Isomerization of butyrate to isobutyrate by Desulforhabdus amnigenus

Abstract The isomerization of butyrate and isobutyrate was investigated for the sulfate reducer Desulforhabdus amnigenus . Nuclear magnetic resonance (NMR) studies with ¹³C-labelled butyrate showed that isobutyrate was formed by migration of the carboxyl group, in conformity with the butyrate isomerization reaction reported for methanogenic consortia. In addition to D. amnigenus , several other butyrate-degrading sulfate reducers ( Desulfobacterium vacuolatum, Desulfoarculus baarsii and Desulfotomaculum sp.) were capable of butyrate isomerization.     

Propionate metabolism in a methanogenic enrichment culture. Direct reductive carboxylation and acetogenesis pathways

Abstract Serial dilutions of methanogenic sludges in propionate medium gave a methanogenic non-acetoclastic enrichment degrading 1 mol of propionate to 1.6 mol of acetate and 0.17 mol of methane, with a transient accumulation of butyrate. NMR recordings showed the conversion of [2-¹³C]- and [3-¹³C]-propionate to [3-¹³C]- and [4-¹³C]-butyrate, respectively, thus demonstrating a reductive carboxylation of propionate to butyrate. The labelling found in the accumulated acetate and fermentation balances also suggested that reductive carboxylation was the major pathway involved in propionate conversion to acetate.     

Anaerobic degradation of isobutyrate by methanogenic enrichment cultures and by a Desulfococcus multivorans strain

Methanogenic enrichment cultures with isobutyrate as sole source of carbon and energy were inoculated with sediment and sludge samples from freshwater and marine origin. Over more than 20 transfers, these cultures fermented 2 mol isobutyrate with 1 mol CO₂ via an intermediate formation of n-butyrate to 4 mol acetate and 1 mol CH₄. The primary isobutyrate-fermenting bacteria could not be purified. From one of the marine enrichment cultures, a sulfate-reducing bacterium was isolated which oxidized isobutyrate with sulfate completely to CO₂. Based on its physiological and morphological properties, this strain was assigned to the known species Desulfococcus multivorans. It also oxidized many other fatty acids without significant release of short-chain intermedeates. The enzymes involved in isobutyrate degradation by this bacterium were assayed in cell-free extracts. The results indicate that isobutyrate is activated to its CoA derivative and oxidized via methylmalonate semialdehyde to propionyl-CoA. Propionyl-CoA is further converted via the methylmalonyl-CoA pathway to acetyl-CoA which is finally cleaved by the CO-dehydrogenase system. It is evident that this is not the pathway used by the fermenting bacteria prevailing in the methanogenic enrichment cultures. There results are discussed on the basis of energetical considerations.     

Anaerobic degradation of long-chain dicarboxylic acids by methanogenic enrichment cultures

Abstract Methanogenic enrichment cultures fermented the long-chain dicarboxylates adipate, pimelate, suberate, azelate, and sebacate (C₆-C₁₀) stoichiometrically to acetate and methane. After several transfers, the cultures contained cells of only a few morphologically distinguishable types. During anaerobic degradation of dicarboxylic acids with even-numbered carbon atoms, propionate accumulated intermediately, and butyrate was the intermediate product of degradation of those with an odd number of carbon atoms. Degradation of the long-chain dicarboxylates depended strictly on the presence of hydrogenotrophic methanogens. The primary attack in these processes was β-oxidation rather than decarboxylation. A general scheme of anaerobic degradation of long-chain dicarboxylic acids has been deduced from these results.     

厌氧消化酸抑制研究进展

厌氧消化工艺目前已广泛应用于各类废水的处理处置过程中,但在实际运行中,受消化条件和物料性质的影响,消化系统经常遭受由挥发性脂肪酸积累过多导致的酸抑制问题,引发产气量下降、产甲烷率降低等问题。近年来,有研究者发现,挥发性脂肪酸的种类和浓度及pH、温度是影响酸抑制的主要因素。基于此,相关研究者分别尝试了添加碱性化学药剂和微量元素及利用生物强化技术与微生物电化学技术来解除酸抑制的尝试,并都取得了不错的效果。本文综述了厌氧消化过程中酸抑制的产生过程、抑制机理及恢复方法,以期为解决厌氧消化酸抑制问题提供参考。     

Laboratory scale examination of the effects of overloading on the anaerobic digestion by glycerol

