Microsoft Word - 20-Bio_revisado_7360.doc Original Article Biosci. J., Uberlândia, v. 27, n. 1, p. 156-161, Jan./Feb. 2011 156 GROWTH OF Aphanothece microscopica NÄGELI ON EXOGENOUS SUGARS CULTIVO DE Aphanothece microscopica NÄGELI A PARTIR DE AÇÚCARES Reinaldo Gaspar BASTOS1, Paola Rizzo de PAIVA1, Mauricio RIGO2, Graziela VEIGA2, Maria Isabel QUEIROZ3 1. Center of Agricultural Sciences - CCA – Federal University of São Carlos – UFSCar, São Carlos, SP, Brasil. reinaldo@cca.ufscar.br; 2. School of Food Engineering - DEALI - West-Center State of University of Paraná –UNICENTRO, Guarapuava, PR, Brasil; 3. School of Chemistry and Food – Federal University of Rio Grande – FURG – Rio Grande do Sul, RS, Brasil. ABSTRACT: Biological processes for wastewater treatment generally produce biomass or active sludge without reuse. In this context, incorporation of organic matter and nutrients from agro industrial effluents into cell mass for single-cell protein allowed application of sustainable process. Cyanobacteria could be used due to its versatile metabolism. So, the aim of this paper was evaluate the growth of cyanobacteria Aphanothece microscopica Nägeli growth on heterotrophic medium with glucose, lactose and sucrose. Growth curves indicated that cultivation of cyanobacterial on the dark depend the type of carbon source and there are different mechanisms for glucose, fructose and sucrose consumption. Results suggest a useful application of cyanobacteria on organic matter removal from wastewater. KEYWORDS: Cyanobacteria. Aphanothece. Wastewater treatment. INTRODUCTION Microalgae are eukaryotic, as the green algae (Chlorophyta), or prokaryotic photosynthetic microorganisms, as the cyanobacteria (Cyanophyceae) (MATA; MARTINS; CAETANO, 2010). Cyanobacteria are photosynthetic organisms with high protein content on biomass, called of single-cell protein (SCP) and capable of simple organic molecules consumption on heterotrophic in the dark (FAY, 1983; FAY, 1992). This particular metabolism provides the organisms with their simple nutritional requirements and the use of microalgae in biotechnology has been increased in food, cosmetic and pharmaceutical industries (MORENO-GARRIDO, 2008; HARUN et al., 2010). Nowadays, microalgae and cyanobacteria have been used also for biodiesel production (MATA; MARTINS; CAETANO, 2010) and carbon dioxide sequestration on photobioreactors (JACOB- LOPES et al., 2009). Furthermore, alternative technologies of bioremediation with simultaneous chemical engineering demand (COD) and nutrients (nitrogen and phosphorus) for incorporation into a microalgal biomass have been studied due to versatile metabolism of these microorganisms (TAM; WONG, 1996; LINCOLN; WILKIE; FRENCH, 1996; BICH; YAZIZ; KAKTI, 1999; MARTINEZ et al., 2000; TAM; WONG, 2000; XING et al., 2000; QUEIROZ; KOETZ; TREPTOW et al., 2001; BASHAN; BASHAN, 2004; ASLAN; KAPLAN, 2006). Aphanothece microscopica Nägeli is a cyanobacteria that has been studied with a view to the valorization of agro-industrial wastewater and the production of SCP (QUEIROZ et al., 2002; BASTOS et al., 2004). Queiroz et al. (2007) reported COD and nitrogen removal from parboiled rice effluent by this microorganism of approximately 83% and 73%, respectively, on batch cultivation. Despite of several papers about biological wastewater treatment by application of microalgae heterotrophic metabolism, literature is very poor concerning organic substrate consumption and oxygen demand for organic matter oxidation (OREN; SHILO, 1979). Literature report that dark endogenous metabolism in cyanobacteria serves mainly the adjustment of photosynthetic period (FAY, 