Fermentative H2 Research Articles

Kinetic studies for the preparation of probiotic cabbage juice: Impact on phytochemicals and bioactivity

A kinetic study for the production of probiotic cabbage juice was carried out under controlled pH and dissolved oxygen using several strains of lactic acid bacteria (LAB). Furthermore, effect of probiotic fermentation on polyphenolic content and antioxidant capacity was investigated. Results showed significant growth in Lactobacillus brevis, Lactobacillus plantarum and Lactobacillus rhamnosus corresponding to 9.19, 9.47 and 10.6logCFU/ml after 24h of fermentation, which is satisfying criteria for a food product to be characterized as probiotic. Lactic acid (LA) was the major end product of the fermented cabbage juice attaining the concentrations of 6.97, 9.69 and 12.2g/l LA for L. plantarum, L. rhamnosus and L. brevis, respectively. LAB fermentation retains more than 75% of total phenolic content (TPC) and total flavonoid content (TFC) of the initial raw material, and similar set of results were observed for antioxidant capacity. First-order kinetics model fitted well with the experimental data with R2 value ranging from 0.92 to 0.96, 0.96 to 0.98 and 82.2 to 97.2 for TPC, TFC and antioxidant capacity, respectively. During refrigerated storage (4°C), all the probiotic cultures met the criterion of maintaining counts greater than 8logCFU/ml; in addition to maintaining bioactive components and antioxidant capacity.

Efficient direct ethanol production from cellulose by cellulase- and cellodextrin transporter-co-expressing Saccharomyces cerevisiae

Efficient degradation of cellulosic biomass requires the synergistic action of the cellulolytic enzymes endoglucanase, cellobiohydrolase, and β-glucosidase. Although there are many reports describing consolidation of hydrolysis and fermentation steps using recombinant Saccharomyces cerevisiae that express cellulolytic enzymes, the efficiency of cellulose degradation has not been sufficiently improved. Although the yeast S. cerevisiae cannot take up cellooligosaccharide, some fungi can take up and assimilate cellooligosaccharide through a cellodextrin transporter. In this study, a S. cerevisiae strain co-expressing genes for several cell surface display cellulases and the cellodextrin transporter was constructed for the purpose of improving the efficiency of direct ethanol fermentation from phosphoric acid swollen cellulose (PASC). The cellulase/cellodextrin transporter-coexpressing strain produced 1.7-fold more ethanol (4.3 g/L) from PASC during a 72-h fermentation than did a strain expressing cellulase only (2.5 g/L). Direct ethanol production from PASC by the recombinant S. cerevisiae strain was improved by co-expression of cellulase display and cellodextrin transporter genes. These results suggest that cellulase- and cellodextrin transporter-co-expressing S. cerevisiae could be a promising technology for efficient direct ethanol production from cellulose.

Alkali-treated sewage sludge as a seeding source for hydrogen fermentation of food waste leachate

In the present work, alkali-treated sewage sludge (SS) was used as a seeding source for H2 fermentation of food waste leachate (FWL). The role of alkaline treatment of SS was to suppress the activity of non-H2-producers in SS and also to enhance the solubility of SS. The effect of pretreatment pH and FWL:SS ratio on H2 production was crucial, by changing the pH conditions and selecting the dominant species. High pretreatment pH and high SS content resulted in high initial pH conditions. The highest H2 yield of 2.1 mol H2/mol hexoseadded was achieved at pretreatment pH 10 and a mixing ratio of FWL:SS = 3:5. At these conditions, the initial pH was 7.9, and cultivation pH was maintained within the reported optimum range of 5.5–6.5 during fermentation. It was found that pretreatment pH 9 was not strong enough to suppress the activity of non-H2-producers in SS, in particular, lactic acid bacteria (LAB). Microbial analysis results confirmed that LAB such as Lactobacillus sp. and Enterococcus sp. were the dominant species at pretreatment pH 9 while Clostridium sp., the main anaerobic H2-producers, were dominant at pretreatment pH 10.

