Microbial conversion of distillery waste to bioenergy: Effects of media enrichment with low chain fatty acids and Candida sp.

During microbial methanogensis of diluted distillery waste or spent wash (initial COD 25,000 mg/l) in the presence of sodium acetate, sodium propionate, or sodium butyrate at the concentration of 2000 mg/l, biogas containing 22.0% to 39.4% methane was produced in 20 days in a semicontinuous fermentation system. The analysis of the volatile fatty acid spectrum of the effluent showed accumulation of 46.3% branched chain fatty acids. When the fermentation medium was supplemented with modified Smith and Mah (SM) medium containing electrolytes and 1% sodium acetate, production of methane went up to 59.0% in 18 days. With the addition of a strain of Candida sp. the coculture produced about the same volume of methane (61%) but the time required was reduced (14 days). COD (16600.8 mg/l) was reduced and the effluent contained only 3% branched chain fatty acids adn 96% straight chain fatty acids. Fortification with the sodium salts of three branched-chain fatty acids as sources of carbon in SM medium (alone) reduced methane production. Only 0.0% to 14.2% methane was recorded. By combining these acids with straight chain fatty acids in SM medium, methane production increased significantly (about 4-fold) in proportion to the concentration of straight chain fatty acids added.     

Effect of pH on Population and Fermentation in a Continuously Cultured Rumen Ecosystem

The effect of pH on rumen fermentation and microbial population was studied in a continuously cultured rumen ecosystem. A marked decrease in the production of volatile fatty acids and methane from alfalfa hay occurred when the cultures were maintained at pH values below 6.0. The decrease in acetate and methane production was greater than that of propionate production. The culture maintained at pH 6.7 contained the types of bacteria often found in high concentration in the rumen, whereas the culture maintained at pH 5.0 had a high percentage of bacteria which could not be identified with the major rumen bacteria found in rumens of animals fed alfalfa hay. Replacement of the bicarbonate-phosphate buffer used to maintain fermentor pH at 6.7 with phosphate alone did not greatly alter the fermentation products produced from a hay-concentrate substrate.     

生物强化污泥厌氧发酵产酸研究进展*

污泥厌氧发酵生产挥发性脂肪酸相较产甲烷,是更具应用价值的污泥稳定途径及资源化利用方式,得到国内外学者的普遍重视。考虑到产酸量低和产酸过程的不稳定性是限制污泥发酵产酸的主要问题,采用生物强化方法实现挥发性脂肪酸的大量积累,与物理和化学方法相比,具有成本低、无二次污染等优点。根据生物强化制剂的类型,归纳了微生物纯培养物、微生物混合培养物及生物酶强化对污泥厌氧发酵产酸的影响,并在此基础上对生物强化技术控制污泥定向产酸、调控奇偶数碳比率等方面的应用进行讨论。此外,分析了影响挥发性脂肪酸产量和组分的因素,如pH、温度、底物、水力停留时间和污泥龄等。最后对生物强化技术的发展方向进行了展望,以期为深入探究污泥资源化利用提供借鉴。     

Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro

AIMS: To assess the effect of protozoal species on rumen fermentation characteristics in vitro. METHODS AND RESULTS: Entodinium caudatum, Isotricha intestinalis, Metadinium medium, and Eudiplodinium maggii from monofaunated wethers and mixed protozoa from conventional wethers were obtained by centrifugation, re-suspended at their normal densities in rumen fluid supernatants from defaunated or conventional wethers and incubated in vitro. The presence of protozoa increased the concentration of ammonia and altered the volatile fatty acids balance with more acetate and butyrate produced at the expense of propionate. Differences among species were observed, notably in the production of methane, which increased with E. caudatum as compared to other ciliates and to defaunated and mixed protozoa treatments (P < 0.05). The increased methanogenesis was not correlated to protozoal biomass indicating that the metabolism of this protozoan and/or its influence on the microbial ecosystem was responsible for this effect. CONCLUSIONS: Entodinium caudatum stimulated the production of methane, a negative effect that was reinforced by a concomitant increase in protein degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of individual species of protozoa highlighted the particular influence of E. caudatum on rumen fermentation. Its elimination (targeted defaunation) from the rumen could reduce methane production without affecting feed degradation.     