1983). Glycogen or exogenous glucose supports a limited dark metabolism, it is being converted to glucose-6-phosphate and metabolized via the respiratory pathway (Figure 1). Cyanobacteria are distinguished from other prokaryotes by their generally low rates of endogenous respiration and by limited ability to utilized organic substances as a source of carbon and energy in the dark. Unlike aerobic heterotrophic bacteria and eukaryotic organisms, cyanobacteria present some of the essential TCA cycle enzymes in extremely low activities. Thus, the incomplete TCA doesn´t function in substrate oxidation, but performs a biosynthesis of various amino acids and lipids. Respiration in cyanobacteria is considered a membrane-bound electron transport process leading to the formation of ATP in the dark. Oxygen is currently known as the terminal oxidant, but the existence of other terminal oxidants cannot be excluded. Amino acid oxidations reduce oxygen and there is no evidence that these reactions are couple Growth of aphanothece… BASTOS, R. G. et al. Biosci. J., Uberlândia, v. 27, n. 1, p. 156-161, Jan./Feb. 2011 157 ATP formation. Reviews in the 90´s years established that the respiration has been found in all cyanobacteria tested for this activity and its probable function is the generation of a minimum amount of energy necessary for survival in the dark (SCHMETTERER, 1994). However, knowledge of cyanobacterial respiration is not yet satisfactory and their application depends of studies on several organic substrates and analysis of oxygen profiles. Figure 1. Schematic pathways of light and dark carbon metabolism in cyanobacteria (adapted by FAY, 1983) In the 1970s, Sansawa and Endo (2004) found a strain of Chlorella regularis that has a high grow heterotrophically in the dark using organic carbon sources as well as autotrophically in the light, with glucose depletion in 6h cultivation at a constant oxygen consumption rate. Queiroz et al. (2007) showed COD removal from parboiled rice effluent with low biomass yield by Aphanothece microscopica Nägeli. It suggests the existence of metabolism capable that assuring slow growth in the dark. In this context, the aim of this paper is to perform studies on cyanobacteria respiration and biomass production using an organic medium in the dark. These tests will evaluate the utilization of glucose, lactose and sucrose as exogenous carbon for the cultivation of Aphanothece microscopica Nägeli. MATERIAL AND METHODS Inoculum of Aphanothece microscopica Nägeli RSMan 92 was gently given from the Biotechnological Laboratory of FURG, Rio Grande, RS, Brazil. Cyanobacterial culture was grown on BG11 medium (RIPKA et al., 1979) for two weeks at 12h:12h (light:darkness) photoperiod, with forced aeration. Experiments were set up with a 5% cyanobacterium inoculum in 200mL Erlenmeyer´s flasks containing BG11 medium and 2.5, 5, 7.5, 10, 15 and 20% of glucose, lactose and sucrose at 1VVM aeration; 30oC; dark. Samples were quantified for biomass concentration through a standard absorbance curve at 556nm (QUEIROZ et al., 2007). The specific growth rates and productivity were calculated from the growth curve. Oxygen and sugar consumption were obtained for maximums productivity. Dissolved oxygen concentration was measured using oxygen analyzer Digimed® DM-4P and glucose concentration was evaluated by glucose- oxidase method after acid treatment of sample (BASTOS, 2006). RESULTS AND DISCUSSION Figures 1a, b and c present biomass profile for Aphanothece’s cultivation on glucose, lactose and sucrose, respectively. Profiles represent higher growth on glucose and sucrose, lower on lactose only 10 