Rapid formation of hydrogen-producing granules in an up-flow anaerobic sludge blanket reactor coupled with high-rate recirculation

Application of an up-flow anaerobic sludge blanket (UASB) reactor to dark fermentative H2 production greatly improves H2 productivity due to the maintenance of high biomass concentration. However, a long start-up HRT and start-up period are required to develop the H2-producing granules (HPGs) and to avoid washing out the suspended sludge at the start of the process. In the present work, a novel strategy to rapidly form HPGs was developed in UASB reactor. To induce highly active mass transfer in the UASB reactor, a high recirculation rate (15 times the influent) was adopted over 10 days, then recirculation was stopped. As the operation progressed, self-flocculation took place and HPGs developed after 90 h of operation. A stable production of H2 was observed after 20 days of operation. The thickness of the HPGs layer in the sole UASB reactor increased progressively, and consequently the average HPG diameter and concentration were 1.86 mm (0.1–3.9 mm) and 52 g/L, respectively, after 60 days of operation. These findings seem to suggest that high-rate recirculation plays a crucial role in accelerating the formation of HPGs in such UASB reactors through high up-flow velocity, providing active mass transfer.

Influence of preprocessing methods and fermentation of adzuki beans on γ-aminobutyric acid (GABA) accumulation by lactic acid bacteria

The effects of pre-processes (immersing, germinating, and cold shock) and fermentation conditions of adzuki beans on γ-aminobutyric acid (GABA) accumulation using mixed cultures of Lactococcus lactis and Lactobacillus rhamnosus were investigated in this study. Among the preprocessing methods, cold shock treatment resulted in the highest observed GABA content (201.2mg/100g); a 150-fold increase compared to the non-treated adzuki beans. The LAB strains grew rapidly in cold-treated adzuki bean substrates and reached 108cfu/ml after 24h of fermentation at 30°C. After optimization, the GABA yield reached 68.2mg/100ml; a 20-fold increase compared to the non-fermentation yield. The viable cell counts of LAB remained above 108cfu/ml after 28days of storage at 4°C. Our results suggest that the combination of cold shock pretreatment and fermentation by LAB may be used for the preparation of adzuki beans with high GABA content, which can then be used as a natural resource of functional foods.

Hydrogen and methane production from untreated rice straw and raw sewage sludge under thermophilic anaerobic conditions

Bioenergy produced from co-digestion of sewage sludge (SS) and rice straw (RS) as raw materials, without pretreatment and additional nutrients, was compared for the one-stage system for producing methane (CH4) and the two-stage system for combined production of hydrogen (H2) and CH4 in batch experiments under thermophilic conditions. In the first stage H2 fermentation process using untreated RS with raw SS, we obtained a high H2 yield (21 ml/g-VS) and stable H2 content (60.9%). Direct utilization of post-H2 fermentation residues readily produced biogas, and significantly enhanced the CH4 yield (266 ml/g-VS) with stable CH4 content (75–80%) during the second stage CH4 fermentation process. Overall, volatile solids removal (60.4%) and total bioenergy yield (8804 J/g-VS) for the two-stage system were 37.9% and 59.6% higher, respectively, than the one-stage system. The efficient production of bioenergy is believed to be due to a synergistically improved second stage process exploiting the well-digested post-H2 generation residues over the one-stage system.

Improvement of acetone, butanol and ethanol production from rice straw by acid and alkaline pretreatments

Rice straw was hydrolyzed and fermented to acetone, butanol, and ethanol by Clostridium acetobutylicum bacterium. Concentrated phosphoric acid and alkaline treatment with NaOH were used for pretreatment of the straw prior to enzymatic hydrolysis using commercial cellulase and β-glucosidase. The enzymatic hydrolysates were then anaerobically fermented by C. acetobutylicum. Hydrolysis of the alkaline pretreated straw resulted in production of 163.5g glucose from each kg of untreated rice straw which was then fermented to 45.2g butanol, 17.7g acetone, and 1.2g ethanol. Additionally, concentrated phosphoric acid pretreatment and subsequent hydrolysis resulted in production of 192.3g glucose from each kg straw from which 44.2g butanol, 18.2g acetone, and 0.6g ethanol were produced after 72h fermentation. Increasing the produced ABE from less than 10g to higher than 62g from each kg straw by the treatments suggested the alkaline and phosphoric acid pretreatments as promising processes for efficient production of ABE from rice straw.