Lessons from the cow: What the ruminant animal can teach us about consolidated bioprocessing of cellulosic biomass

Consolidated bioprocessing (CBP) of cellulosic biomass is a promising source of ethanol. This process uses anaerobic bacteria, their own cellulolytic enzymes and fermentation pathways that convert the products of cellulose hydrolysis to ethanol in a single reactor. However, the engineering and economics of the process remain questionable. The ruminal fermentation is a very highly developed natural cellulose-degrading system. We propose that breakthroughs developed by cattle and other ruminant animals in cellulosic biomass conversion can guide future improvements in engineered CBP systems. These breakthroughs include, among others, an elegant and effective physical pretreatment; operation at high solids loading under non-aseptic conditions; minimal nutrient requirements beyond the plant biomass itself; efficient fermentation of nearly all plant components; efficient recovery of primary fermentation end-products; and production of useful co-products. Ruminal fermentation does not produce significant amounts of ethanol, but it produces volatile fatty acids and methane at a rapid rate. Because these alternative products have a high energy content, efforts should be made to recover these products and convert them to other organic compounds, particularly transportation fuels.     

Shifts in xylanases and the microbial community associated with xylan biodegradation during treatment with rumen fluid

Treatment with rumen fluid improves methane production from non-degradable lignocellulosic biomass during subsequent methane fermentation; however, the kinetics of xylanases during treatment with rumen fluid remain unclear. This study aimed to identify key xylanases contributing to xylan degradation and their individual activities during xylan treatment with bovine rumen microorganisms. Xylan was treated with bovine rumen fluid at 37°C for 48 h under anaerobic conditions. Total solids were degraded into volatile fatty acids and gases during the first 24 h. Zymography showed that xylanases of 24, 34, 85, 180, and 200 kDa were highly active during the first 24 h. Therefore, these xylanases are considered to be crucial for xylan degradation during treatment with rumen fluid. Metagenomic analysis revealed that the rumen microbial community’s structure and metabolic function temporally shifted during xylan biodegradation. Although statistical analyses did not reveal significantly positive correlations between xylanase activities and known xylanolytic bacterial genera, they positively correlated with protozoal (e.g., Entodinium, Diploplastron, and Eudiplodinium) and fungal (e.g., Neocallimastix, Orpinomyces, and Olpidium) genera and unclassified bacteria. Our findings suggest that rumen protozoa, fungi, and unclassified bacteria are associated with key xylanase activities, accelerating xylan biodegradation into volatile fatty acids and gases, during treatment of lignocellulosic biomass with rumen fluid.     

End product yields from the extraruminal fermentation of various polysaccharide, protein and nucleic acid components of biofuels feedstocks

"Extraruminal" fermentations employing in vitro incubation of mixed ruminal bacterial consortia, are capable of converting a complex array of biomass materials to mixtures of volatile fatty acids (VFA), methane, and carbon dioxide. Most of the potential energy in the biomass feedstock is retained in the VFA products, which are potential reactants for electrochemical conversion to hydrocarbon fuels. Quantitative data on VFA yields and proportions from biomass components are necessary for determining industrial feasibility, but such measurements have not been systematically reported. VFA yields and proportions were determined for a variety of carbohydrates, proteins and nucleic acids. Carbohydrates yielded primarily acetic and propionic acids, while proteins also yielded a more favorable product mix (longer average chain length and branched chain VFAs). Addition of certain co-substrates (e.g., glycerol) favorably improved the VFA product mix. The results have implications for hydrocarbon fuel generation from biomass materials by hybrid fermentation/chemical processes.     

On-line extraction of volatile fatty acids in acidogenic chemostat culture using a supported liquid membrane

An on-line extraction of volatile fatty acids (acetic and butyric acids) from acidogenic fermentation in chemostat cultures using a supported liquid membrane (SLM) was investigated in order to overcome end-product inhibition. By using SLM, the high-cell-density retaining dilution rate of the chemostat could be increased, thus enhancing the microbial acidogenesis. To further understand this phenomenon, the growth and extraction kinetics were studied. The effect of substrate concentration was found to obey the Haldane equation. Regarding the inhibition by volatile fatty acids, it turned out that undissociated butyric acid rather than acetic acid severely inhibited the growth, corresponding to non-competitive kinetics. The extraction rates of the acids were proportional to their undissociated concentration as well as to the SLM area/broth volume, and butyric acid extraction was easier than that of acetic acid.     

