The solvent-producing clostridia are well known for their ability to produce acetone, butanol and isopropanol in industrial fermentation. Production of these compounds occurs in cells that have completed a metabolic switch under specific growth conditions. Knowledge of the regulation of the metabolic switch will make the industrial process more reliable. From an isopropanol-producing strain Clostridium beijerinckii NRRL B593, a gene which encodes a putative NtrC-like regulatory protein was cloned and sequenced. The gene codes for a polypeptide of 632 amino acids and has been designated the stc gene. Expression of the stc gene was confirmed by RT-PCR. The co-presence of the stc gene with the adh gene which encodes a primary/secondary alcohol dehydrogenase in isopropanol-producing clostridia suggests that the stc gene may be functionally related to isopropanol production. From C. beijerinckii NRRL B592, a region which encompassed the solvent-production genes ald (aldehyde dehydrogenase), ctfA and ctfB (acetoacetate: butyrate/acetate CoA-transferase) and part of adc (acetoacetate decarboxylase) was cloned and sequenced. The organization of these genes was similar to that in C. beijerinckii NRRL B593. Northern analysis indicated that these four genes were co-transcribed on the same messenger RNA in C. beijerinckii NRRL B593. Therefore, in C. beijerinckii, the sol operon consists of the ald -ctfA-ctfB-adc genes, which differs from the sol operon in Clostridium acetobutylicum. / Master of Science

No description available.

O hidrogênio (H2) tem sido considerado uma fonte de energia limpa bastante promissora, pois sua combustão origina apenas moléculas de água, sendo uma alternativa ao uso de combustíveis fósseis. Entretanto, os métodos atuais de produção de H2 demandam matérias-primas finitas e uma grande quantidade de energia, tornando a sua obtenção não sustentável. Mais recentemente, a via fermentativa tem sido considerada para a produção de H2, utilizando como matérias-primas efluentes industriais, materiais lignocelulósicos e biomassa de algas, denominado de bio-hidrogênio de primeira, segunda e terceira geração, respectivamente. Neste trabalho foi isolada uma bactéria anaeróbia a partir de uma cultura mista (lodo) de um sistema de tratamento de vinhaça, após pré-tratamento do lodo a pH 3 por 12 horas. Este microrganismo foi identificado com 99% de similaridade como Clostridium beijerinckii com base na sequência do gene RNAr 16S denominado de C. beijerinckii Br21. A temperatura e o pH mais adequados para o crescimento e produção de H2 por esta cultura foi 35 °C e pH inicial 7,0. A bactéria possui a capacidade de utilizar ampla variedade de fontes de carbono para a produção de H2 por fermentação, especialmente, monossacarídeos resultantes da hidrólise de biomassa de algas, tais como glicose, galactose e manose. Foram realizados ensaios em batelada para a produção de H2 com a bactéria isolada empregando diferentes concentrações de glicose e galactose, visando a sua futura utilização em hidrolisados de alga. Os parâmetros cinéticos dos ensaios de fermentação estimados pelo modelo de Gompertz modificado, como a velocidade máxima de produção (Rm), a quantidade máxima de hidrogênio produzido (Hmáx) e o tempo necessário para o início da produção de hidrogênio (fase lag) para a glicose (15 g/L) foram de: 58,27 mL de H2/h, 57,68 mmol de H2 e 8,29 h, respectivamente. Para a galactose (15 g/L), a Rm, Hmáx e foram de 67,64 mL de H2/h, 47,61 mmol de H2 e 17,22 horas, respectivamente. O principal metabólito detectado ao final dos ensaios de fermentação, foi o ácido butírico, seguido pelo ácido acético e o etanol, tanto para os ensaios com glicose, como com galactose. C. beijerinckii é um candidato bastante promissor para a produção de H2 por fermentação a partir de glicose e galactose e, consequentemente, a partir de biomassa de algas como substratos. / Hydrogen (H2), considered an alternative to fossil fuels, is a promising source of clean energy because its combustion originates water molecules only. However, the current H2 production methods require finite