Clostridial Fermentation Research Articles

Simultaneous production of renewable biohydrogen, biobutanol and biopolymer from phytogenic CoNPs-assisted Clostridial fermentation for sustainable energy and environment

Considering the environmental impacts, rapid fossil fuel depletion and production costs, sustainable production of clean biofuels from alternative sources is required to meet the increasing demand for energy while avoiding environmental pollution. In this study, phytogenic cobalt nanoparticles (CoNPs)-assisted dark fermentation process was developed for the simultaneous production of biohydrogen, biobutanol and biopolymer from glucose using Clostridium acetobutylicum NCIM 2337. The maximum biohydrogen yield of 2.89 mol H2/mol glucose was achieved at 1.5 mg of CoNPs, which is 1.6 folds higher than that of the control experiment. The high level of soluble metabolites, specifically acetate and butyrate, confirmed the production of biohydrogen through acetate/butyrate pathways. The modified Gompertz model fitted well with experimental results of CoNPs-assisted biohydrogen production. The CoNPs could act as an electron carrier in intracellular metabolism to enhance the activity of ferredoxin and hydrogenase enzymes, thus improving biohydrogen production. Furthermore, biobutanol and biopolymer yields of 975 ± 2.5 mg/L and 1182 ± 1.4 mg/L were achieved, with 2.0 mg and 2.5 mg of CoNP, respectively, which were 1.27 and 1.19 folds higher than the control values. Hence, the inclusion of CoNPs in the fermentation medium seems to be a promising technique for the enhanced simultaneous production of biohydrogen, biobutanol and biopolymer. The environmental perspectives of the obtained renewable biohydrogen, biobutanol and biopolymer are also discussed.

Polycationic Surfaces Promote Whole-Cell Immobilization and Induce Microgranulation of Clostridium saccharoperbutylacetonicum N1-4 for Enhanced Biobutanol Production.

Immobilization is a common strategy used to protect microbial cells to improve the performance of bioprocesses. However, the interaction mechanism between the cells and the immobilization material is generally poorly understood. In this study, we employed natural polysaccharide-based materials as immobilization carriers for clostridial fermentation in an attempt to enhance the production of butanol (a valuable biofuel/biochemical but highly toxic to the host cells) and meanwhile elucidate the interaction mechanisms related to immobilization. The utilization of chitosan powder as the immobilization carrier enhanced butanol productivity by 97% in the fermentation with Clostridium saccharoperbutylacetonicum N1-4 and improved butanol titer by 21% in the fermentation with Clostridium beijerinckii NCIMB 8052. Additionally, analogue derivatives using microcrystalline cellulose (MCC) and cotton cationized on the surface with 3-chloro-2-hydroxypropyltrymethylammonium (CHPTA) and 2-chloro-N,N-diethylaminoethyl chloride (DEAEC) were prepared and used as immobilization carriers for similar fermentation conditions. The CHPTA derivatives showed slightly increased production of butanol and total solvent with C. saccharoperbutylacetonicum. Overall, our results indicated that the interaction between the cell and the carrier material occurs through a double mechanism involving adsorption immobilization and induced aggregation. This work provides insights concerning the effects of the chemical properties of the carrier material (such as the cation density and surface area) on fermentation performance, enabling a better understanding of the interaction between bacterial cells and the cationic materials. The derivatization strategies employed in this study can be applied to most cellulosic materials to modulate the properties and enhance the interaction between the cell and the carrier material for immobilization, thus improving the bioprocess performance.

Nutritive value, silage fermentation characteristics, and aerobic stability of grass-legume round-baled silages at differing moisture concentrations with and without manure fertilization and microbial inoculation.