Anaerobic Degradation of Carrageenan from the Red Macroalga Eucheuma cottonii

Anaerobic degradation of the sulfated polysaccharide carrageenan was investigated by batch digestion of the red macroalga Eucheuma cottonii. During a 10-week incubation, ca. 60% of the starting E. cottonii biomass was fermented to CO₂, methane, and volatile fatty acids (predominantly acetate). Carrageenan degradation paralleled the loss of total biomass, suggesting no preferential degradation or preservation. After 10 weeks of incubation, the carrageenan content of the remaining biomass was 51%, as opposed to 61% of the original E. cottonii biomass. Carrageenan recovered after 10 weeks of digestion had a lower average molecular weight (319,000 versus 510,000) and formed solutions with considerably lower viscosities than did intact carrageenan. The percent C and percent N content of the particulate material in the digestors increased over time, probably as a result of microbial growth. In contrast, the percent S content decreased continuously; the loss of sulfur was most likely a result of the hydrolysis of carrageenan. Results from this study indicate that it is not economically viable to process E. cottonii simultaneously for hydrocolloids and methane.     

餐厨垃圾协同剩余污泥发酵产酸的生物过程与影响因素研究进展

资源化利用是应对餐厨垃圾(Kitchen waste,KW)和剩余污泥(Excess sludge,ES)快速增加的有效方法,而厌氧发酵获得挥发性脂肪酸(Volatile fatty acids,VFAs)是其中的重要方式之一,但单一底物限制了VFAs的高效生产.近年来,不同底物厌氧共发酵产生VFAs被广泛研究与应用,...     

The effect of plant alkaloids on in vitro rumen microbial fermentation

Crude alkaloid extracts from green Italian Ryegrass differed from those extracted from the ensiled grass with particular respect to the perloline moiety. Free perloline and crude alkaloid extracts from both silage and grass inhibited the production of volatile fatty acids in the fermentation of glucose by rumen microbes. With silage as fermentation substrate alkaloid extracts from silage and from grass (0.200 mg/l) caused a decrease in both substrate utilization ( P< < 0.01) and proportion of methane in the gas phase ( P< < 0.01). Molar proportions of acetate were also significantly decreased ( P < 0.01) with a corresponding increase in the proportion of propionate ( P < 0.01). These effects were not observed when grass nuts were used as the fermentation substrate. The results suggest that it is not the alkaloids per se that affect rumen microbial metabolism.     

Quantitative Method for the Gas Chromatographic Analysis of Short-Chain Monocarboxylic and Dicarboxylic Acids in Fermentation Media

A method for the preparation and gas chromatographic analysis of the butyl esters of volatile (C-1-C-7) and nonvolatile (lactic, succinic, and fumaric) acids in microbial fermentation media is presented. Butyl esters were prepared from the dry salts of the acids. The esters were separated by temperature programming on a column of Chromosorb W coated with Dexsil 300 GC liquid phase and analyzed with a flame ionization detector. Apparent recoveries with butanol-HCl or butanol-H2SO4 as butylating agents were 80 to 90% for most acids. Chromatographic profiles of the butyl esters demonstrated that both volatile and nonvolatile acids can be detected and separated in 24 min on a single column. Standard calibration curves (peak area versus concentration) of the butyl esters were linear in the range of 5 to 40 mumol of acid per ml. The advantages of using an internal standard (heptanoic acid) for quantitating fatty acids in a mixture are given. Chromatograms of butylated fermentation media in which rumen anaerobic bacteria were grown illustrated that this method is useful for determining short-chain volatile and nonvolatile acids of toxonomic significance.     

Early diagenesis of organic matter in lacustrine sediments in terms of methane fermentation

The amounts of organic compounds such as amino acids (proteinaceous substances), fatty acids, and sterols in lacustrine sediments were found to decrease greatly from the mud‐water interface to depths of 20 cm and less at greater core depths. The amounts of methane produced by microbial activity in subsamples incubated for 40 days at 27°C were also found to decrease with core depth, apparently limited by the exhaustion of organic substrates subject to methane fermentation. Decomposition rates of certain organic compounds added to sediment samples were determined by incubation experiments at different temperatures for 40 to 450 days. Experimental results with mixed cultures of microorganisms in lacustrine sediments indicated the following: (a) The marked decomposition of organic compounds such as amino acids, fatty acids, and sterols in the lacustrine sediments resulted mainly in the formation of methane, (b) Unsaturated compounds were more readily decomposed with the ultimate formation of methane than saturated compounds, (c) The susceptibility of saturated fatty acids to methane formation decreased in proportion to their molecular weights.     