raw materials and a large amount of energy, which makes them unsustainable. The fermentative pathway has been considered for H2 production from renewable raw materials such as industrial wastewater, lignocellulosic materials, and algal biomass, the so-called first, second, and third bio-hydrogen generation, respectively. In this work, after pre-treatment at pH 3 for 12 h, a H2-producing bacterium was isolated from a mixed culture (sludge) collected from an anaerobic bioreactor used to treat sugarcane vinasse. The microorganism was identified as Clostridium beijerinckii based on the sequence of the 16S rRNA gene; it was named C. beijerinckii Br21. The most appropriate temperature and initial pH to achieve H2 production by this strain was 35 °C and 7, respectively. The bacterium was able to use a wide variety of carbon sources, especially the monosaccharides glucose, galactose, and mannose resulting from hydrolysis of algal biomass. Batch assays using different concentrations of glucose and galactose were performed to produce H2. The kinetic parameters of the tests were estimated by the Gompertz modified model. The maximum production rate (Rm), the maximum amount of produced H2 (Hmáx), and the phase lag () for glucose and galactose, both at 15 g/L, were 58.27 and 67.64 mL of H2/h, 57.68 and 47.61 mmol of H2, and 8.29 and 17.22 h, respectively. The main metabolite detected at the end of fermentation tests was butyric acid, followed by acetic acid and ethanol. The results indicated that the new C. beijerinckii isolate is a promising candidate for fermentative H2 production from algal biomass.

O hidrogênio (H2) tem sido considerado uma fonte de energia limpa bastante promissora, pois sua combustão origina apenas moléculas de água, sendo uma alternativa ao uso de combustíveis fósseis. Entretanto, os métodos atuais de produção de H2 demandam matérias-primas finitas e uma grande quantidade de energia, tornando a sua obtenção não sustentável. Mais recentemente, a via fermentativa tem sido considerada para a produção de H2, utilizando como matérias-primas efluentes industriais, materiais lignocelulósicos e biomassa de algas, denominado de bio-hidrogênio de primeira, segunda e terceira geração, respectivamente. Neste trabalho foi isolada uma bactéria anaeróbia a partir de uma cultura mista (lodo) de um sistema de tratamento de vinhaça, após pré-tratamento do lodo a pH 3 por 12 horas. Este microrganismo foi identificado com 99% de similaridade como Clostridium beijerinckii com base na sequência do gene RNAr 16S denominado de C. beijerinckii Br21. A temperatura e o pH mais adequados para o crescimento e produção de H2 por esta cultura foi 35 °C e pH inicial 7,0. A bactéria possui a capacidade de utilizar ampla variedade de fontes de carbono para a produção de H2 por fermentação, especialmente, monossacarídeos resultantes da hidrólise de biomassa de algas, tais como glicose, galactose e manose. Foram realizados ensaios em batelada para a produção de H2 com a bactéria isolada empregando diferentes concentrações de glicose e galactose, visando a sua futura utilização em hidrolisados de alga. Os parâmetros cinéticos dos ensaios de fermentação estimados pelo modelo de Gompertz modificado, como a velocidade máxima de produção (Rm), a quantidade máxima de hidrogênio produzido (Hmáx) e o tempo necessário para o início da produção de hidrogênio (fase lag) para a glicose (15 g/L) foram de: 58,27 mL de H2/h, 57,68 mmol de H2 e 8,29 h, respectivamente. Para a galactose (15 g/L), a Rm, Hmáx e foram de 67,64 mL de H2/h, 47,61 mmol de H2 e 17,22 horas, respectivamente. O principal metabólito detectado ao final dos ensaios de fermentação, foi o ácido butírico, seguido pelo ácido acético e o etanol, tanto para os ensaios com glicose, como com galactose. C. beijerinckii é um candidato bastante promissor para a produção de H2 por fermentação a partir de glicose e galactose e, consequentemente, a partir de biomassa de algas como substratos. / Hydrogen (H2), considered an alternative to fossil fuels, is a promising source of clean energy because its combustion originates water