Two stage biodiesel and hydrogen production from molasses by oleaginous fungi and Clostridium acetobutylicum ATCC 824

In the present study biodiesel was produced by various fungal species isolated from Egypt using sugarcane molasses as substrate. In the first stage 6 oleaginous fungi, namely, Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, Fusarium oxysporum, Aspergillus parasiticus and Emericella nidulans var. lata were used for lipid production. Subsequent to fungal cultivation on sugarcane molasses the cultures were filtered and biodiesel was prepared by direct esterification of dry fungal biomass. Methyl esters of palmitic, stearic, linoleic and elaidic represented the major components while palmitoleic represented a minor component of biodiesel produced from tested oleaginous fungi. In the second stage, the spent medium of fungal culture was used as the fermentation medium for hydrogen production by Clostridium acetobutylicum ATCC 824. The maximum total H₂ yield was obtained with the spent medium of E. nigrum and A. alternata. The results presented in this study suggest a possibility of interlinking the biodiesel production technology by fungi with hydrogen production by C. acetobutylicum ATCC 824 to exploit the residual sugars in the spent media and therefore increase the economic feasibility of the biofuel production from molasses.     

Dark and photofermentation H2 production from hydrolyzed biomass of the potent extracellular polysaccharides producing cyanobacterium Nostoc commune and intracellular polysaccharide (glycogen) enriched Anabaena variabilis NIES-2095

The biological H₂ production industry would be independent from other industries if it has its own supply of organic materials especially in non-agricultural countries. In this study, acid hydrolyzed biomass of the potent extracellular polysaccharides (EPSs) producing cyanobacterium Nostoc commune and glycogen (as intracellular polysaccharide) enriched Anabaena variabilis NIES-2095 were used as a cheap organic carbon feedstock for biological H₂ production by two stages dark fermentation by Escherichia coli strain MWW and Clostridium acetobutylicum DSM-792 or Clostridium beijerinckii DSM-1820 and photofermentation by Rhodobacter capsulatus JCM-21090 under anaerobic conditions. Acid hydrolysis of air dried cyanobacterial biomass was conducted at optimum conditions of 4 M HCl at 120 °C in an autoclave for 30 min and subsequently neutralized and used as an organic carbon source for first stage dark fermentation followed by a second stage photofermentation. The facultative anaerobe Escherichia coli strain MWW was used for maintaining anaerobiosis. Escherichia coli strain MWW was isolated and identified by morphological and biochemical characterizations as well as molecular biological phylogenetic analysis of its 16S rDNA sequence. Nostoc commune was identified by morphological and microscopic characterizations and by 16S rDNA sequence phylogenetic analysis. The two stages dark fermentation by Escherichia coli and Clostridium acetobutylicum or Clostridium beijerinckii and photofermentation by Rhodobacter capsulatus produced in total 5.9 and 5.6 mol H₂/mole reducing sugars of acid hydrolyzed Nostoc commune EPSs/biomass, respectively and 5.43 and 5 mol H₂/mole reducing sugars of acid hydrolyzed biomass of glycogen enriched Anabaena variabilis, respectively. These results indicate a high potency of using cyanobacterial polysaccharides/biomass (extracellular polysaccharides and intracellular glycogen) as an organic carbon source for H₂ production which would be of importance for non-agricultural countries.     

Mathematical modelling for biohydrogen production by Clostridium beijerinckii

Hydrogen is an energy source that can be produced by Clostridium sporogenes microorganism. In the present work, modeling of dark fermentation using Clostridium beijerinckii and dextrose as substrate was performed to evaluate how the gases and liquid by-products affect the biological process. A mathematical model was developed according to ADM1. The developed model takes into account biochemical reactions, physicochemical equilibrium as well as mass transfer processes during dark fermentation. Findings revealed that Clostridium beijerinckii reached a yield as high as 3.58 mol of H₂/mol of dextrose and generates by-products in the aqueous phase that may either be used as raw materials in a chemical process. Clostridium beijerinckii is very sensitive to acid media (pH < 5.0) and shows a low rate of biohydrogen production (even the absence of metabolic activity) at pH lower than 4.5. The developed model is able to predict (R² > 0.95) dextrose consumption profile, cumulative biohydrogen production and the maximum concentrations of liquid by-products.     