盐单胞菌利用短链脂肪酸合成聚羟基脂肪酸酯

盐单胞菌(Halomonas)能够利用多种底物为碳源生长,由于其能在高盐条件下进行不灭菌的开放发酵,已被开发用作下一代生物技术的底盘细胞.包括乙酸、丙酸和丁酸在内的短链挥发性脂肪酸能够以生物质为原料制备,有望成为用于微生物发酵的新型碳源.利用10-50g/L浓度的丁酸为碳源对Halomonas sp.TD01和TD08...     

High-Rate two-phase process for the anaerobic degradation of cellulose, employing rumen microorganisms for an efficient acidogenesis

A novel two-stage anaerobic process for the microbial conversion of cellulose into biogas has been developed. In the first phase, a mixed population of rumen bacteria and ciliates was used in the hydrolysis and fermentation of cellulose. The volatile fatty acids (VFA) produced in this acidogenic reactor were subsequently converted into biogas in a UASB-type methanogenic reactor.A stepwise increase of the loading rate from 11.9 to 25.8 g volatile solids/L reactor volume/day (g VS/L/day) did not affect the degradation efficiency in the acidogenic reactor, whereas the methanogenic reactor appeared to be overloaded at the highest loading rate. Cellulose digestion was almost complete at all loading rates applied. The two-stage anaerobic process was also tested with a closed fluid circuit. In this instance total methane production was 0.438 L CH(4)g VS added, which is equivalent to 98% of the theoretical value. The application of rumen microorganisms in combination with a high-rate methane reactor is proposed as a means of efficient anaerobic degradation of cellulosic residues to methane. Because this newly developed two-phase system is based on processes and microorganisms from the ruminant, it will be referred to as "Rumen Derived Anaerobic Digestion" (RUDAD-) process.     

Effect of propionate toxicity on methanogenesis of night soil at phychrophilic temperature

The effect of propionate concentrations on biodegradation of human waste (night soil) was studied at 10 degrees C. Propionate was toxic for the biomethanation at all the pH tested (6.0, 7.0 and 8.0). The maximum reduction in biogas production in presence of 200 mM propionate was observed at pH 7.0 followed by 8.0. The methane content in biogas also followed a similar trend and at pH 7.0 an 11.5% decrease was observed. Propionate caused the reduction of methanogenic count by an approximately 2log value. Total volatile fatty acids increased with the increase in propionate concentration and particularly accumulation of propionate was observed. The results were also compared with the 30 degrees C fermentation.     

Microbial biomass and community structure of a phase-separated methanogenic reactor determined by lipid analysis

Summary An anaerobic phase-separation biomass reactor was established on cellulose with the hydrolysis and fermentation steps occurring in the first stage, and acetogenesis and methanogenesis in the second stage. Based upon lipid biomarker analysis, eubacterial and eukaryotic cells accounted for approximately 6% of the volatile solids of the first stage and 17% of the second, while methanogens were approximately 1% of the volatile solids in the first stage and 9% of the second. Clustering the polar lipid fatty acids into groups based upon their distributions between the two stages of the reactor clarified the differences in community structure caused by phase-separated operation. Although inoculated from the same source, the two stages maintained very different microbial communities. Signature fatty acids known as indicators of unbalanced growth in eubacteria were significantly higher in the first stage of the reactor.     

Dietary starch and rhubarb supplement increase ruminal dissolved hydrogen without altering rumen fermentation and methane emissions in goats