molecules only. However, the current H2 production methods require finite raw materials and a large amount of energy, which makes them unsustainable. The fermentative pathway has been considered for H2 production from renewable raw materials such as industrial wastewater, lignocellulosic materials, and algal biomass, the so-called first, second, and third bio-hydrogen generation, respectively. In this work, after pre-treatment at pH 3 for 12 h, a H2-producing bacterium was isolated from a mixed culture (sludge) collected from an anaerobic bioreactor used to treat sugarcane vinasse. The microorganism was identified as Clostridium beijerinckii based on the sequence of the 16S rRNA gene; it was named C. beijerinckii Br21. The most appropriate temperature and initial pH to achieve H2 production by this strain was 35 °C and 7, respectively. The bacterium was able to use a wide variety of carbon sources, especially the monosaccharides glucose, galactose, and mannose resulting from hydrolysis of algal biomass. Batch assays using different concentrations of glucose and galactose were performed to produce H2. The kinetic parameters of the tests were estimated by the Gompertz modified model. The maximum production rate (Rm), the maximum amount of produced H2 (Hmáx), and the phase lag () for glucose and galactose, both at 15 g/L, were 58.27 and 67.64 mL of H2/h, 57.68 and 47.61 mmol of H2, and 8.29 and 17.22 h, respectively. The main metabolite detected at the end of fermentation tests was butyric acid, followed by acetic acid and ethanol. The results indicated that the new C. beijerinckii isolate is a promising candidate for fermentative H2 production from algal biomass.

The onset of solvent production by the clostridia involves regulation at the transcriptional level. The signal triggering the onset has not been identified, but redox and energetic states have been suggested as possible factors. Because several solvent-producing clostridia, including Clostridium acetobutylicum and Clostridium beijerinckii, are nitrogen-fixing organisms and both nitrogen-fixation and alcohol production (n-butanol, isopropanol and ethanol) are reductant-dependent processes, the effect of nitrogen fixation on the onset and progression of solvent production in C. beijerinckii NRRL B593 and vice versa was investigated. For this purpose, a defined growth medium containing three amino acids was developed for C. beijerinckii NRRL B593, and this medium was used for growing solvent-producing and nitrogen-fixing cultures. The nitrogen-fixing cultures produced solvents with a solventogenic shift, which appeared to coincide with a decrease in nitrogen-fixing activity. Measurement of specific activities of acetoacetate decarboxylase and aldehyde dehydrogenase and Northern blot analysis of the mRNA of the solvent-producing genes in samples harvested periodically from a nitrogen-fixing culture of C. beijerinckii showed the presence of both enzyme activities and the mRNA carrying the solvent-production genes throughout incubation. A 2.5-fold increase in the specific activity of acetoacetate decarboxylase and a 4.5-fold increase in the specific activity of aldehyde dehydrogenase were observed when the activities in the latest cell-free extract was compared with the activities in the earliest cell-free extract. When C. beijerinckii was grown in the medium containing 4 mM ammonium acetate, the onset of nitrogen fixation coincided with the onset of solvent production and prevented accumulation of solvents to high levels, which suggested competition between alcohol-producing enzymes and nitrogenase for the reductant. Recently, a 20-kb region of the genomic DNA of C. beijerinckii NRRL B593 that contained the nif genes and ORFs with other putative functions was sequenced in our laboratory. An examination of the nif clusters of C. beijerinckii, C. acetobutylicum and C. pasteurianum revealed apparent differences in the intervening ORFs which suggested differences in the regulation of nitrogen fixation in these organisms. Transcriptional analysis of genes in the nif cluster of C. beijerinckii by