and 15%. Biomass concentration maximum was obtained for glucose (400mg/L at 10 and 2.5%), sucrose (200mg/L at 5%) and lactose (500mg/L at 10 and 15%). Maximums biomass productivity was 5,5mg/L.h for 10% glucose, 6,6mg/L.h for 10% Received: 22/04/10 Accepted: 2009/10 Growth of aphanothece… BASTOS, R. G. et al. Biosci. J., Uberlândia, v. 27, n. 1, p. 156-161, Jan./Feb. 2011 158 lactose and 2,9mg/L.h for 5% sucrose. High biomass productivity for lactose suggest rapid assimilation this sugar and enzymatic mechanism suitable for this exogenous sugar. However, the lactose profile presented different pattern, suggesting a different incorporation mechanism with a long lasting lag phase, specific growth rate and biomass productivity (Table 1). According to the results, there was a slow growth in the dark, with consumption of sugars, featuring the respiratory metabolism. This results showed growth higher than heterotrophic experiments on BG 11 medium with Aphanothece microscopica Nägeli RSMan92 (BASTOS et al., 2004). Others authors have reported that the only objective of the respiration of cyanobacteria is to generate minimal energy for growth in the dark (FAY, 1992; ANAND, 1998). Indeed, minimal energy represents a slow growth on heterothophic metabolism comparing the growth profile with previous papers about Aphanothece sp. on photosynthetic medium. Pelroy and Basshan (1973a) reported that dark growth of blue-green algae Aphanocapsa 6714 is slow in comparison to photosynthetic growth, decreasing by almost an order of magnitude. (b) Figure 1. Biomass profile for heterotrophic Aphanothece’s growth on 2.5% (� ), 5% (○), 7,5% (∆), 10% (∇), 15% (◊) e 20% (†) of glucose (a), lactose (b) and sucrose (c). 0 20 40 60 80 0 50 100 150 200 250 300 350 400 450 B io m as s (m g /L ) Time (hours) (a) 0 20 40 60 80 0 100 200 300 400 500 600 B io m as s (m g /L ) Time (hours) (b) 0 20 40 60 80 0 20 40 60 80 100 120 140 160 180 200 220 240 B io m as s (m g/ L ) Time (hours) (c) Growth of aphanothece… BASTOS, R. G. et al. Biosci. J., Uberlândia, v. 27, n. 1, p. 156-161, Jan./Feb. 2011 159 The assay on sucrose present slight growth for concentrations higher than 2.5%, in contrast to other sugars. This observation in concern of the profiles presented suggests different mechanisms of sugars assimilation, which support dark growth. Studies on the enzymatic mechanism of disaccharides and monosaccharides by cyanobacteria must be further developed to the better knowledge of this bioreaction. Analysis of enzymes in cell-free extracts revealed that ribulose- 1,5 biphosphate was a strong inhibitor of glucose-6- phosphate dehydrogenase, the first enzyme of the oxidative pathway (PELROY; BASSHAN, 1973b). Moreover, dark incubation of cells led to the immediate disappearance of this metabolite, at the same time as the oxidative pathway was being activated. Besides, these authors suggests that the potential for heterotrophic growth by microalgae is probably due to the permeability of the cell membrane for organic molecules rather than a fundamental biochemical difference between Aphanothece or Aphanocapsa and strictly photoautotrophic others microalgae. Moreover, transport across the cell membrane may depend on the presence of specific carriers, which mediate the uptake of a particular substance (FAY, 1983). This information supports our data growth by Aphanothece on glucose and lactose, higher than on sucrose. Another consideration is a low oxygen demand by Aphanothece in Table 1, indicating the oxidative pathway different the aerobic microorganisms. It is an important engineering parameter for design of heterotrophic bioreactors using