For baled silages, production of clostridial fermentation products can be exacerbated by exceeding normal moisture targets (45% to 55%), and/or by the application of dairy slurry before harvest. Our objectives were to test a microbial inoculant as a mitigant of clostridial products in high-moisture, grass-legume (52% ± 13.8% cool-season grasses, 44.0% ± 14.0% legumes [predominately alfalfa]) baled silages in swards that were fertilized with dairy slurry. A secondary objective was to examine the effects of bale moisture and inoculation on the aerobic stability of these fermented silages following exposure to air. After the first-cutting was removed, three manure treatments were applied as a whole-plot factor: 1) control (no manure); 2) slurry applied immediately to stubble (63,250 L/ha); or 3) slurry applied after a 1-wk delay (57,484 L/ha). An interactive arrangement of bale moisture (64.1% or 48.4%) and inoculation (yes or no) served as a subplot term in the experiment. The inoculant contained both homolactic (Lactococcus lactis 0224) and heterolactic (Lactobacillus buchneri LB1819) bacteria. The experimental design was analyzed as a randomized complete block with four replications, and the study included 48 experimental units (1.2 × 1.2-m round bales). Total fermentation acids were affected (P ≤ 0.021) by slurry application strategies, but this was likely related to inconsistent bale moisture across slurry-application treatments. Concentrations of butyric acid were low, and there were no detectable contrasts comparing manure treatments (mean = 0.05%; P ≥ 0.645). Bale moisture affected all measures of fermentation, with bales made at 64.1% moisture exhibiting a more acidic final pH (4.39 vs. 4.63; P < 0.001), less residual water-soluble carbohydrates (2.1% vs. 5.1%; P < 0.001), as well as greater lactic acid (4.64% vs. 2.46%; P < 0.001), acetic acid (2.26% vs. 1.32%; P < 0.001), and total fermentation acids (7.37% vs. 3.97%; P < 0.001). Inoculation also reduced pH (4.47 vs. 4.56; P = 0.029), and increased acetic acid (1.97% vs. 1.61%; P < 0.001) and 1,2-propanediol (1.09% vs. 0.72%; P < 0.001) compared to controls. During a 34-d aerobic exposure period, maximum surface bale temperatures were not affected (P ≥ 0.186) by any aspect of treatment, likely due to the prevailing cool ambient temperatures; however, yeast counts were numerically lower in response to greater (P < 0.001) production of acetic acid that was stimulated by both high bale moisture and inoculation.

Effects of wilting during a cloudy day and storage temperature on the fermentation quality and microbial community of Napier grass silage.

Wilting affects the chemical compositions and epiphytic microorganisms of grasses to some extent, thereby influencing the fermentation characteristics of silage. The present study investigated the effects of wilting during a cloudy day and ensiling temperature (20, 30 and 40 °C) on the fermentation of Napier grass (Pennisetum purpureum). Wilting during a cloudy day significantly decreased the water-soluble carbohydrate content (P < 0.05) and significantly increased pH, counts of lactic acid bacteria and aerobic bacteria in grass (P < 0.05). Wilting increased the relative abundance of the Enterobacteriaceae family, as well as genera such as Acinetobacter, Pantoea and Lactococcus, and also decreased that of Sphingomonas and Methylobacterium, in fresh grass. Although ensiling increased the relative abundance of Lactobacillus, a Enterobacteriaceae genus was the dominant microorganism in Napier grass before ensiling and after ensiling. Wilting increased pH, acetic acid, butyric acid and NH3 -N contents, and also reduced the lactic acid content, of Napier grass silage. Additionally, the fermentation quality of silages at 30 °C was poorer, irrespective of wilting, as indicated by high pH, high butyric acid and NH3 -N contents, high relative abundance of Clostridium and Bifidobacterium, and a low lactic acid content. Ensiling at 40 °C enhanced lactic acid bacteria fermentation and also suppressed clostridial fermentation and the growth of microorganisms in silages. Napier grass should be ensiled immediately after being harvested during a cloudy day and ensiling at 30 °C should be avoided to prevent poor fermentation. © 2022 Society of Chemical Industry.