Northern blotting revealed four different transcripts. The absence of mRNAs of the nif-associated ORFs in RNA samples isolated from non-nitrogen-fixing cells indicated that the nif-associated ORFs are regulated in parallel to the nif genes. By studying the effect of ammonia addition on nitrogen-fixing activities of C. beijerinckii and C. pasteurianum, significant differences in the regulation of nitrogen-fixation in the two species were observed. C. beijerinckii NRRL B593, but not C. pasteurianum, showed a rapid decrease in nitrogen-fixing activity in vivo upon ammonium acetate addition. However, measurement of nitrogen-fixing activities in vitro before and after ammonium acetate addition showed the presence of active nitrogenase throughout growth in both organisms. The results suggest that the nitrogenase activity in C. beijerinckii NRRL B593 is inhibited when ammonia is available. A second nifH-hybridizing mRNA was detected in Northern blots during studies of the expression of nifH1 in C. pasteurianum. The mRNA was identified as that from either the nifH2 or nifH6 gene after sequencing the cDNA strands, which were generated by RT (Reverse Transcriptase)-PCR. In addition, Western blot analysis of the cell-free extracts of nitrogen-fixing cells of C. pasteurianum indicated the presence of a second NifH-related polypeptide. The two NifH-related polypeptides were separated by preparative gel electrophoresis and characterized by MALDI-TOF (Matrix-assisted Laser Desorption Ionization Time-Of-Flight) mass spectrometry. The results suggested the expression of NifH2/H6 protein in nitrogen-fixing cells of C. pasteurianum. The physiological significance of the expression of the nifH2 or nifH6 gene or both is yet to be determined. / Ph. D.

In acetone/butanol-producing clostridia, the metabolic intermediate acetoacetyl-CoA can be directed toward butyrate or butanol formation by the reaction catalyzed by 3-hydroxybutyryl-CoA dehydrogenase, or toward acetone formation by the reaction catalyzed by acetoacetate:acetate/butyrate CoA-transferase. 3-Hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35 or 1.1.1.157) has been purified 45-fold to apparent homogeneity from the solvent-producing anaerobe Clostridium beijerinckii strain NRRL B593. The identities of 34 of the 35 N-terminal amino acid residues have been determined. The enzyme exhibited a native M_r of 213,000 and a subunit M_r of 30,800. It is specific for the (S)-enantiomer of 3-hydroxybutyryl-CoA. Michaelis constants for NADH and acetoacetyl-CoA were 8.6 and 14 µM, respectively. The maximum velocity of the enzyme was 540 µmol/(min mg) for the reduction of acetoacetyl-CoA with NADH. The enzyme could use either NAD(H) or NADP(H) as cosubstrate; however, NAD(H) appeared to be the physiological substrate. In the presence of 9.5 µM NADH, the enzyme was inhibited by acetoacetyl-CoA at concentrations as low as 20 µM, but the inhibition was relieved as the concentration of NADH was increased, suggesting a possible mechanism for modulating the energy efficiency during growth. Acetoacetate:acetate/butyrate CoA-transferase (EC 2.8.3.9) has been purified 308-fold to apparent homogeneity from the same organism. The enzyme exhibited a native M_r of 89,100. The subunits of the enzyme were separated by preparative SDS-PAGE, and exhibited M, values of 28,400 and 25,200. The identities of the 34 N-terminal amino acids of the large subunit and 38 of the 39 N-terminal amino acids of the small subunit were determined. The N-terminal region of the two subunits showed significant similarity with several other CoA transferase enzymes. Michaelis constants for butyrate and acetoacetyl-CoA were 11.7 mM and 107 µM, respectively, while those for acetate and acetoacetyl-CoA were 424 mM and 118 µM, respectively. The value of k_cat/K_m was approximately 100 times higher with butyrate than with acetate. Implications of the properties of these two enzymes for the acetone-butanol fermentation are discussed, and a model for the induction of the enzymes responsible for solvent production is suggested. / Ph. D.

No description available.