Hydrogen production by Clostridium acetobutylicum ATCC 824 and megaplasmid-deficient mutant M5 evaluated using a large headspace volume technique

Biohydrogen production is measured using a variety of techniques, ranging from low cost intermittent gas release methods where yields are usually reduced due to high partial pressures of hydrogen, to expensive respirometers that can eliminate pressure buildup. A new large headspace volume technique was developed that reduces the potential for hydrogen gas inhibition without the need for a respirometer. We tested this method with two strains of clostridia, Clostridium acetobutylicum ATCC 824 and its mutant M5 that lacks a megaplasmid responsible for butanol and acetone production, and a mixed culture (heat-treated sludge). The hydrogen yield using M5 (2.64 mol-H₂/mol-glucose) was 47% higher than that of the parent strain (1.79 mol-H₂/mol-glucose), and 118% larger than that obtained in tests with the sludge inoculum (1.21 mol-H₂/mol-glucose). The increased yield for M5 was primarily due to a decrease in biomass synthesis (38%) compared to the parent strain. Hydrogen yields measured using this new method were on average 14% higher than those obtained using a conventional respirometric method. These findings indicate enhanced biohydrogen production from the megaplasmid-deficient mutant of C. acetobutylicum ATCC 824, and that an intermittent gas-sampling technique can effectively measure high hydrogen gas by using a large headspace volume.     

Molecular characterization and homologous overexpression of [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1

The H₂-evoving [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1 was isolated to elucidate molecular characterization and modular structure of the hydrogenase. Then, homologous overexpression of the hydrogenase gene was for the first time performed to enhance hydrogen production. The hydA open reading frame (ORF) was 1734-bp, encodes 577 amino acids with a predicted molecular mass of 63,970 Da, and presents 80% and 75% identity at the amino acid level with the [FeFe]-hydrogenase genes of Clostridium kluyveri DSM 555 and Clostridium acetobutylicum ATCC 824, respectively. One histidine residue and 19 cysteine residues, known to fasten one [2Fe–2S] cluster, three [4Fe–4S] clusters and one H-cluster, were conserved in hydA of C. tyrobutyricum.     

Fermentative production of hydrogen from cassava processing wastewater by Clostridium acetobutylicum

This work reports on the effect of initial substrate concentration on COD consumption, pH, and H₂ production during cassava processing wastewater fermentation by Clostridium acetobutylicum ATCC 824. Five initial COD wastewater concentrations, namely 5.0, 7.5, 10.7, 15.0, and 30.0 g/L, were used. The results showed that higher substrate concentrations (30.0 and 15.0 COD/L) led to lower H₂ yield as well as less efficient substrate conversion into H₂. On the other hand, initial COD concentrations of 10.7, 7.5 and 5 g/L furnished 1.34, 1.2 and 2.41 mol H₂/mol glucose, with efficiency of glucose conversion into H₂ of 34, 30, and 60% (mol/mol), respectively. These results demonstrate that cassava processing wastewater, a highly polluting effluent, can be successfully employed as substrate for H₂ production by C. acetobutylicum at lower COD concentrations.     

Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization

This work evaluated the effects of individual alkaline, sodium carbonate (Na₂CO₃ denoted as; NaC), sodium sulfide (Na₂SO₃ denoted as; NaS) and combination of NaC + NaS pretreatment for the saccharification of sugarcane bagasse (SCB). The effects of different pretreatments on chemical composition and structural complexity of SCB in relation with its saccharification were investigated. For enzymatic hydrolysis of pretreated SCB we have utilized the produced crude enzymes by Streptomyces sp. MDS to make the process more cost effective. A enzyme dose of 30 filter paperase (FPU) produced a maximum reducing sugar (RS) 592 mg/g with 80.2% hydrolysis yield from NaC + NaS pretreated SCB under optimized conditions. The resulted enzymatic hydrolysates of each pretreated SCB were applied for hydrogen production using Clostridium beijerinckii KCTC1785. NaC + NaS pretreated SCB hydrolysates exhibited maximum H₂ production relative to other pretreatment methods. Effects of temperature, initial pH of culture media and increasing NaC + NaS pretreated SCB enzymatic hydrolysates concentration (2.5–15 g/L) on bioH₂ production were investigated. Under the optimized conditions, the cumulative H₂ production, H₂ production rate, and H₂ yield were 1485 mL/L, 61.87 mL/L/h and 1.24 mmol H₂/mol of RS (0.733 mmol H₂/g of SCB), respectively. The efficient conversion of the SCB hydrolysate to H₂ without detoxification proves the viability of process for cost-effective hydrogen production.     

Distinct effects of furfural,hydroxymethylfurfural and its mixtures on dark fermentation hydrogen production and microbial structure of a mixed culture

Past research has suggested that furfural and hydroxymethylfurfural (HMF) present in lignocellulosic hydrolyzates exert a synergistic effect on dark fermentative H₂ production, which has not adequately proven in microbial consortia. It is possible that some members of a consortium experience less inhibition than others, helping to the entire consortium to overcome the inhibition. To elucidate the type of inhibitory effect that these agents exert, the objective of this study was to contrast the individual impacts of furfural and HMF with the corresponding mixtures at the same concentration threshold on the hydrogen production and the relative abundance of different members in the consortium. Heat-treated anaerobic granules served as inoculum to ferment xylose for hydrogen production in presence of furfural (0.10, 0.50, and 1.00 g/L), HMF (0.02, 0.09, 0.19 g/L) and the corresponding mixtures in comparison with a control in absence of these agents. Furfural alone did not inhibit the hydrogen production; indeed, the inoculum completely degraded furfural at all its concentrations with the presence of furoic acid.HFM was partially degraded in the treatments with the lowest/middle concentrations, resulting in higher hydrogen production than the control. In contrast, at the highest HMF concentration, the inoculum was unable to remove it resulting in the strongest inhibition of hydrogen production. All the F/HMF mixtures had an inhibitory effect on the hydrogen production. A log2 fold change analysis of pyrosequencing reads evidenced that these agents promoted the growth of Lactobacillus, and clostridia species such as Clostridium butyricum, Clostridium bifermentans, and Clostridium sartagoforme, suggesting their active participation in the detoxification process.     

Glycerol and mixture of carbon sources conversion to hydrogen by Clostridium beijerinckii DSM791 and effects of various heavy metals on hydrogenase activity

Hydrogen is a carbon-neutral energy feedstock which is produced during fermentation of various carbon sources. The genomes of clostridia encode mainly [Fe-Fe]-hydrogenases. Clostridium beijerinckii DSM791 performed anaerobic fermentation of glycerol in batch culture at pH 7.5 and pH 5.5 and produced H₂. At pH 7.5, the glycerol consumption rate was 3.7 g/g cell mass/h, which was higher than that at pH 5.5. H₂ production reached 5 mmol/h/g cell mass at pH 7.5. The specific hydrogenase activity was ～1.4 fold higher if cells were grown on glycerol compared to cells grown on glucose. Single (Fe²⁺, Fe³⁺, Ni²⁺) or mixed supply of metals (Fe²⁺ and Ni²⁺) increased the specific hydrogenase activity by ～50%. These results suggest that C. beijerinckii DSM791 could be used as a potential H₂ producer. It may help to further enhance H₂ production using different industrial or agricultural wastes where glycerol and other carbon sources are present.     