Impact of Long-Term Storage on Alfalfa Leaf and Stem Silage Characteristics

Fractionation of alfalfa (Medicago sativa L.) into leaves and stems can reduce cutting frequency while producing a high-value feedstuff. A difficulty with fractionation is the higher moisture content at ensiling due to the inability to wilt leaves without substantial dry matter loss or financial cost. To evaluate whether the silage quality of fractionated alfalfa is competitive with conventional methods under long-term storage conditions, high-moisture leaves (250–280 g·kg−1 dry matter, DM) and stems (190–240 g·kg−1 DM) were ensiled for 21 and 350 days and compared to wilted whole-plant silage for two alfalfa developmental stages. At 21 days, leaf and whole-plant silage fermentation characteristics indicated suitable lactic acid bacterial fermentation through decreased pH, high lactic acid–to-acetic acid ratio, and a lack of clostridial fermentation. At 350 days, leaf silage fermentation and nutritional quality decreased due to sustained proteolysis, but true protein still exceeded that of whole-plant silage. High-moisture stem fractions fermented poorly; at 21 days, stage 3 stems had significant amounts of butyric acid, while stage 5 stems became clostridial at 350 days. Long-term storage of high-moisture leaf silages can produce good-quality silage despite exceeding moisture contents typically recommended for alfalfa, while wilting is required for stem silages.

Co-feeding glucose with either gluconate or galacturonate during clostridial fermentations provides metabolic fine-tuning capabilities

Clostridiumacetobutylicum ATCC 824 effectively utilizes a wide range of substrates to produce commodity chemicals. When grown on substrates of different oxidation states, the organism exhibits different recycling needs of reduced intracellular electron carrying co-factors. Ratios of substrates with different oxidation states were used to modulate the need to balance electron carriers and demonstrate fine-tuned control of metabolic output. Three different oxidized substrates were first fed singularly, then in different ratios to three different strains of Clostridium sp. Growth was most robust when fed glucose in exclusive fermentations. However, the use of the other two more oxidized substrates was strain-dependent in exclusive feeds. In glucose-galacturonate mixed fermentation, the main products (acetate and butyrate) were dependant on the ratios of the substrates. Exclusive fermentation on galacturonate was nearly homoacetic. Co-utilization of galacturonate and glucose was observed from the onset of fermentation in growth conditions using both substrates combined, with the proportion of galacturonate present dictating the amount of acetate produced. For all three strains, increasing galacturonate content (%) in a mixture of galacturonate and glucose from 0 to 50, and 100, resulted in a corresponding increase in the amount of acetate produced. For example, C.acetobutylicum increased from ~ 10 mM to ~ 17 mM, and then ~ 23 mM. No co-utilization was observed when galacturonate was replaced with gluconate in the two substrate co-feed.

Microbial community dynamics during alfalfa silage with or without clostridial fermentation

This study was conducted to examine the effects of Lactobacillus plantarum (LP) and sucrose (S) on clostridial community dynamics and correlation between clostridia and other bacteria in alfalfa silage during ensiling. Fresh alfalfa was directly ensiled without (CK) or with additives (LP, S, LP + S) for 7, 14, 28 and 56 days. Clostridial and bacterial communities were evaluated by next-generation sequencing. Severe clostridial fermentation occurred in CK, as evidenced by the high contents of butyric acid, ammonia nitrogen, and clostridia counts, whereas all additives, particularly LP + S, decreased silage pH and restrained clostridial fermentation. Clostridium perfringens and Clostridium butyricum might act as the main initiators of clostridial fermentation, with Clostridium tyrobutyricum functioning as the promoters of fermentation until the end of ensiling. Clostridium tyrobutyricum (33.5 to 98.0%) dominated the clostridial community in CK from 14 to 56 days, whereas it was below 17.7% in LP + S. Clostridium was negatively correlated with the genus Lactobacillus, but positively correlated with the genera Enterococcus, Lactococcus and Leuconostoc. Insufficient acidification promoted the vigorous growth of C. tyrobutyricum of silage in later stages, which was mainly responsible for the clostridial fermentation of alfalfa silage.

Effects of processing, moisture, and storage length on the fermentation profile, particle size, and ruminal disappearance of reconstituted corn grain.