Application of a simple method to reduce the start-up period in a H2-producing UASB reactor using xylose

The major objective of dark fermentative H2 production (DFHP) is the significant enhancement of the H2 production performance from economic and engineering perspectives. An up-flow anaerobic sludge blanket (UASB) reactor based on H2-producing granules (HPGs) has been introduced, which results in the efficiency of the H2 production being improved significantly. However, the long start-up period remains a substantial obstacle in the stable operation of the UASB reactor. In the present study, a newly proposed operational strategy was employed to form the HPG using xylose as the feedstock, and this resulted in a successful reduction of the start-up period of the UASB reactor. After the mixed liquor in the continuously stirred tank reactor was transferred to the UASB reactor as a seeding source, the height of the HPG in the UASB reactor was progressively increased. In the UASB reactor, a maximum H2 yield of 2.98 mol H2/mol xylose and a stable H2 production rate of 9.98 L H2/L/h were observed at a hydraulic retention time of 6 h and a substrate concentration of 30 g COD/L. In this novel method, HPGs with an average particle size of 2.32 mm (0.1–4.7 mm) were successfully developed after 120 days and this is the first report documenting the successful formation of HPGs using xylose as feedstock in a UASB reactor.

Collection of some microbial consortia producing hydrogen from anaerobic wastes

The preparation of hydrogen-producing microbial consortia from three anaerobic digested sludges were carried out by four different pretreatment methods (heat – shock, acid, base and aeration treatment) as well as untreatment. The obtained microbial seeds have been estimated for their stability in fermentative hydrogen production by three consecutive batch fermentations under the same conditions of pH 6.5, room temperature and cultivation time and also investigated the H2 fermentation from different concentrations of glucose and xylose. Three microbial seeds have the most effective H2 production at 5 g/l of glucose or xylose after 48 h cultivation time. The sewage sludge pretreated at 80oC for 30 minutes shows the hydrogen yield of 1.27 mol/mol glucose and 0.82 mol/mol xylose. The sludge in the biogas tank pretreated at 60oC for 30 minutes has the hydrogen yield of 1.27 mol/mol glucose and 0.71 mol/mol xylose. The sludge of the Hoa Binh waste treatment plant pretreated at 60oC for 30 minutes presents the hydrogen yield of 1.31 mol/mol glucose and 0.66 mol/mol xylose.

The influence of slaughterhouse waste on fermentative H2 production from food waste: Preliminary results

The aim of this study was to evaluate the influence of slaughterhouse waste (SHW; essentially the skin, fats, and meat waste of pork, poultry, and beef) in a fermentative co-digestion process for H2 production from pre-selected organic waste taken from a refectory (food waste [FW]). Batch tests under mesophilic conditions were conducted in stirred reactors filled with different proportions of FW and SHW. The addition of 60% and 70% SHW to a mixture of SHW and FW improved H2 production compared to that in FW only, reaching H2-production yields of 145 and 109mlgVS0-1, respectively, which are 1.5–2times higher than that obtained with FW alone. Although the SHW ensured a more stable fermentative process due to its high buffering capacity, a depletion of H2 production occurred when SHW fraction was higher than 70%. Above this percentage, the formation of foam and aggregated material created non-homogenous conditions of digestion. Additionally, the increasing amount of SHW in the reactors may lead to an accumulation of long chain fatty acids (LCFAs), which are potentially toxic for anaerobic microorganisms and may inhibit the normal evolution of the fermentative process.