O H2 pode ser obtido por processos biológicos, como a fermentação, conduzidos à temperatura e pressão ambientes. Para tal são utilizadas matérias-primas renováveis, ricas em carboidratos, como as biomassas lignocelulósicas e de algas. Estas biomassas têm estrutura química complexa e requerem uma etapa de pré-tratamento e/ou hidrólise antes da sua utilização na fermentação. Processos de hidrólise podem liberar, tanto monossacarídeos, quanto substâncias potencialmente inibidoras de fermentação. Esse estudo avaliou o efeito de 3 potenciais inibidores de fermentação (5-hidroximetilfurfural -HMF, ácido levulínico AL e ácido fórmico AF), derivados da hidrólise de biomassas. Ensaios cinéticos de fermentação em batelada foram realizados com o microrganismo produtor de H2, Clostridium beijerinckii Br21, utilizando glicose como fonte de carbono e diferentes concentrações de cada inibidor. O efeito do HMF, do AL e do AF foram avaliados nas faixas de concentração de 0,5 a 2,5 g/L, de 1,0 a 4,0 g/L, e de 0,5 a 2,0 g/L, respectivamente. Retiraram-se amostras do gás produzido e do líquido para estimar as velocidades específicas de produção de H2, do crescimento celular e de consumo de glicose, nos ensaios com e sem a presença de inibidor (controle). Foi observada inibição de todos os parâmetros avaliados, comparados ao controle. Houve um aumento do tempo para início da produção e diminuição do rendimento de H2 com o aumento da concentração de todos os inibidores. Os resultados das fermentações permitiram estimar a concentração dos compostos que inibem 50% a produção de H2, o crescimento celular e o consumo do substrato (CI50). Os valores de CI50 obtidos para a produção de H2 pelo HMF, AL e AF foram 0,89, 2,50 e 1,15 g/L, respectivamente. Para o crescimento celular a CI50 do HMF, AL e AF foram 1,42, 2,08 e 1,46 g/L, respectivamente. Para o consumo de substrato a CI50 foi 3,23, 3,79 e 0,43 g/L, para o HMF, AL e AF, respectivamente. As concentrações de CI50 para a produção de H2 foram testados em 2 microrganismos distintos, o C. beijerinckii Br21 e o Clostridium acetobutylicum ATCC 824, para fins comparativos. Assim pode-se verificar a inibição na produção de H2 no C. beijerinckii Br21 de 49,3, 48,7 e 51,3%, enquanto que o C. acetobutylicum ATCC 824 apresentou inibição de 45,5, 61,3 e 59,6%, para o HMF, AL e AF, respectivamente. Foi estimada também a concentração de compostos que inibem 25% a produção de H2, a CI25, a fim de realizar misturas com os inibidores e testá-las em ambos os microrganismos. Os valores obtidos de CI25 para HMF, AL e AF foram 0,66, 2,15 e 0,89 g/L, respectivamente. A partir desses valores foram feitas 4 misturas distintas: HMF+AL, HMF+AF, AL+AF e HMF+AL+AF. A inibição da produção de H2 a partir dessas misturas em C. beijerinckii Br21foram de 58,9, 58,4, 49 e 85,9%, enquanto que para o C. acetobutylicum ATCC 824 obteve-se os valores de 67,6, 66,6, 55,5 e 88,8%, para HMF+AL, HMF+AF, AL+AF e HMF+AL+AF, respectivamente. Portanto, pode-se notar que o C. acetobutylicum ATCC 824 mostrou ser mais sensível aos efeitos causados pelos inibidores, sendo que o HMF parece atuar mais sobre a produção de H2, enquanto que os ácidos têm efeito mais global no metabolismo da bactéria. Esses estudos mostraram os limites destes compostos, quando se deseja utilizar hidrolisados de biomassas como matéria-prima para a produção fermentativa do H2.pelas espécies de Clostridium estudadas. / H2 can be obtained by biological processes, such as fermentation, conducted at ambient temperature and pressure. Renewable raw materials like lignocellulosic and algae biomass, which are rich in carbohydrates, can be used for this purpose. These types of biomass have complex chemical structures and require a pretreatment and/or hydrolysis step before they are used in fermentation. Hydrolysis may release not only monosaccharides but also potentially