Performance of batch solid state fermentation for hydrogen production using ground wheat residue

Ground wheat (21 g) was subjected to batch solid state dark fermentation for bio-hydrogen production. Clostridium acetobutylicum (B-527) was used as the culture of dark fermentation bacteria at mesophilic conditions. Effects of moisture content on the rate and yield of bio-hydrogen formation were investigated. The highest CHF (1222 ml), hydrogen yield (63 ml H₂ g^?1 starch), formation rate (10.64 ml H₂ g^?1 starch h^?1) and specific hydrogen formation rate (0.28 ml H₂ g^?1 biomass h^?1) were obtained with a moisture content of 80%. Nearly complete starch hydrolysis and glucose fermentation were achieved with more than 80% moisture content and the highest substrate conversion rate (21.9 mg L^?1 h^?1) was obtained with 90% moisture content at batch solid state fermentation producing volatile fatty acids (VFA) and H₂.     

Enhancement of Escherichia coli bacterial biomass and hydrogen production by some heavy metal ions and their mixtures during glycerol vs glucose fermentation at a relatively wide range of pH

Escherichia coli growth and H₂ production were followed in the presence of heavy metal ions and their mixtures during glycerol or glucose fermentation at pH 5.5–7.5. Ni²⁺ (50 μM) with Fe²⁺ (50 μM) but not sole metals stimulated bacterial biomass during glycerol fermentation at pH 6.5. Ni²⁺+Fe³⁺ (50 μM), Ni² +Fe³⁺+Mo⁶⁺ (20 μM) and Fe³⁺+Mo⁶⁺ (20 μM) but not sole metals enhanced up to 3-fold H₂ yield but Cu⁺ or Cu²⁺ (100 μM) inhibited it. At pH 7.5 stimulating effect on biomass was observed by Ni²⁺+Fe²⁺+Mo⁶⁺. H₂ production was enhanced 2.7 fold particularly by Ni²⁺+Fe³⁺+Mo⁶⁺ at the late stationary growth phase. Whereas at pH 5.5 increased biomass was when Fe²⁺+Mo⁶⁺ or Mo⁶⁺ were added. H₂ yield was decreased compared with that at pH 6.5, but metal ions again enhanced it. During glucose fermentation at pH 6.5 biomass was increased by the mixtures of metal ions, and 1.2 fold increased H₂ yield was observed. At pH 7.5 Ni²⁺+Fe²⁺ increased biomass but Cu⁺ or Cu²⁺ had suppressing effect; Fe³⁺+Mo⁶⁺ stimulated H₂ production. At pH 5.5 biomass also was raised by Ni²⁺+Fe²⁺+Mo⁶⁺; H₂ yield was increased upon Mo⁶⁺ and Mo⁶⁺+Fe²⁺ or Mo⁶⁺+Fe³⁺ additions. The results point out the importance of Ni²⁺, Fe²⁺, Fe³⁺ and Mo⁶⁺ and some of their combinations for E. coli bacterial growth and H₂ production mostly during glycerol but not glucose fermentation and at acidic conditions (pH 5.5 and 6.5). They can be used for optimizing fermentation processes on glycerol, controlling bacterial biomass and developing H₂ production biotechnology.     

Consolidated bioprocessing of hydrogen production from agave biomass by Clostridium acetobutylicum and bovine ruminal fluid

The biological production of H₂ represents a renewable and eco-friendly energy alternative compared to fossil fuels. However, its production from lignocellulose involves the use of expensive enzymatic complexes. In the present work, the production of H₂ from pretreated agave biomass was evaluated by means of a Consolidated Bioprocess (CBP). This strategy was carried through the interaction of cellulose-degrading microorganisms obtained from bovine ruminal fluid (BRF) capable of enhancing H₂ production by Clostridium acetobutylicum. The results obtained show the capacity of BRF to hydrolyze the acid pretreated agave, improving the production of H₂ in the experiments where the inoculum of Clostridium was greater. According to the results, production of H₂ is significantly affected by the increase of the solids loading, obtaining a maximum H₂ production at a 10% of solids loading, pH 5.5 and 35 °C, representing a yield of 150 L of H₂ per Kg of biomass in 264 h.     