Multiple process integrations for broad perspective analysis of fermentative H2 production from wastewater treatment: Technical and environmental considerations

The functional certainty associated with secondary or tertiary process integration towards enhancing the viability of fermentative biohydrogen (H2) production from wastewater stabilization was investigated with eleven diverse combinations of dark-fermentation (acidogenic, HA), photo-fermentation (HP for H2) and methanogenic (for CH4) processes. Based on the specificity of individual process, the biocatalyst and the feeding pH were selected and operated at uniform hydraulic retention time (48h). Individually, HP operation showed higher H2 production (4.10mmol H2) and yield (16.02mol H2/kg CODR) than of HA (3.38mmol H2, 11.33mol H2/kg CODR) which was found contrary to the observed substrate degradation. Two-stage process integration showed marked improvement in both H2 production and substrate degradation. Integration of HP with HA showed maximum H2 production while HA with HP evidenced maximum H2 yield. Integration of methanogenic process with HP documented both higher biogas production and yield. Maximum substrate degradation was evidenced with three stage sequential integration of dark-fermentation, methanogenic and photo-fermentation processes. Three-stage integration contributed for higher substrate degradation rather than energy generation, especially with HP as the terminal process. Organic flux, energy efficiency and carbon footprint analyses were used to comprehensively delineate the practical consideration of the integrated processes.

Sub-dominant bacteria as keystone species in microbial communities producing bio-hydrogen

Nowadays mixed cultures are considered as a serious alternative to pure cultures in biotechnological processes. Mixed cultures can use various organic substrates and be operated under unsterile and continuous conditions. Although most of studies dealing with fermentative hydrogen production have focused on dominant species, sub-dominant bacteria can also have a significant effect despite their low abundance. The determination of their exact ecological role is essential for better understanding microbial metabolic networks in mixed cultures.In this work, the contribution of sub-dominant bacteria to fermentative H2 production was investigated using chemostats continuously fed with a glucose-based medium. Interestingly, Clostridium pasteurianum was dominant in six assays on seven at steady state, and only bacterial populations in low abundance differed. Acting as keystone species, these bacteria impacted substantially the microbial metabolic network of the overall ecosystem despite their low abundance. While Bacillus spp. and Lactobacillus spp. lowered the H2 yields by diverting a part of the H2 potential to lactate production, the presence of Escherichia coli increased the H2 yield by redirecting the metabolic network to acetate and butyrate hydrogen-producing pathways.

Development and calibration of a model for biohydrogen production from organic waste

Existing models for H2 production are capable of predicting digester failure caused by a specific disturbance. However, they are based on studies using simple sugars, while it is known that H2 production and fermentation kinetics vary with the composition and characteristics of the substrate used. Because the behaviour of biological processes may differ significantly when the digesting material is a complex matrix, such as organic waste, the aim of this study was to develop and calibrate a mathematical model for the prediction of hydrogen production on the basis of the results obtained from a laboratory scale experimental study using source-selected organic waste. The calibration was carried out for the most important kinetic parameters in mesophilic anaerobic digestion processes and also served as a sensitivity analysis for the influence of both the specific growth rate (μmax and the half velocity constant (ks), both of which are strongly dependant on the substrate used. High values of μmax led to a shorter lag-time and to an overestimate of the cumulative final H2 production relative to the experimentally measured production. Additionally, high values of ks associated with amino acid and sugar fermentation corresponded to a lower rate of substrate consumption and to a greater lag-time for growth of hydrogen-producing microorganisms. In this case, a lower final H2 production was predicted than that which was experimentally observed. Because the model development and calibration provided useful information concerning the role of the kinetic constants in the analysis of a fermentative H2 production process from organic wastes, they may also represent a good foundation for the analysis of fermentative H2 production from organic waste for pilot and full-scale applications.

Optimization of dark fermentative H2 production from microalgal biomass by combined (acid+ultrasonic) pretreatment

In this study, individual pretreatments (acid and ultrasonic) and a combination of these pretreatments were optimized to enhance the efficiency of dark fermentative hydrogen production (DFHP) from microalgal biomass. The experimental results show that the maximum H₂ production performance of 42.1 mL H₂/g dry cell weight (dcw) was predicted at 0.79% (v/w) HCl and at a specific energy input (SEI) of 49,600 kJ/kg dcw in the combined pretreatment, while it was limited in both individual pretreatments. Repeated batch testing of the predicted optimal conditions revealed that the combined pretreatment conditions for DFHP from microalgal biomass were successfully optimized by increasing the solubilization of the feedstock and by reducing the formation of the toxic 5-hydroxymethylfurfural (HMF).