fermentation-inhibiting substances. This study evaluates how three potential fermentation inhibitors (5-hydroxymethylfurfural (HMF), levulinic acid-(LA), and formic acid (FA) derived from algal biomass hydrolysis affect H2 production. Kinetic batch fermentation assays were performed by using the H2-producing microorganism Clostridium beijerinckii Br21, glucose as carbon source, and different concentrations of each inhibitor. The effect of HMF, LA, and FA on H2 production was evaluated for inhibitor concentrations ranging from 0.5 to 2.5 g/L, 1.0 to 4.0 g/L, and 0.5 to 2.0 g/L, respectively. Samples of the produced gas and liquid were taken to estimate the specific rates of H2 production, cell growth, and glucose consumption in the assays conducted in the presence or in the absence (control) of an inhibitor. Increasing inhibitor concentration delayed the onset of H2 production and diminished the H2 yield. The fermentation results allowed us to estimate the inhibitor concentration that inhibited 50% of the H2 production, cell growth, and substrate consumption rates, designated IC50. Concerning the H2 production rate, IC50 was 0.89, 2.50, and 1.15 g/L for HMF, LA, and FA, respectively. As for the cell growth rate, IC50 was 1.42, 2.08, and 1.46 g/L for HMF, LA, and FA, respectively. Regarding the substrate consumption rate, IC50 was 3.23, 3.79, and 0.43 g/L for HMF, LA, and FA, respectively. IC50 was also tested in the presence of C. beijerinckii Br21 or Clostridium acetobutylicum ATCC 824 and one of the inhibitors. The H2 production rate decreased by 49.3, 48.7, and 51.3% in the presence of C. beijerinckii Br21 and of HMF, AL, or AF, respectively. In the presence of C. acetobutylicum ATCC 824 and of HMF, AL, or AF, the H2 production rate reduced by 45.5, 61.3, and 59.6%, respectively. The inhibitor concentration that inhibited 25% of H2 production, IC25, was also determined so that mixtures of the inhibitors could be prepared and tested in the presence of the microorganisms. HMF, LA, and FA afforded IC25 of 0.66, 2.15, and 0.89 g/L, respectively. On the basis of these values, four different mixtures were prepared: HMF+LA, HMF+FA, LA+FA, and HMF+LA+FA. In the presence of C. beijerinckii Br21, HMF+LA, HMF+FA, LA+FA, and HMF+LA+FA inhibited H2 production by 58.9, 58.4, 49, and 85.9%, respectively, whereas in the presence of C. acetobutylicum ATCC 824, inhibitions were 67.6, 66.6, 55.5, and 88.8% respectively. Therefore, C. acetobutylicum ATCC 824 was more sensitive to the effects caused by inhibitors. HMF seemed to affect the H2 production rate more, whereas acids appeared to act more globally on bacterial metabolism. These results reveal the concentration limits of the tested inhibitors when biomass hydrolysates are employed as raw material for fermentative H2 production.

O H2 pode ser obtido por processos biológicos, como a fermentação, conduzidos à temperatura e pressão ambientes. Para tal são utilizadas matérias-primas renováveis, ricas em carboidratos, como as biomassas lignocelulósicas e de algas. Estas biomassas têm estrutura química complexa e requerem uma etapa de pré-tratamento e/ou hidrólise antes da sua utilização na fermentação. Processos de hidrólise podem liberar, tanto monossacarídeos, quanto substâncias potencialmente inibidoras de fermentação. Esse estudo avaliou o efeito de 3 potenciais inibidores de fermentação (5-hidroximetilfurfural -HMF, ácido levulínico AL e ácido fórmico AF), derivados da hidrólise de biomassas. Ensaios cinéticos de fermentação em batelada foram realizados com o microrganismo produtor de H2, Clostridium beijerinckii Br21, utilizando glicose como fonte de carbono e diferentes concentrações de cada inibidor. O efeito do HMF, do AL e do AF foram avaliados nas faixas de concentração de 0,5 a 2,5 g/L, de 1,0 a 4,0 g/L, e de 0,5 a 2,0 g/L, respectivamente. Retiraram-se amostras