Effect of acid-pretreatment on hydrogen fermentation of food waste: Microbial community analysis by next generation sequencing

This work presents the effect of acid-pretreatment on H₂ fermentation of food waste with detailed microbial information by next generation sequencing. The pretreated food waste at pH 1.0–4.0 was cultivated under mesophilic conditions without external inoculum addition. From the food waste acid-pretreated at pH 1–3, H₂ yields in the range of 1.37–1.74 mol H₂/mol hexose_added were achieved, attaining the highest value at pH 2. Clostridium sp. such as Clostridium acetobutylicum ATCC 824 and Clostridium perfringens occupied more than 70% of total number of sequences at pH 1–3. On the other hand, in the control (no pretreatment) and at pH 4, lactic acid bacteria such as Lactobacillus and Streptococcus were found to be the dominant genus (>90% of total number of sequences), resulting in a low H₂ yield. In addition, the effect of substrate concentration on H₂ fermentation was investigated, and the maximum H₂ productivity was estimated to be 27.2 L H₂/L/d by Andrew's model.     

Biological hydrogen production of the genus Clostridium: Metabolic study and mathematical model simulation

The biochemical hydrogen potential (BHP) tests were conducted to investigate the metabolism of glucose fermentation and hydrogen production performance of four Clostridial species, including C. acetobutylicum M121, C. butyricum ATCC19398, C. tyrobutyricum FYa102, and C. beijerinckii L9. Batch experiments showed that all the tested strains fermented glucose, reduced medium pH from 7.2 to a value between 4.6 and 5.0, and produced butyrate (0.37–0.67 mmol/mmol-glucose) and acetate (0.34–0.42 mmol/mmol-glucose) as primary soluble metabolites. Meanwhile, a significant amount of hydrogen gas was produced accompanied with glucose degradation and acid production. Among the strains examined, C. beijerinckii L9 had the highest hydrogen production yield of 2.81 mmol/mmol-glucose. A kinetic model was developed to evaluate the metabolism of glucose fermentation of those Clostridium species in the batch cultures. The model, in general, was able to accurately describe the profile of glucose degradation as well as production of biomass, butyrate, acetate, ethanol, and hydrogen observed in the batch tests. In the glucose re-feeding experiments, the C. tyrobutyricum FYa102 and C. beijerinckii L9 isolates fermented additional glucose during re-feeding tests, producing a substantial amount of hydrogen. In contrast, C. butyricum ATCC19398 was unable to produce more hydrogen despite additional supply of glucose, presumably due to the metabolic shift from acetate/butyrate to lactate/ethanol production.     

Investigation of the interaction between intermediates from gasification of biomass in supercritical water: Formaldehyde/formic acid mixtures

Supercritical water gasification (SCWG) is a promising technology for converting wet biomass and waste into renewable energy. While the fundamental mechanism involved in SCWG of biomass is not completely understood, especially hydrogen (H₂) production produced from the interaction among key intermediates. In the present study, formaldehyde mixed with formic acid as model intermediates were tested in a batch reactor at 400 °C and 25 MPa for 30 min. The gas and liquid phases were collected and analyzed to determine a possible mechanism for H₂ production. Results clearly showed that both gasification efficiency (GE) and hydrogen efficiency (HE) increased with addition of formic acid, and the maximum H₂ yield reached 17.92 mol/kg with a relative formic acid content of 66.67% in the mixtures. The total organic carbon removal rate and formaldehyde conversion rate also increased to 67.33% and 89.81% respectively. The reaction pathways for H₂ formation form mixtures was proposed and evaluated, formic acid promoted self-decomposition of formaldehyde to generate H₂, and induced a radical reaction of generated methanol to produce more H₂.     