Lime mud from paper-making process addition to food waste synergistically enhances hydrogen fermentation performance

The effect of lime mud from paper-making process (LMP) addition on the H2 fermentation of food waste (FW) was investigated. It was found that a slight addition of LMP (1.0–4.0 g in 200 g FW) significantly enhanced the H2 fermentation performance, not only increasing the total amount of H2 produced but also accelerating the whole reaction, shortening the lag period, and increasing the H2 production rate. Fermentation stability and microbial germination were also facilitated by LMP addition. This was attributed to the existence of Ca, Fe, Mn and alkaline substances such as CaCO3 and NaOH. The batch process treating a mixture of FW and LMP was showed that the highest hydrogen production of 137.6 mL H2/g VS was achieved at final pH 5.0, adding 3 g LMP (in 200 g FW) to the fermentation process, which lag-phase time was about 2.5 h.

Evaluation of different supplementary nutrients for enhanced biohydrogen production by Enterobacter aerogenes NRRL B 407 using waste derived crude glycerol

Crude glycerol (CG) has several advantages over a range of conventional substrates used for biohydrogen production by Enterobacter aerogenes NRRL B 407. Meanwhile, high process cost due to requirement of expensive supplementary media component is a concern. Therefore, different less expensive (or wastes) materials have been evaluated as supplementary nutrient for H2 production by CG (meat processing and restaurant waste based biodiesel derived) bioconversion. Among the materials selected, slaughterhouse liquid waste (SL), brewery waste biomass (BWB) and urea was found to improve the production by 18.81 ± 3.56, 27.30 ± 3.54 and 38.57 ± 3.66%, respectively. Further, in the case of urea (10 mg/L), cumulative production as high as 116.41 ± 3.72 mmol H2/L media has been achieved; which is comparable to other reports available on CG bioconversion. Thus, present study demonstrates successful replacement of large amount (∼5–6 g/L) of expensive nutrients/buffering agents by negligible amount (∼10 mg/L) of different waste materials, without compromising the cumulative H2 yield. Further, the strain used in the present study was found to grow at an acidic pH as low as 3.3, indicating its prospective application for dark fermentative H2 production.

Linking genome content to biofuel production yields: a meta-analysis of major catabolic pathways among select H2and ethanol-producing bacteria

BackgroundFermentative bacteria offer the potential to convert lignocellulosic waste-streams into biofuels such as hydrogen (H2) and ethanol. Current fermentative H2 and ethanol yields, however, are below theoretical maxima, vary greatly among organisms, and depend on the extent of metabolic pathways utilized. For fermentative H2 and/or ethanol production to become practical, biofuel yields must be increased. We performed a comparative meta-analysis of (i) reported end-product yields, and (ii) genes encoding pyruvate metabolism and end-product synthesis pathways to identify suitable biomarkers for screening a microorganism’s potential of H2 and/or ethanol production, and to identify targets for metabolic engineering to improve biofuel yields. Our interest in H2 and/or ethanol optimization restricted our meta-analysis to organisms with sequenced genomes and limited branched end-product pathways. These included members of the Firmicutes, Euryarchaeota, and Thermotogae.ResultsBioinformatic analysis revealed that the absence of genes encoding acetaldehyde dehydrogenase and bifunctional acetaldehyde/alcohol dehydrogenase (AdhE) in Caldicellulosiruptor, Thermococcus, Pyrococcus, and Thermotoga species coincide with high H2 yields and low ethanol production. Organisms containing genes (or activities) for both ethanol and H2 synthesis pathways (i.e. Caldanaerobacter subterraneus subsp. tengcongensis, Ethanoligenens harbinense, and Clostridium species) had relatively uniform mixed product patterns. The absence of hydrogenases in Geobacillus and Bacillus species did not confer high ethanol production, but rather high lactate production. Only Thermoanaerobacter pseudethanolicus produced relatively high ethanol and low H2 yields. This may be attributed to the presence of genes encoding proteins that promote NADH production. Lactate dehydrogenase and pyruvate:formate lyase are not conducive for ethanol and/or H2 production. While the type(s) of encoded hydrogenases appear to have little impact on H2 production in organisms that do not encode ethanol producing pathways, they do influence reduced end-product yields in those that do.ConclusionsHere we show that composition of genes encoding pathways involved in pyruvate catabolism and end-product synthesis pathways can be used to approximate potential end-product distribution patterns. We have identified a number of genetic biomarkers for streamlining ethanol and H2 producing capabilities. By linking genome content, reaction thermodynamics, and end-product yields, we offer potential targets for optimization of either ethanol or H2 yields through metabolic engineering.