these microalgae. Table 1. Biomass productivity and specific growth rate at the best conditions at different sugars Experiment Biomass productivity (mg/L.h) Specific growth rate (h-1) Oxygen consumption rate (mmol/L.min) Glucose 10% 5.5 0.013 0.025 Lactose 10% 6.6 0.113 0.023 Sucrose 5% 2.9 0.054 0.053 Heterotrophic growth of Aphanothece microscopica Nägeli on sugar medium explains the occurrence of cyanobacterial blooms in niches with high organic load (QUEIROZ; KOETZ; TREPTOW, 2001). So, the growth data from these sources of organic carbon is a reasonable prospect in the possible use of these microorganisms in the treatment of agro-industrial effluents, which are rich in soluble organic matter. CONCLUSION Cyanobaterial growth on darkness depends of carbon source; there are different mechanisms of glucose, fructose and sucrose consumption. The result of this research suggests a possible application of cyanobacteria to the removal of organic matter from wastewater. ACKNOWLEDGMENTS The authors acknowledge the support given by UNICENTRO, FURG and ProPq/UFSCar. RESUMO: Os processos biológicos de tratamento de águas residuárias produzem grandes quantidades de biomassa geralmente sem utilização posterior. Neste contexto, a incorporação de matéria orgânica e nutrientes de efluentes agroindustriais em células microbianas visando a produção de proteínas unicelulares corresponderia a um processo sustentável. Nesse sentido, as cianobactérias poderiam ser aplicadas devido ao seu metabolismo versátil. Sendo assim, o trabalho teve como objetivo avaliar o cultivo heterotrófico da cianobactéria Aphanothece microscopica Nägeli em meios contendo glicose, lactose e sacarose. As curvas de crescimento indicaram que o cultivo heterotrófico depende do tipo de fonte de carbono, sugerindo diferentes mecanismos de incorporação e consumo da glicose, lactose sacarose. Os resultados indicam uma possível aplicação desta cianobactéria na remoção destas moléculas orgânicas em águas residuárias. PALAVRAS-CHAVE: Cianobactéria. Aphanothece. Tratamento de águas residuárias. Growth of aphanothece… BASTOS, R. G. et al. Biosci. J., Uberlândia, v. 27, n. 1, p. 156-161, Jan./Feb. 2011 160 REFERENCES ANAND, N. Cyanobacterial taxonomic: classical concepts and modern trends. In: SUBRAMANIAN, G.; KAUSHIK, B.D.; VENKATARAMAN, G.S. (Ed.). Cyanobacterial biotechnology. Enfield: Science Publishers Inc., 1998. p. 337–340, ASLAN, S.; KAPLAN, I. K. Batch kinetics of nitrogen and phosphorus removal from synthetic wastewater by algae. Ecological Engineering, Oxford, v. 28, p. 64-70, 2006. BASHAN, L., BASHAN, Y. Recent advances in removing phosphorus from wastewater and its future use as fertilizer (1997–2003). Water Research, New York, v. 38, p. 4222–4246, 2004. BASTOS, R. G.; QUEIROZ, M. I.; ALMEIDA, T. L.; BENERI, R.L.; ALMEIDA, R.. V.; PADILHA, M. Remoção de nitrogênio e materia orgánica do efluente da parboilização do arroz por Aphanothece microscopica Nägeli na ausencia de luminosidade. Revista da Engenharia Sanitária e Ambiental, Rio de Janeiro, v. 9, n. 2, p. 112-116, 2004. BASTOS, R. G. Transferência de oxigênio no cultivo em estado sólido de Drechslera (Helminthosporium) monoceras. 2006. 165f. Tese (Doutorado) - Faculdade de Engenharia Química, Universidade Estadual de Campinas, Campinas, 2006. BICH, N. N., YAZIZ, M. I., KAKTI, N. Combination of Chlorella vulgaris Andeichhornia crassipes for wastewater nitrogen removal. Water Research, New York, v. 33, n. 10, p. 2357–2362, 1999. FAY, P. Oxygen relations of nitrogen fixation in cyanobacteria. Microbiological Reviews, Washington, v. 56, n. 2, p. 340-373, 1992. FAY, P. The blue-greens (Cyanophyta-cyanobacteria). 