Our objective was to examine the effects of processing, moisture, and anaerobic storage length of reconstituted corn grain (RCG) on the fermentation profile, geometric mean particle size (GMPS), and ruminal dry matter disappearance (DMD). Dry corn kernels were ground (hammer mill, 5-mm screen) or rolled, then rehydrated to 30%, 35%, or 40% moisture, and stored for 0, 14, 30, 60, 90, 120, or 180 d in laboratory silos. Rolled corn had an increased GMPS compared with ground corn (2.24 and 1.13 mm, respectively, at ensiling). However, there was a trend for an interaction between processing and moisture concentration to affect particle size, with GMPS increasing with increased moisture concentration, especially in ground corn. Longer storage periods also slightly increased GMPS. Processing, moisture, and storage length interacted to affect the fermentation pattern (two- or three-way interactions). Overall, pH decreased, whereas lactic acid, acetic acid, ethanol, and NH3-N increased with storage length. RCG with 30% moisture had less lactic acid than corn with 35% and 40% moisture, indicating that fermentation might have been curtailed and also due to the clostridial fermentation that converts lactic acid to butyric acid. Ensiling reconstituted ground corn with 30% of moisture led to greater concentrations of ethanol and butyric acid, resulting in greater DM loss than grain rehydrated to 35% or 40% of moisture. Ammonia-N and in situ ruminal DMD were highest for reconstituted ground corn with 35% or 40% of moisture, mainly after 60 d of storage. Therefore, longer storage periods and greater moisture contents did not offset the negative effect of greater particle size on the in situ ruminal DMD of rolled RCG. Nonetheless, RCG should be ensiled with more than 30% moisture and stored for at least 2 mo to improve the ruminal DMD and reduce the formation of ethanol and butyric acid.

Milling byproducts are an economically viable substrate for butanol production using clostridial ABE fermentation

Butanol is a platform chemical that is utilized in a wide range of industrial products and is considered a suitable replacement or additive to liquid fuels. So far, it is mainly produced through petrochemical routes. Alternative production routes, for example through biorefinery, are under investigation but are currently not at a market competitive level. Possible alternatives, such as acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia are not market-ready to this day either, because of their low butanol titer and the high costs of feedstocks. Here, we analyzed wheat middlings and wheat red dog, two wheat milling byproducts available in large quantities, as substrates for clostridial ABE fermentation. We could identify ten strains that exhibited good butanol yields on wheat red dog. Two of the best ABE producing strains, Clostridium beijerinckii NCIMB 8052 and Clostridium diolis DSM 15410, were used to optimize a laboratory-scale fermentation process. In addition, enzymatic pretreatment of both milling byproducts significantly enhanced ABE production rates of the strains C. beijerinckii NCIMB 8052 and C. diolis DSM 15410. Finally, a profitability analysis was performed for small- to mid-scale ABE fermentation plants that utilize enzymatically pretreated wheat red dog as substrate. The estimations show that such a plant could be commercially successful.Key points• Wheat milling byproducts are suitable substrates for clostridial ABE fermentation.• Enzymatic pretreatment of wheat red dog and middlings increases ABE yield.• ABE fermentation plants using wheat red dog as substrate are economically viable.Graphical abstract

Metagenome analyses reveal the role of Clostridium perfringens in alfalfa silage anaerobic deterioration.

The clostridial fermentation caused by the outgrowth of Clostridia was mainly responsible for the silage anaerobic deterioration. Our previous results showed that Clostridium perfringens dominated the clostridial community in poor-fermented alfalfa silage. This study was conducted to further examine the role of C. perfringens in silage anaerobic deterioration through fermentation products and the microbial community analyses. Direct-cut alfalfa was ensiled with C. perfringens contamination (CKC) or with the addition of Lactobacillus plantarum, sucrose and C. perfringens (LSC). Contamination with C. perfringens enhanced the clostridial fermentation in CKC silage, as indicated by high contents of butyric acid, ammonia nitrogen and Clostridia, while LSC silage was well preserved. The genera Bifidobacterium, Garciella and Clostridium dominated the bacterial community in CKC silage, while predominate genus was replaced by Lactobacillus in LSC silage. The clostridial community in CKC silage was dominated by Garciella sp. (26.9 to 58.1%) and C. tyrobutyricum (24.4 to 48.6%), while the relative abundance of C. perfringens was below 5.0%. Therefore, the effect of Clostridia contamination on ensiling fermentation was dependent on the ensilability of the silage material. Garciella sp. and C. tyrobutyricum, rather than C. perfringens, played dominant role in the clostridial fermentation in CKC silage.