Hydrogen is an important intermediate that is produced during carbohydrate fermentation to volatile fatty acid and utilized by methanogens to produce methane in the rumen. Ruminal volatile fatty acid and dissolved methane concentrations are more than 500 times greater than dissolved hydrogen concentration. Therefore, we hypothesized that dissolved hydrogen might have a higher sensitivity in response to dietary changes compared with volatile fatty acid and dissolved methane. Using goats, we investigated the effects of increasing dietary starch content (maize replaced with wheat bran) and supplementing with rhubarb rhizomes and roots on the relationships among dissolved hydrogen, dissolved methane and other fermentation end products. The study was conducted in a replicated 4×4 Latin square with a 2×2 factorial arrangement of four treatments: two starch levels (220 v. 320 g/kg dry matter (DM)), without and with rhubarb supplement (0% v. 2.8% of total mixed ration). Increased dietary starch and rhubarb supplementation did not alter volatile fatty acid concentrations or methane emissions in terms of g/day, g/g DM intake and g/g organic matter digested. However, goats fed the high-starch diet had greater dissolved hydrogen (P=0.005) and relative abundance of Selenomonas ruminantium (P<0.01), and lower (P=0.02) copy number of protozoa than those fed the low-starch diet. Rhubarb increased ruminal dissolved H₂ (P=0.03) and total volatile fatty acid concentration (P<0.001), but decreased copies of bacteria (P=0.002). In conclusion, dissolved hydrogen appears to be more sensitive to dietary changes with starch content and rhubarb supplementation, when compared with volatile fatty acid concentrations and methane production.     

Effects of culture conditions on Pectinatus cerevisiiphilus and Pectinatus frisingensis metabolism: a physiological and statistical approach

The genus Pectinatus has been often reported in beer spoilage with off-flavours. The bacteria are strictly anaerobic, Gram-negative rods. Propionate and acetate are the main fermentation products from glucose in the two species belonging to the genus, P. cerevisiiphilus and P. frisingensis. Amino acids routinely present at a high level in beer were not growth substrates for both species, and a significant accumulation of succinate was observed with lactate as growth substrate. Both Pectinatus ssp. showed almost identical fermentation balances on glucose. Growth kinetics of both glucose-grown species were unchanged under a N₂, H₂ or 20% CO₂-containing atmosphere. Combinations of culture medium pH values from pH 3·9 to pH 7·2, of glucose levels between 5 and 55 mmol l^-1, and of lactate concentrations varied from 4 to 40 mmol l^-1 demonstrated that biomass and volatile fatty acids production were proportional to glucose concentration for both Pectinatus species. A significant increase of volatile fatty acid production was measured for both species at the lowest pH values with a lactate or a glucose concentration increase. The maximum biomass production was observed at pH 6·2 for P. cerevisiiphilus , and between pH 4·5 and pH 4·9 for P. frisingensis. Glucose and lactate or pH value were dependent with regard to propionate and acetate production in P. frisingensis. On the other hand, the variations of these three parameters were independent with regard to biomass production for both strains, and to volatile fatty acids production for P. cerevisiiphilus. Addition of ethanol to glucose-grown cultures completely inhibited growth at 1·3 mol l^-1 ethanol for P. cerevisiiphilus , and at 1·8 mol l^-1 for P. frisingensis.     

In Vitro Study and Comparison of Caecal Methanogenesis and Fermentation Pattern in the Brown Hare (Lepus europaeus) and Domestic Rabbit (Oryctolagus cuniculus)

The brown hare and the domestic rabbit are mid-sized herbivorous mammals and hindgut fermenters, though their digestive physiologies differ in some traits. The objective of this study was to estimate and compare the caecal microbial activity in hares and rabbits via an analysis of the following end-products of in vitro caecal fermentation: methane, total gas production, short chain fatty acids and ammonia concentration. Hare caecal methanogenesis occurred at a much lower level (0.25 mmol/kg for samples incubated without substrate and 0.22 mmol/kg for samples incubated with substrate) than that of the rabbit (15.49 and 11.73 mmol/kg, respectively) (P<0.001). The impact of the substrate’s presence on caecal methanogenesis was not significant, though its presence increased the total gas production during fermentation (P<0.001). Hare caecal microflora produced a lower short chain fatty acids concentration than did rabbit microorganisms (P<0.05). In unincubated hare samples, the short chain fatty acids concentration was 28.4 mmol/kg, whereas in unincubated rabbit samples, the short chain fatty acids concentration was 51.8 mmol/kg. The caecal fermentation pattern of the hare was characterised by higher propionate and isobutyrate molar proportions compared with those observed in rabbit caecum (P<0.01). No significant changes in the ammonia concentration in either rabbit or hare caecum were found. The results obtained indicate some differences in the activity of the microbial populations colonising the hare and rabbit caecum, particularly in regards to methanogenic Archaea.