5th. ed. London: Edward Arnold Publishers, 1983. 88p. HARUN, R.; SINGH, M.; FORDE, G.; DANQUAH, M. K. Bioprocess engineering of microalgae to produce a variety of consumer products. Renewable and sustainable energy reviews, Golden, v. 14, p. 1037-1047, 2010. JACOB-LOPES, E.; REVAH, S.; HERNÁNDEZ, S.; SHIRAI, K.; FRANCO, T. T. Development of operational strategies to remove carbon dioxide in photobioreactors. Chemical Engineering Journal, Lausanne, v. 153, p. 120-126, 2009. LINCOLN, E. P.; WILKIE, A. C.; FRENCH, B. T. Cyanobacterial process for renovating dairy wastewater. Biomass and Bioenergy, Oxford, v. 10, p. 63-68, 1996. MATA, T. M.; MARTINS, A. A.; CAETANO, N. S. Microalgae for biodiesel production and others applications: A review. Renewable and sustainable energy reviews, Golden, v. 14, p. 212-232, 2010. MORENO-GARRIDO, I. Microalgae immobillization: current techniques and uses. Bioresource Technology, Essex, v. 99, p. 3949-3964, 2008. OREN, A.; SHILO, M. Anaerobic heterotrophic dark metabolism in the cyanobacterium Oscillatoria limnetica: Sulfur respiration and lactate fermentation. Archives of Microbiology, Berlin, v. 122, n. 1, p. 77-84, 1979. PELROY, R. A.; BASSHAN, J. A. Efficiency of energy conversion by aerobic glucose metabolism in Aphanocapsa 6714. Journal of Bacteriology, Washington, v. 115, n. 3, p. 937-942, 1973a. PELROY, R. A.; BASSHAN, J. A. Kinetics of glucose incorporation by Aphanocapsa 6714. Journal of Bacteriology, Washington, v. 115, n. 3, p. 943-948, 1973b. Growth of aphanothece… BASTOS, R. G. et al. Biosci. J., Uberlândia, v. 27, n. 1, p. 156-161, Jan./Feb. 2011 161 QUEIROZ, M. I., BASTOS, R. G., BENERI, R. L.; ALMEIDA, R. V. Evaluación del crecimiento de la Aphanothece microscopica Nägeli en las aguas residuales de la parbolización del arroz. Información Tecnologica, La Serena, v. 13, n. 1, p. 61-65, 2002. QUEIROZ, M. I.; KOETZ, P. R.; TREPTOW, R. O. The Nagele microscocal Aphanothece potential in the production of the single-cell protein from the remaining water. In: INTERNATIONAL CONGRESS ON ENGINEERING AND FOOD, 8., 2001, Pennsylvania. Proceedings … Lancaster: Technomic Publishing Co., 2001. p. 2027-2031. QUEIROZ, M. I.; LOPES, E.J.; ZEPKA, L. Q.; BASTOS, R. G.; GOLDBECK, R. The kinetics of the removal of nitrogen and organic matter from parboiled rice effluent by cyanobacteria in a stirred batch reactor. Bioresource Technology, Essex, v. 98, n. 11, p. 2163-2169, 2007. RIPKA, R.; DERUELLES, J.; WATERBURY, J. B.; HERDMAN, M.; STANIER, R. Y. Generic assignments strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology, London, v. 111, p. 1-61, 1979. SANSAWA, H.; ENDO, H. Production of intracellular phytochemicals in Chlorella under heterotrophic conditions. Journal of Bioscience and Bioengineering, Osaka, v. 98, p. 437-444, 2004. SCHMETTERER, G. Cyanobacterial respiration. In: BRYANT, D. A. The molecular biology of cyanobacteria. Dordrecht: Kluwer Academic Plubishers, 1994. p. 409-435. TAM, N. F. Y.; WONG, Y. S. Effect of ammonia concentration on growth of Chlorella vulgaris and nitrogen removal from media. Bioresource Technology, Essex, v. 57, p. 45-50, 1996. TAM, N.F.Y.; WONG, Y.S. Effect of immobilized microalgal bead concentrations on wastewater nutrient removal. Environmental Pollution, Barking, v. 107, p. 145-151, 2000. XING, X. H., JUN, B. M., YANAGIDA, M., TANJI, Y.; UNNO, H. Effect of C/N values on microbial simultaneous removal of carbonaceous and nitrogenous substances in wastewater by single continuous-flow fluidized-bed bioreactor containing porous carrier particles. Biochemical Engineering Journal, Amsterdam, v. 5, p. 29-37, 2000.