do gás produzido e do líquido para estimar as velocidades específicas de produção de H2, do crescimento celular e de consumo de glicose, nos ensaios com e sem a presença de inibidor (controle). Foi observada inibição de todos os parâmetros avaliados, comparados ao controle. Houve um aumento do tempo para início da produção e diminuição do rendimento de H2 com o aumento da concentração de todos os inibidores. Os resultados das fermentações permitiram estimar a concentração dos compostos que inibem 50% a produção de H2, o crescimento celular e o consumo do substrato (CI50). Os valores de CI50 obtidos para a produção de H2 pelo HMF, AL e AF foram 0,89, 2,50 e 1,15 g/L, respectivamente. Para o crescimento celular a CI50 do HMF, AL e AF foram 1,42, 2,08 e 1,46 g/L, respectivamente. Para o consumo de substrato a CI50 foi 3,23, 3,79 e 0,43 g/L, para o HMF, AL e AF, respectivamente. As concentrações de CI50 para a produção de H2 foram testados em 2 microrganismos distintos, o C. beijerinckii Br21 e o Clostridium acetobutylicum ATCC 824, para fins comparativos. Assim pode-se verificar a inibição na produção de H2 no C. beijerinckii Br21 de 49,3, 48,7 e 51,3%, enquanto que o C. acetobutylicum ATCC 824 apresentou inibição de 45,5, 61,3 e 59,6%, para o HMF, AL e AF, respectivamente. Foi estimada também a concentração de compostos que inibem 25% a produção de H2, a CI25, a fim de realizar misturas com os inibidores e testá-las em ambos os microrganismos. Os valores obtidos de CI25 para HMF, AL e AF foram 0,66, 2,15 e 0,89 g/L, respectivamente. A partir desses valores foram feitas 4 misturas distintas: HMF+AL, HMF+AF, AL+AF e HMF+AL+AF. A inibição da produção de H2 a partir dessas misturas em C. beijerinckii Br21foram de 58,9, 58,4, 49 e 85,9%, enquanto que para o C. acetobutylicum ATCC 824 obteve-se os valores de 67,6, 66,6, 55,5 e 88,8%, para HMF+AL, HMF+AF, AL+AF e HMF+AL+AF, respectivamente. Portanto, pode-se notar que o C. acetobutylicum ATCC 824 mostrou ser mais sensível aos efeitos causados pelos inibidores, sendo que o HMF parece atuar mais sobre a produção de H2, enquanto que os ácidos têm efeito mais global no metabolismo da bactéria. Esses estudos mostraram os limites destes compostos, quando se deseja utilizar hidrolisados de biomassas como matéria-prima para a produção fermentativa do H2.pelas espécies de Clostridium estudadas. / H2 can be obtained by biological processes, such as fermentation, conducted at ambient temperature and pressure. Renewable raw materials like lignocellulosic and algae biomass, which are rich in carbohydrates, can be used for this purpose. These types of biomass have complex chemical structures and require a pretreatment and/or hydrolysis step before they are used in fermentation. Hydrolysis may release not only monosaccharides but also potentially fermentation-inhibiting substances. This study evaluates how three potential fermentation inhibitors (5-hydroxymethylfurfural (HMF), levulinic acid-(LA), and formic acid (FA) derived from algal biomass hydrolysis affect H2 production. Kinetic batch fermentation assays were performed by using the H2-producing microorganism Clostridium beijerinckii Br21, glucose as carbon source, and different concentrations of each inhibitor. The effect of HMF, LA, and FA on H2 production was evaluated for inhibitor concentrations ranging from 0.5 to 2.5 g/L, 1.0 to 4.0 g/L, and 0.5 to 2.0 g/L, respectively. Samples of the produced gas and liquid were taken to estimate the specific rates of H2 production, cell growth, and glucose consumption in the assays conducted in the presence or in the absence (control) of an inhibitor. Increasing inhibitor concentration delayed the onset of H2 production and diminished the H2 yield. The fermentation results allowed us to estimate the inhibitor concentration that inhibited 50% of the H2 production, cell growth, and