Butanol production by Saccharomyces cerevisiae: perspectives, strategies and challenges.

The search for gasoline substitutes has grown in recent decades, leading to the increased production of ethanol as viable alternative. However, research in recent years has shown that butanol exhibits various advantages over ethanol as a biofuel. Furthermore, butanol can also be used as a chemical platform, serving as an intermediate product and as a solvent in industrial reactions. This alcohol is naturally produced by some Clostridium species; however, Clostridial fermentation processes still have inherent problems, which focuses the interest on Saccharomyces cerevisiae for butanol production, as an alternative organism for the production of this alcohol. S. cerevisiae exhibits great adaptability to industrial conditions and can be modified with a wide range of genetic tools. Although S. cerevisiae is known to naturally produce isobutanol, the n-butanol synthesis pathway has not been well established in wild S. cerevisiae strains. Two strategies are most commonly used for of S. cerevisiae butanol production: the heterologous expression of the Clostridium pathway or the amino acid uptake pathways. However, butanol yields produced from S. cerevisiae are lower than ethanol yield. Thus, there are still many challenges needed to be overcome, which can be minimized through genetic and evolutive engineering, for butanol production by yeast to become a reality.

Laboratory scale production of hydrocarbon motor fuel components from lignocellulose: Combination of new developments of membrane science and catalysis

Lignocellulose pretreated with dilute alkali and hydrolysed with cellulases was used as substrate for the processing of acetone–butanol–ethanol (ABE) clostridial fermentation followed by the vapour-phase membrane concentration of two alcohols and acetone with up to 99% degree of recovery. ABE concentrate was used for the catalytic synthesis of fuel components over a zeolite catalyst with aliphatic and aromatic hydrocarbons as the main products with up to 60% conversion. The zeolite catalyst was effectively regenerated from coking by hot steam treatment with up to 100% recovery in catalytic activity. The main novelties of the process are in demonstration the effectiveness of vapor-phase ABE concentration, its effective conversion to hydrocarbons followed by of catalyst’ regeneration. The process described paves the way to developing renewable technologies for the production of motor fuel components.

Effects of sodium nitrite treatment on the fermentation quality of red clover-grass silage harvested at two dry matter concentrations and inoculated with clostridia

Legumes are particularly susceptible to clostridial fermentation when ensiled because of their high buffering capacity and water-soluble carbohydrate contents. The aim of the study was to investigate if a sodium nitrite treatment (900 g t-1 herbage in fresh matter [FM]) impairs butyric acid fermentation of red clover-timothy-meadow fescue silage compared with formic acid-treated (4 l t-1 FM) and untreated silage. The sward was harvested after wilting at low dry matter (DM) (LDM, 194 g kg-1) and high DM (HDM, 314 g kg-1) concentrations and half of the herbage batches were inoculated with Clostridium tyrobutyricum spores before additive treatments. No butyric acid fermentation was observed in HDM silages probably because of the relatively high DM and nitrate contents of the herbage mixture. In LDM silage butyric acid was detected only in formic acid-treated silage, and the number of clostridia copies was higher in formic acid-treated than in sodium nitrite treated silage. Sodium nitrite treatment was superior to FA treatment in suppressing clostridial fermentation in the LDM silages.

Engineering solventogenic clostridia for commercial production of bio‐chemicals

The manufacture of bio-chemicals through the use of microbial fermentation and renewable feedstock has a number of well-known advantages linked to sustainability and reduced impacts on the environment. Markets for molecules produced with greener credentials are growing as consumers become more aware of what is in the formulated products they use every day. The use of solventogenic clostridia has now been re-commercialised for the production of bio-acetone and bio-n-butanol. The different impurity profiles of these bio-based molecules compared with petro-versions results in performance advantages in downstream derivatisation chemistry, giving an added benefit alongside sustainability advantages. Advances in genome editing now enable us to take the benefits observed with clostridial fermentation and apply them to the production of the next generation of bio-molecules.

