RESUMO Nos últimos anos há um interesse crescente no uso de diversos resíduos agroindustriais. No Brasil, as indústrias de alimentos acumulam uma enorme quantidade de resíduos, principalmente as de processamento de frutos cítricos.Entre as possibilidades de valorização destes resíduos, a digestão anaeróbia é uma alternativa promissora na geração de energia renovável e controle da poluição. Nesse sentido, esse estudo avaliou a digestão anaeróbia de água residuária citrícola, visando à produção biológica de hidrogênio a partir de três inóculos: (1) Clostridium Acetobutylicum ATCC 824,(2) Clostridium Beijerinckii ATCC 10132 e (3) consórcio anaeróbio obtido da água residuária citrícola. O experimento foi realizado em 51h, em triplicatas de reatores anaeróbios em batelada, alimentados com 2 fontes distintas de carbono: glicose (10,7 g DQO L-1) e água residuária citrícola (10,0 g DQO L-1), headspace preenchido com N2 (99,99%), pH inicial 7,0 e mantidos a 37°C, sob modo estático. Nos reatores alimentados com glicose foram verificados geração de hidrogênio apenas com os inóculos 1 e 2, que foram, respectivamente, de 36,8 mmol L-1 e 64,1 mmol L-1. Nos reatores operados com água residuária citrícola foram obtidos 21,2; 15,7 e; 37,6 mmol H2 L-1 com os inóculos 1; 2 e 3, respectivamente. Além disso, foram verificadas gerações expressivas de etanol para todos os inóculos testados nos reatores alimentados com glicose. Na caracterização do consórcio anaeróbio citrícola foi verificado predomínio de bacilos Gram + e quantificados os gêneros de bactérias anaeróbias geradoras de H2 (UFC mL-1): Clostridium sp. (3x105), Bacteroides sp. (4x105), Lactobacillus sp. (4x105), Streptococcus sp. (5x104), e Veillonella sp. (3x105), revelando diversidade elevada de bactérias anaeróbias produtoras de hidrogênio presentes na água residuária agroindustrial. Tais resultados ressaltam que a geração de hidrogênio pode ocorrer em vias metabólicas distintas com consequente favorecimento na geração de biocombustíveis como etanol ou butanol.

ABSTRACT In recent years there has been a growing interest in the use of various agro-industrial wastes. In Brazil, the food industries accumulate an enormous amount of waste, especially those that process citrus fruits. Among the possibilities for valuing these residues, anaerobic digestion is a promising alternative in the generation of renewable energy and pollution control. In this sense, this study evaluated the anaerobic digestion of citrus wastewater, aiming at the biological production of hydrogen from three inocula: (1) Clostridium Acetobutylicum ATCC 824, (2) Clostridium Beijerinckii ATCC 10132 and (3) anaerobic consortium obtained from citrus wastewater. The experiment was carried out in 51h, in triplicates of anaerobic batch reactors, fed with 2 different carbon sources: glucose (10.7 g COD L-1) and citrus wastewater (10.0 g COD L-1), headspace filled with N2 (99.99%), initial pH 7.0 and maintained at 37 ° C, under static mode. In the reactors fed with glucose, hydrogen generation was verified only with the inocula 1 and 2, which were, respectively, 36.8 mmol L-1 and 64.1 mmol L-1. In the reactors operated with citrus wastewater, were obtained 21.2; 15.7 e; 37.6 mmol H2 L-1 with inocula 1; 2 and 3, respectively. In addition, expressive generations of ethanol were verified for all inocula tested in the reactors fed with glucose. In the characterization of the citrus anaerobic consortium, a predominance of Gram + bacilli was verified and the genera of anaerobic H2-generating bacteria (UFC mL-1) were quantified: Clostridium sp. (3x105), Bacteroides sp. (4x105), Lactobacillus sp. (4x105), Streptococcus sp. (5x104), and Veillonella sp. (3x105), revealing a high diversity of anaerobic hydrogen-producing bacteria present in agro-industrial wastewater. Such results emphasize that the generation of hydrogen can occur in different metabolic pathways with consequent favor in the generation of biofuels such as ethanol or butanol.