Prolongation of H2 production during mixed carbon sources fermentation in E. coli batch cultures: New findings and role of different hydrogenases

Hydrogen (H₂) gas production in batch cultures was studied upon utilization of the mixture of glucose, glycerol and formic acid by Escherichia coli BW25113 wild type (wt) at pH of 5.5–7.5. At pH 7.5H₂ was continuously produced during 240 h but at pH 6.5 and 5.5 it was detected till 168 h and 120 h, respectively. Specific growth rate (μ) of wt was the highest (1.05 h^?1) at pH 6.5. Moreover, at pH 5.5 in hycE μ decreased by ～4.14 fold compared to wt, suggesting major role of Hyd-3 in cell growth. H₂ yield (8.8 mmol H₂ L^?1) was the highest at pH 7.5. In hybC H₂ yield was increased ～1.62 fold than in wt. These data might be applied for biomass and biohydrogen production from various organic wastes where mixtures of carbon sources are present.     

Hydrogen production from acid and enzymatic oat straw hydrolysates in an anaerobic sequencing batch reactor: Performance and microbial population analysis

Feasibility of hydrogen production from acid and enzymatic oat straw hydrolysates was evaluated in an anaerobic sequencing batch reactor at 35 °C and constant substrate concentration (5 g chemical oxygen demand/L). In a first experiment, hydrogen production was replaced by methane production. Selective pressures applied in a second experiment successfully prevented methane production. During this experiment, initial feeding with glucose/xylose, as model substrates, promoted biomass granulation. Also, the highest hydrogen molar yield (HMY, 2 mol H₂/mol sugar consumed) and hydrogen production rate (HPR, 278 mL H₂/L-h) were obtained with these model substrates. Gradual substitution of glucose/xylose by acid hydrolysate led to disaggregation of granules and lower HPR and HMY. When the model substrates were completely substituted by enzymatic hydrolysate, the HMY and HPR were 0.81 mol H₂/mol sugar consumed and 29.6 mL H₂/L-h, respectively. Molecular analysis revealed a low bacterial diversity in the stages with high hydrogen production and vice versa. Furthermore, Clostridium pasteurianum was identified as the most abundant species in stages with a high hydrogen production. Despite that feasibility of hydrogen production from hydrolysates was demonstrated, lower performance from hydrolysates than from model substrates was obtained.     

Anthrahydroquinone-2,6-disulfonate increases the rate of hydrogen production during Clostridium beijerinckii fermentation with glucose,xylose, and cellobiose

Fermentative hydrogen production is considered a reasonable alternative for generating H₂ as an energy carrier for electricity production using hydrogen fuel cells. The kinetics of hydrogen production from glucose, xylose and cellobiose were investigated using pure culture Clostridium beijerinckii NCIMB 8052. Adding anthrahydroquinone-2,6-disulfonate (AH₂QDS) at concentrations ranging from 100 μM to 500 μM increased the hydrogen production rates from 0.80 to 1.35 mmol/L-hr to 1.20–2.70 mmol/L-hr with glucose, xylose, or cellobiose as the primary substrates. AH₂QDS amendment also increased the substrate utilization rate and biomass growth rate by at least two times. These findings suggest that adding hydroquinone reducing equivalents influence cellular metabolism with hydrogen production rate, substrate utilization rate, and growth rate being simultaneously affected. Resting cell suspensions were conducted to investigate the influence of AH₂QDS on the hydrogen production rate from glyceraldehyde 3-phosphate, which is a shared intermediate in both glycolysis and pentose phosphate pathway. Data demonstrated that hydrogen production rate increased by 1.5 times when glyceraldehyde 3-phosphate was the sole carbon source, suggesting that the hydroquinone may alter reactions starting with or after glyceraldehyde 3-phosphate in central metabolism. These data demonstrate that adding hydroquinones increased overall metabolic activity of C. beijerinckii. This will eventually increase the efficiency of industrial scale production once appropriate hydroquinone equivalents are identified that work well in large-scale operations, since fermentation rate is one of the two critical factors (production rate and yield) influencing efficiency and cost.