Analysis of metabolite profiles of Saccharomyces cerevisiae strains suitable for butanol production.

Butanol has advantages over ethanol as a biofuel. Although butanol is naturally produced by some Clostridium species, clostridial fermentation has inherent characteristics that prevent its industrial application. Butanol-producing Saccharomyces cerevisiae strains may be a solution to this problem. The aim of this study was to evaluate the ability of wild-type and industrial Brazilian strains of S. cerevisiae to produce n-butanol using glycine as co-substrate and evaluate the relationship between the production of this alcohol and other metabolites in fermented broth. Of the 48 strains analyzed, 25 were able to produce n-butanol in a glycine-containing medium. Strains exhibited different profiles of n-butanol, isobutanol, ethanol, glycerol and acetic acid production. Some wild-type strains showed substantial n-butanol production capability, for instance UFMG-CM-Y267, which produced about 12.7 mg/L of butanol. Although this concentration is low, it demonstrates that wild-type S. cerevisiae can synthesize butanol, suggesting that selection and genetic modification of this microorganism could yield promising results. The findings presented here may prove useful for future studies aimed at optimizing S. cerevisiae strains for butanol production.

CRISPR-Cas, a highly effective tool for genome editing in Clostridium saccharoperbutylacetonicum N1-4(HMT).

ABSTRACTThe solventogenic clostridia have long been known for their ability to convert sugars from complex feedstocks into commercially important solvents. Although the acetone-butanol-ethanol process fell out of favour decades ago, renewed interest in sustainability and ‘green’ chemistry has re-established our appetite for reviving technologies such as these, albeit with 21st century improvements. As CRISPR-Cas genome editing tools are being developed and applied to the solventogenic clostridia, their industrial potential is growing. Through integration of new pathways, the beneficial traits and historical track record of clostridial fermentation can be exploited to generate a much wider range of industrially relevant products. Here we show the application of genome editing using the endogenous CRISPR-Cas mechanism of Clostridium saccharoperbutylacetonicum N1-4(HMT), to generate a deletion, SNP and to integrate new DNA into the genome. These technological advancements pave the way for application of clostridial species to the production of an array of products.

Hydrolytic pre-treatment methods for enhanced biobutanol production from agro-industrial wastes

Brewery industry liquid waste (BLW), brewery spent grain (BSG), apple pomace solid wastes (APS), apple pomace ultrafiltration sludge (APUS) and starch industry wastewater (SIW) have been considered as substrates to produce biobutanol. Efficiency of hydrolysis techniques tested to produce fermentable sugars depended on nature of agro-industrial wastes and process conditions. Acid-catalysed hydrolysis of BLW and BSG gave a total reducing sugar yield of 0.433 g/g and 0.468 g/g respectively. Reducing sugar yield from microwave assisted hydrothermal method was 0.404 g/g from APS and 0.631 g/g from APUS, and, 0.359 g/g from microwave assisted acid-catalysed SIW dry mass. Parameter optimization (time, pH and substrate concentration) for acid-catalysed BLW hydrolysate utilization using central composite model technique produced 307.9 g/kg glucose with generation of inhibitors (5-hydroxymethyl furfural (20 g/kg), furfural (1.6 g/kg), levulinic acid (9.3 g/kg) and total phenolic compound (0.567 g/kg)). 10.62 g/L of acetone-butanol-ethanol was produced by subsequent clostridial fermentation of the substrate.