RESUMO O esgotamento das reservas de petróleo e a crescente preocupação com as mudanças climáticas têm acelerado novas pesquisas para obtenção de energias renováveis. A produção de biogás através da digestão anaeróbia vem se mostrando uma ótima opção, não só para produção de energia, como também para manejo adequado de resíduos orgânicos. O biodiesel teve sua produção aumentada em diversos países por ser um combustível renovável e biodegradável. Entretanto, é largamente obtido da transesterificação de óleos vegetais e animais, onde seu rendimento é 9 litros de biodiesel para 1 litro de glicerol. O glicerol bruto possui grau reduzido de pureza, se tornando uma matéria-prima barata com potencial para produção de energia renovável, principalmente gás hidrogênio (H2) e metano (CH4). Nesse sentido, os objetivos deste estudo foram: operar um reator horizontal de leito fixo (RAHLF), com alimentação contínua e determinar a partida do mesmo na produção de H2 a partir de glicerol bruto residual de biodiesel, co-digerido em esgoto sanitário nas fases de: partida, ensaio 1 e ensaio 2. Durante a fase de partida e Ensaio 1, o afluente foi constituído de 99% esgoto 1% glicerol bruto (v/v) e 1,5% de glicerol bruto no Ensaio 2. Na partida e ensaio 1 foram verificadas médias de produção de 1 mol H2 m3 d-1 e 3,2 mols H2 m3 d-1, respectivamente. A maior produção diária atingida foi 11,34 mols H2 m3 d-1 no Ensaio 2 com geração média de ácidos graxos voláteis (AGV) de 3414,0 mg L-1. Tais resultados foram promissores e novos avanços com proporções mais elevadas do glicerol bruto e agentes tamponantes poderão evitar grandes variações de pH, viabilizando assim gerações mais elevadas e mais estáveis de H2, em reatores contínuos.

ABSTRACT The depletion of oil reserves and the growing concern about climate change have accelerated new research to obtain renewable energy. The production of biogas through anaerobic digestion has proved to be a great option, not only for energy production, but also for the proper management of organic waste. Biodiesel had its production increased in several countries as it is a renewable and biodegradable fuel; moreover, it does not require major modifications for its use in diesel engines. However, it is largely obtained from the transesterification of vegetable oils and animal fat, where its yield is 9 volumes of biodiesel to 1 volume of glycerol. The crude glycerol has a low degree of purity, becoming an inexpensive raw material with the potential to produce renewable energy, mainly hydrogen gas (H2) and methane (CH4). In this sense, the objectives of this study were: to operate a horizontal anaerobic reactor with fixed bed (HARFB), with continuous feeding and to determine its start in the production of H2 from residual biodiesel glycerol co-digested in sanitary sewage in the phases from: Startup, Test 1 and Test 2. During the Startup and Test 1 assay, the affluent was constituted by 99% sewage 1% crude glycerol (v/v) and 1.5% crude glycerol in Test 2. At Startup and Test 1, production averages of 1 mol H2 m3 d-1 and 3.2 moles H2 m3 d-1 were verified, respectively. The highest daily production achieved was 11.34 moles H2 m3 d-1 in Test 2 with an average generation of volatile fatty acids (VFA) of 3414.0 mg L-1. Such results were promising and new advances with higher proportions of crude glycerol and buffering agents could avoid large pH variations, thus enabling higher and more stable generations of H2 in continuous reactors.

The inhibition effect of limonene on the organisms involved in anaerobic reactors may affect obtaining H2 in this process. Rapid and precise limonene quantification is desirable to characterize wastewaters. The objective of this research was to develop a Gas Chromatography Coupled to Mass Spectrometry (GC-MS) method to determine limonene concentration in liquid samples from batch reactors applied to H2 production at 30 ºC. Method validation was carried out through calibration curves (3 to 20 mg L-1of limonene), in addition to evaluating its precision (CV =0.1%), linearity (R2=0.979), limits of detection (1.7 mg L-1) and quantification (4.2 mg L-1). Its inhibitory effect was evaluated with increasing concentrations of limonene (without addition to 2000 mg L-1). Limonene affected the starting time of H2 production (λ), since λ was 8.7 h in control assays and 15 h with 2000 mg L-1 of limonene. The same was observed for obtaining H2, since 19.5 mmol H2 L-1 was obtained in control assays and 8.7 mmol H2 L-1 was obtained in assays with 2000 mg L-1 of limonene. This method was effective for limonene determination in liquid samples from batch reactors and could be used for characterization and control of anaerobic reactors.

The aim of this work was to identify groups of microorganisms that are capable of degrading organic matter utilizing sulfate as an electron acceptor. The assay applied for this purpose consisted of running batch reactors and monitoring lactate consumption, sulfate reduction and sulfide production. A portion of the lactate added to the batch reactors was consumed, and the remainder was converted into acetic, propionic and butyric acid after 111 hours of operation These results indicate the presence of sulfate-reducing bacteria (SRB) catalyzing both complete and incomplete oxidation of organic substrates. The sulfate removal efficiency was 49.5% after 1335 hours of operation under an initial sulfate concentration of 1123 mg/L. The SRB concentrations determined by the most probable number (MPN) method were 9.0x10(7) cells/mL at the beginning of the assay and 8.0x10(5) cells/mL after 738 hours of operation.