The anaerobic digestion of pure glycerol, which produces a baseline acetic acid to propionic acid ratio of 0.2, was studied in laboratory scale reactors (3 l working volume) at mesophilic temperature (37 °C) with 3000 mg chemical oxygen demand (COD) l⁻¹d⁻¹. During the experiment tVFA and C2-C6 VFA analysis and daily biogas yield measurement were carried out. Following 10 days of a 15% d⁻¹ increase in the organic loading rate (OLR) of 3.0-10.5 g COD l⁻¹d⁻¹, the concentration of propionic acid increased to 6200-8000 mg l⁻¹. Then the inoculum was divided into three parts feeding with 100% glycerol, 50% glycerol + 50% acetic acid, and 50% glycerol + 50% thick stillage, (presented in % of 2.60 g COD l⁻¹d⁻¹ OLR), respectively. The application of co-substrates reduced the recovery period by 5 days compared to feeding with pure glycerol. When the reactors were loaded with glycerol again (10% OLR raise per day) the previously applied co-substrates had a positive effect on the VFA composition and the biogas yield as well.     

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。     

Volatile fatty acids accumulation and rhamnolipid generation in situ from waste activated sludge fermentation stimulated by external rhamnolipid addition

The solubilization and acidification of waste activated sludge (WAS) were apparently enhanced by external rhamnolipid (RL) addition. The maximum solute carbohydrate concentrations increased linearly from 48 ± 5 mg COD L⁻¹ in the un-pretreated WAS (blank) to 566 ± 19 mg COD L⁻¹, and protein increased from 1050 ± 8 to 3493 ± 16 mg COD L⁻¹ at RL dosage of 0.10 g g⁻¹ TSS. The highest VFAs concentration peaked at 3840 mg COD L⁻¹ at RL dosage of 0.04 g g⁻¹ TSS, which was 4.24-fold higher than the blank test. RL was generated in situ during WAS fermentation when external RL was added. It was detected that RL concentration was increased from initial 880 ± 92 mg L⁻¹ to 1312 ± 7 mg L⁻¹ at the end of 96 h with RL dosage of 0.04 g g⁻¹ TSS, which was increased to 1.49-fold. Meanwhile, methane production was notably reduced to a quite low level of 2.0 mL CH₄ g⁻¹ VSS, showing effective inhibition of methanogens by RL (58.8 mL CH₄ g⁻¹ VSS in the blank). In addition, the activity of hydrolytic enzymes (protease and α-glucosidase) was enhanced accordingly. VFAs accumulation and RL generation in situ demonstrated that the additional RL substantially performed enhanced biological effects for waste activated sludge fermentation.     

Uptake of 13C-Tracer Arachidonate and γ-Linolenate by the Brain and Liver of the Suckling Rat Observed Using 13C-NMR

Arachidonic acid (AA; 20:4n-6) is one of the principal components of the phosphoglycerides in neural cell membranes. During the critical period of postnatal development in mammals, AA is supplied preformed, directly from the milk or derived from precursor fatty acids such as gamma-linolenic acid (GLA; 18:3n-6). In this study, 13C-NMR spectroscopy was applied to investigate the incorporation of [1-(13)C]AA and [3-(13)C]GLA into liver and brain lipids of 7-15-day-old rats. The main objective was to establish the importance of dietary GLA for tissue AA accretion relative to the contribution from preformed dietary AA. [1-(13)C]AA and [3-(13)C]GLA were injected into the stomach of 7-day-old rats as a mixture. 13C-NMR spectroscopy of lipid extracts revealed incorporation of [1-(13)C]AA and [5-(13)C]AA (the latter derived from metabolism of the injected [3-(13)C]GLA) into phosphoglycerides and triacylglycerols. Preformed AA was 10 (liver)-17 (brain) times more efficient in contributing to tissue AA than AA derived from precursor GLA. In separate experiments, NMR spectroscopy was used to assess uptake of [1-(13)C]AA directly in living rats and intact organs. Results showed that intact liver and brain contain an appreciable amount of NMR-detectable lipids. The in vivo/in vitro information obtained from organs provided details on the mobility and turnover of tissue lipids.     

Batch culture enrichment of ANAMMOX populations from anaerobic and aerobic seed cultures

Discharge of nitrate and ammonia rich wastewaters into the natural waters encourage eutrophication, and contribute to aquatic toxicity. Anaerobic ammonium oxidation process (ANAMMOX) is a novel biological nitrogen removal alternative to nitrification-denitrification, that removes ammonia using nitrite as the electron acceptor. The feasibility of enriching the ANAMMOX bacteria from the anaerobic digester sludge of a biomethanation plant treating vegetable waste and aerobic sludge from an activated sludge process treating domestic sewage is reported in this paper. ANAMMOX bacterial activity was monitored and established in terms of nitrogen transformations to ammonia, nitrite and nitrate along with formation of hydrazine and hydroxylamine.     