Effects of pretreatment on the microbial community and l-lactic acid production in vinasse fermentation

Microwave–alkali and steam–alkali coupled pretreatments were carried out to improve the yield and optical purity of l-lactic acid produced using vinasse fermentation. Lactobacillus casei was inoculated into the system to initiate fermentation. Polymerase chain reaction denaturing gradient gel electrophoresis was used to analyze the microbial community during fermentation with and without the pretreatments. The original bacterial genus in vinasse was essentially inactivated, whereas L. casei became the dominant genus after 24h of fermentation. The system subjected to microwave–alkali coupled pretreatment released more reducing sugars and produced more lactic acid (up to 30.32g/L), which is twice that without pretreatment. In addition, the proportion of lactic acid in the organic acids also increased. The optical purity of the l-lactic acid produced under the microwave–alkali coupled pretreatment reached 91%, which is 2% higher than that under the steam–alkali coupled pretreatment and 7% higher than that under the control conditions. Therefore, the microwave–alkali coupled pretreatment is an effective method for the highly efficient bioconversion of vinasse into bioenergy.

Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase l-isoleucine production in Corynebacterium glutamicum

Threonine dehydratase and acetohydroxy acid synthase are critical enzymes in the l-isoleucine biosynthesis pathway of Corynebacterium glutamicum, but their activities are usually feedback-inhibited. In this study, we characterized a feedback-resistant threonine dehydratase and an acetohydroxy acid synthase from an l-isoleucine producing strain C. glutamicum JHI3-156. Sequence analysis showed that there was only a single amino acid substitution (Phe383Val) in the feedback-resistant threonine dehydratase, and there were three mutated amino acids (Pro176Ser, Asp426Glu, and Leu575Trp) in the big subunit of feedback-resistant acetohydroxy acid synthase. The mutated threonine dehydratase over-expressed in E. coli not only showed completely resistance to l-isoleucine inhibition, but also showed enhanced activity. The mutated acetohydroxy acid synthase over-expressed in E. coli showed more resistance to l-isoleucine inhibition than the wild type. Over-expression of the feedback-resistant threonine dehydratase or acetohydroxy acid synthase in C. glutamicum JHI3-156 led to increase of l-isoleucine production; co-expression of them in C. glutamicum JHI3-156 led to 131.7% increase in flask cultivation, and could produce 30.7g/L l-isoleucine in 72-h fed-batch fermentation. These results would be useful to enhance l-isoleucine production in C. glutamicum.

Evaluation of γ- aminobutyric acid, phytate and antioxidant activity of tempeh-like fermented oats (Avena sativa L.) prepared with different filamentous fungi.

Tempeh is a popular traditional fermented food in Asia. Many tempeh-like foods are made from cereal grains. However, the information of γ-aminobutyric acid (GABA) accumulation in those tempeh-like cereal grains during fermentation is lacking. Meanwhile, little information is available on the anti-nutrient contents and antioxidant activity of tempeh-like fermented oats. The aim of the present work was to study the changes of GABA, phytate, natural antioxidants and antioxidant activity of tempeh-like fermented oats. As fermentation time progressed, the GABA, total phenolics content (TPC) and flavonoids increased rapidly. The Aspergillus oryzae-fermented oats had the highest GABA, whereas Rhizopus oryzae-fermented oats had the highest TPC. Phytate, an anti-nutrient component, was dramatically reduced in the fermented oats, especially those by A. oryzae (reduced by about 63%). The antioxidant activities of fermented oats were also significantly enhanced after 72h fermentation (p < 0.05). This study demonstrated that oats fermented by generally recognized as safe (GRAS) fungi can be recommended as tempeh-like functional foods with higher GABA, more natural antioxidants and lower phytate compared with native oats.