substrate consumption rates, designated IC50. Concerning the H2 production rate, IC50 was 0.89, 2.50, and 1.15 g/L for HMF, LA, and FA, respectively. As for the cell growth rate, IC50 was 1.42, 2.08, and 1.46 g/L for HMF, LA, and FA, respectively. Regarding the substrate consumption rate, IC50 was 3.23, 3.79, and 0.43 g/L for HMF, LA, and FA, respectively. IC50 was also tested in the presence of C. beijerinckii Br21 or Clostridium acetobutylicum ATCC 824 and one of the inhibitors. The H2 production rate decreased by 49.3, 48.7, and 51.3% in the presence of C. beijerinckii Br21 and of HMF, AL, or AF, respectively. In the presence of C. acetobutylicum ATCC 824 and of HMF, AL, or AF, the H2 production rate reduced by 45.5, 61.3, and 59.6%, respectively. The inhibitor concentration that inhibited 25% of H2 production, IC25, was also determined so that mixtures of the inhibitors could be prepared and tested in the presence of the microorganisms. HMF, LA, and FA afforded IC25 of 0.66, 2.15, and 0.89 g/L, respectively. On the basis of these values, four different mixtures were prepared: HMF+LA, HMF+FA, LA+FA, and HMF+LA+FA. In the presence of C. beijerinckii Br21, HMF+LA, HMF+FA, LA+FA, and HMF+LA+FA inhibited H2 production by 58.9, 58.4, 49, and 85.9%, respectively, whereas in the presence of C. acetobutylicum ATCC 824, inhibitions were 67.6, 66.6, 55.5, and 88.8% respectively. Therefore, C. acetobutylicum ATCC 824 was more sensitive to the effects caused by inhibitors. HMF seemed to affect the H2 production rate more, whereas acids appeared to act more globally on bacterial metabolism. These results reveal the concentration limits of the tested inhibitors when biomass hydrolysates are employed as raw material for fermentative H2 production.

Statistically based experimental designs were applied to optimise medium constituent for biogas production utilizing N-­‐acetylglucosamine as a carbon source for Clostridium beijerinckii and Clostridium paraputrificum. The important medium constituents influencing total biogas produced, identified by the Plackett and Burman method, were FeSO4.7H2O and initial pH for C. beijerinckii cultures whilst for C. paraputrificum cultures N-­‐acetylglucosamine, L-­‐ cysteine.HCl.H2O and MgCl2. A one factor L-­‐cysteine.HCl.H2O optimization design was applied to investigate the ideal concentration of L-­‐cysteine.HCl.H2O required to achieve an anaerobic environment for optimum C. beijerinckii total biogas production. The Method of Steepest Ascent was then employed to locate the optimal area of the significant medium variables. Using the Box-­‐behnken method, experimental results showed that there were significant linear effects of independent variables, N-­‐acetylglucosamine for C. beijerinckii cultures and for C. paraputrificum cultures N-­‐acetylglucosamine, L-­‐cysteine.HCl.H2O and MgCl2 on total biogas volume. Significant curvature or quadratic effects of N-­‐ acetylglucosamine and L-­‐cysteine.HCl.H2O were identified for C. paraputrificum cultures. There were no significant interaction effects between medium constituent variables on resulting biogas volume. The optimal conditions for the maximum volume of biogas produced for C. beijerinckii cultures were 21 g/l of N-­‐ acetylglucosamine, 0.1 g/l of FeSO4.7H2O and initial pH of 6.11 and for C. paraputrificum were 29 g/l of N-­‐acetylglucosamine, 0.27 g/l of L-­‐ cysteine.HCl.H2O and 0.4 g/l of MgCl2. Using this statistical optimization strategy, the total biogas volume from N-­‐acetylglucosamine utilization increased from 150 ml/l to 6533 ml /l in the C. beijerinckii cultures and 100 ml/l to 5350 ml/l in the C. paraputificum cultures. The maximum yield of bio-­‐hydrogen by C. paraputrificum from N-­‐acetylglucosamine was 2.55 mol of H2 / mol of N-­‐ acetylglucosamine and by C. beijerinckii was 2.43 mol of H2 / mol of N-­‐ acetylglucosamine.