Anaerobic organic acid production of food waste in once-a-day feeding and drawing-off bioreactor

Acidogenesis of food waste was studied in a 2-L reactor with semi-continuous mode operation (once-a-day feeding and draw-off) for maximum 65 days to examine optimal volatile acid compositions for biological nitrogen removal (BNR) and enhanced biological phosphorus removal (ENPR). Various operational parameters of hydraulic retention time (HRT), organic loading rate (ORL), pH and temperature were investigated for soluble chemical oxygen demand (SCOD), volatile fatty acid composition, nitrogen and phosphate. The yields (gTVFA/g VS) and the volumetric productivity (gTVFA/d L) increased with HRT from 0.26-0.32, 1.25-1.50 (at 4 days) to 0.36-0.39, 1.71-1.83 (at 12 days). However, the acetate fraction (%) decreased with HRT from 35.7-37.5 at 4 days to 23.5-25 at 12 days. The yields decreased with increase of organic loading from 0.34-0.37 at 5 g/L d to 0.29-0.30 at 13 g/L d and the productivity increased from 1.63-1.65 to 3.61-3.75. The yield and productivity were highest at 35 degrees C among 25, 35 and 45 degrees C. The yield and productivity at pH 5.5 and 6.0 were best and very similar to each other. The condition of 35 degrees C, pH 6.0, HRT 8 days, ORL 9 g/L d resulted in TVFA, SCOD, acetate and butyrate of 25, 39.5, 12 and 5.25 g/L, respectively.     

Maximum removal rate of propionic acid as a sole carbon source in UASB reactors and the importance of the macro- and micro-nutrients stimulation

The maximum propionic acid (HPr) removal rate (R_HPr) was investigated in two lab-scale Upflow Anaerobic Sludge Bed (UASB) reactors. Two feeding strategies were applied by modifying the hydraulic retention time (HRT) in the UASB_HRT and the influent HPr concentration in the UASB_HPr, respectively. The experiment was divided into three main phases: phase 1, influent with only HPr; phase 2, HPr with macro-nutrients supplementation and phase 3, HPr with macro- and micro-nutrients supplementation. During phase 1, the maximum R_HPr achieved was less than 3 g HPr-COD L⁻¹ d⁻¹ in both reactors. However, the subsequent supplementation of macro- and micro-nutrients during phases 2 and 3 allowed to increase the R_HPr up to 18.1 and 32.8 g HPr-COD L⁻¹ d⁻¹, respectively, corresponding with an HRT of 0.5 h in the UASB_HRT and an influent HPr concentration of 10.5 g HPr-COD L⁻¹ in the UASB_HPr. Therefore, the high operational capacity of these reactor systems, specifically converting HPr with high throughput and high influent HPr level, was demonstrated. Moreover, the presence of macro- and micro-nutrients is clearly essential for stable and high HPr removal in anaerobic digestion.     

A phylogenetic analysis of Wadi el Natrun soda lake cellulase enrichment cultures and identification of cellulase genes from these cultures

Samples of sediments and surrounding soda soils (SS) from the extremely saline and alkaline lakes of the Wadi el Natrun in the Libyan Desert, Egypt, were obtained in October 2000. Anaerobic enrichment cultures were grown from these samples, DNA isolated, and the bacterial diversity assessed by 16S rRNA gene clone analysis. Clones derived from lake sediments (LS) most closely matched Clostridium spp., Natronoincola histidinovorans, Halocella cellulolytica, Bacillus spp., and the Cytophaga–Flexibacter–Bacteroides group. Similar clones were identified in the SS, but Bacillus spp. predominated. Many of the clones were most closely related to organisms already identified in alkaline or saline environments. Two genomic DNA libraries were made from the pooled LS enrichments and a single SS-enrichment sample. A novel cellulase activity was identified and characterized in each. The lake cellulase ORF encoded a protein of 1,118 amino acids; BLASTP analysis showed it was most closely related to an endoglucanase from Xanthomonas campestris. The soil-cellulase ORF encoded a protein of 634 amino acids that was most closely related to an endoglucanase from Fibrobacter succinogenes.     

Specific removal of chlorine from the ortho-position of halogenated benzoic acids by reductive dechlorination in anaerobic enrichment cultures

Anaerobic enrichment cultures catalysing the reductive dechlorination of chlorinated benzoic acids were obtained from three fresh-water sediments collected from seven different locations. Sub-cultures from these enrichments specifically removed ortho-substituted chlorine from 2,3,6-, 2,3,5- and 2,4,6-trichlorobenzoic acid, yielding chloride and 2,5-, 3,5-, and 2,4-dichlorobenzoic acids, respectively. These reductive dehalogenations were stimulated by the addition of benzoate and/or volatile organic acids. In one of these enrichments dehalogenation of ortho- and/or para-chlorine substituents was also observed from 2,3-, 2,4-, 2,5-, and 3,4-dichlorobenzoic acid, yielding 3- and 4-chlorobenzoate. Removal of meta-chlorines was not observed in any of the enrichments.     