The effect of cultivar, wilting and storage period on fermentation and the clostridial community of alfalfa silage

The objective of this study was to gain deeper insights into chemical transformations and the clostridial community dynamics during the ensiling of alfalfa. A factorial experiment was conducted with four alfalfa cultivars [Sanditi (A1), AC Caribou (A2), WL319HQ (A3) and 4030 (A4)] × three wilting durations [0 h (direct-cut), 2 h and 4 h] × three storage periods (14, 28 and 56 days). The clostridial community was examined using denaturing gradient gel electrophoresis. High butyric acid and ammonia nitrogen (NH3–N) contents and clostridia numbers were observed in direct-cut silage, with higher values observed in A4 than in the other three cultivars. However, butyric acid and NH3–N contents and clostridia numbers decreased with wilting regardless of the cultivar. Although Clostridium ghonii and Clostridium sartagoforme were common to all direct-cut and wilted silages, bands for these species were faint. Differences in the appearing time of these species were observed, bands for C. ghonii were found after 14 and 28 days of ensiling, while those for C. sartagoforme were only found after 56 days. In addition, in direct-cut silage, distinct bands for Clostridium perfringens were detected in A1, A2 and A3, while those for Clostridium sporogenes were detected in A4. The inactive spores of clostridia could be observed in 4-h wilted silage as no butyric acid was detected. It was concluded that the enhanced clostridial fermentation of direct-cut silage was attributed mainly to C. perfringens and C. sporogenes, while C. ghonii and C. sartagoforme were involved in the restricted clostridial fermentation of 2-h wilted silage.

A parametric response surface study of fermentative hydrogen production from cheese whey

Batch factorial experiments were performed on cheese whey+wastewater sludge mixtures to evaluate the influence of pH and the inoculum-to-substrate ratio (ISR) on fermentative H2 production and build a related predictive model. ISR and pH affected H2 potential and rate, and the fermentation pathways. The specific H2 yield varied from 61 (ISR=0, pH=7.0) to 371L H2/kg TOCwhey (ISR=1.44gVS/g TOC, pH=5.5). The process duration range was 5.3 (ISR=1.44gVS/g TOC, pH=7.5) − 183h (ISR=0, pH=5.5). The metabolic products included mainly acetate and butyrate followed by ethanol, while propionate was only observed once H2 production had significantly decreased. The multiple metabolic products suggested that the process was governed by several fermentation pathways, presumably overlapping and mutually competing, reducing the conversion yield into H2 compared to that expected with clostridial fermentation.

Dynamics of microbial community during ensiling direct-cut alfalfa with and without LAB inoculant and sugar.

To gain deeper insights into the clostridial community dynamics and chemical transformations during the ensiling of alfalfa. Direct-cut alfalfa silage (with the dry matter content of 240gkg-1 ) was prepared with or without the addition of a lactic acid bacterial inoculant and sucrose. Silages were sampled at 0, 3, 7, 14, 28 and 56days after ensiling and their bacterial community was determined using high-throughput sequencing with a special focus on the clostridial community. A clostridial fermentation occurred in the control silage, with high contents of acetic acid, butyric acid and ammonia nitrogen and Clostridia counts; while the inoculated silage was well preserved, with low pH and high lactic acid content. Lactic acid bacteria dominated the bacterial community during the ensiling process. In the control silage, Weissella confusa, Lactobacillus brevis, Enterococcus mundtii and Pediococcus acidilactici were identified at the beginning of the fermentation. Thereafter, W. confusa, Lactobacillus helsingborgensis and Bifidobacterium asteroides appeared and quickly prevailed. In the inoculated silage, Lactobacillus plantarum dominated the whole ensiling process. The genus Clostridium dominated the clostridial community, and was depressed with the inoculated treatment. Clostridium perfringens, Garciella sp. and Clostridium baratii were the main initiators of the clostridial fermentation of the control silage, while Clostridium tyrobutyricum became the most abundant Clostridia with prolonged ensiling. Overall in the inoculated silage, little changes in the clostridial community were observed throughout the ensiling period. Treating alfalfa silage with a homolactic acid-based bacterial inoculant prevented a clostridial fermentation resulting in more efficient fermentation. Distinct changes in the bacterial community with a special focus on the clostridial community were associated with the development of the clostridial fermentation during the ensiling of alfalfa. High-throughput sequencing based on a novel Clostridia-specific primer set proved a potentially useful tool to study the clostridial community dynamics, and could aid to elucidate the mechanism by which the clostridial fermentation develops during the ensiling of alfalfa.