytical Chemists. Official Methods of Analysis. (2000). Washington D.C.: AOAC, Avancini, S.R.P.; Faccin, G.L.; Vieira, M.A.; Rovaris A.A.; Podesta, R.; Tramonte, R.; Souza, N.M.A.; Amante, E.R. (2007). Cassava starch fermentation wastewater: Characterization and preliminary toxicological studies. Food and Chemical Toxicology, 45 (11), Bligh, E.G.; Dyer, J. W. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37 (8), Castanha, R. F. Morais, L. A. S.; Mariano, A. P.; Monteiro, R. T. R. (2013). Comparison of two lipid extraction methods produced by yeast in cheese whey. Brazilian Archives of Biology and Technology, 56 (4), Cazetta, M. L.; Antonia, M.; Colabone, P. (2005). Use of sugar cane molasses and vinasse for proteic and lipidic biomass production by yeast and bacteria: Semina: Exact and Technological Sciences, 26 (2), Frengova, G.; Simova, E.; Pavlova, K.; Beshkova, B.; Grigorova, D. (1994). Formation of carotenoids by Rhodotorula glutinis in whey ultrafiltrate. Biotechnology and Bioengineering, 44 (8), Miller, G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analitical Chemistry, 31 (3), Nitschke, M.; Pastore, G.M. (2006). Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater. Bioresource Technology, 97 (2), Reyna-Martínez, R.; Gomez-Flores, R.; López-Chuken, U. J.; González-González, R.; Fernández- Delgadillo, S.; Balderas-Rentería, I. (2014). Lipid production by pure and mixed cultures of Chlorella pyrenoidosa and Rhodotorula mucilaginosa isolated in Nuevo Leon, Mexico. Applied Biochemistry and Biotechnology, 175 (1), Santos, L.; Kotovicz, V.; Barana, A. C.; Almeida, M. M. (2013). Utilização de resíduos agroindustriais para produção de amiloglucosidase por Aspergillus awamori (agroindustrial wastes utilization to glucoamylase production by Aspergillus awamori. Brazilian Journal Agroindustrial Technology, 6, (1), Spier, F. (2014). Microbial lipid production from raw glycerol in biodiesel synthesis. (Thesis). Federal University of Rio Grande, Rio Grande-RS. Xue, F.; Miao, J.; Zhang, X.; Luo, H.; Tan, T. (2008). Studies on lipid production by Rhodotorula glutinis fermentation using monosodium glutamate wastewater as culture medium. Bioresource Technology, 99 (13),