Growth media in anaerobic fermentative processes: The underestimated potential of thermophilic fermentation and anaerobic digestion

Fermentation and anaerobic digestion of organic waste and wastewater is broadly studied and applied. Despite widely available results and data for these processes, comparison of the generated results in literature is difficult. Not only due to the used variety of process conditions, but also because of the many different growth media that are used. Composition of growth media can influence biogas production (rates) and lead to process instability during anaerobic digestion. To be able to compare results of the different studies reported, and to ensure nutrient limitation is not influencing observations ascribed to process dynamics and/or reaction kinetics, a standard protocol for creating a defined growth medium for anaerobic digestion and mixed culture fermentation is proposed. This paper explains the role(s) of the different macro- and micronutrients, as well as the choices for a growth medium formulation strategy. In addition, the differences in nutrient requirements between mesophilic and thermophilic systems are discussed as well as the importance of specific trace metals regarding specific conversion routes and the possible supplementary requirement of vitamins. The paper will also give some insight into the bio-availability and toxicity of trace metals. A remarkable finding is that mesophilic and thermophilic enzymes are quite comparable at their optimum temperatures. This has consequences for the trace metal requirements of thermophiles under certain conditions. Under non-limiting conditions, the trace metal requirement of thermophilic systems is about 3 times higher than for mesophilic systems.     

LDL particle core enrichment in cholesteryl oleate increases proteoglycan binding and promotes atherosclerosis

Several studies in humans and animals suggest that LDL particle core enrichment in cholesteryl oleate (CO) is associated with increased atherosclerosis. Diet enrichment with MUFAs enhances LDL CO content. Steroyl O-acyltransferase 2 (SOAT2) is the enzyme that catalyzes the synthesis of much of the CO found in LDL, and gene deletion of SOAT2 minimizes CO in LDL and protects against atherosclerosis. The purpose of this study was to test the hypothesis that the increased atherosclerosis associated with LDL core enrichment in CO results from an increased affinity of the LDL particle for arterial proteoglycans. ApoB-100-only Ldlr^−/− mice with and without Soat2 gene deletions were fed diets enriched in either cis-MUFA or n-3 PUFA, and LDL particles were isolated. LDL:proteogylcan binding was measured using surface plasmon resonance. Particles with higher CO content consistently bound with higher affinity to human biglycan and the amount of binding was shown to be proportional to the extent of atherosclerosis of the LDL donor mice. The data strongly support the thesis that atherosclerosis was induced through enhanced proteoglycan binding of LDL resulting from LDL core CO enrichment.     

Transport and metabolism of exogenous fumarate and 3-phosphoglycerate in vascular smooth muscle

The keto (linear) form of exogenous fructose 1,6-bisphosphate, a highly charged glycolytic intermediate, may utilize a dicarboxylate transporter to cross the cell membrane, support glycolysis, and produce ATP anaerobically. We tested the hypothesis that fumarate, a dicarboxylate, and 3-phosphoglycerate (3-PG), an intermediate structurally similar to a dicarboxylate, could support contraction in vascular smooth muscle during hypoxia. To assess ATP production during hypoxia we measured isometric force maintenance in hog carotid arteries during hypoxia in the presence or absence of 20 mM fumarate or 3-PG. 3-PG improved maintenance of force (p < 0.05) during the 30-80 min period of hypoxia. Fumarate decreased peak isometric force development by 9.5% (p = 0.008) but modestly improved maintenance of force (p < 0.05) throughout the first 80 min of hypoxia. 13C-NMR on tissue extracts and superfusates revealed 1,2,3,4-13C-fumarate (5 mM) metabolism to 1,2,3,4-13C-malate under oxygenated and hypoxic conditions suggesting uptake and metabolism of fumarate. In conclusion, exogenous fumarate and 3-PG readily enter vascular smooth muscle cells, presumably by a dicarboxylate transporter, and support energetically important pathways.     

Selecting for lactic acid producing and utilising bacteria in anaerobic enrichment cultures

Lactic acid-producing bacteria are important in many fermentations, such as the production of biobased plastics. Insight in the competitive advantage of lactic acid bacteria over other fermentative bacteria in a mixed culture enables ecology-based process design and can aid the development of sustainable and energy-efficient bioprocesses. Here we demonstrate the enrichment of lactic acid bacteria in a controlled sequencing batch bioreactor environment using a glucose-based medium supplemented with peptides and B vitamins. A mineral medium enrichment operated in parallel was dominated by Ethanoligenens species and fermented glucose to acetate, butyrate and hydrogen. The complex medium enrichment was populated by Lactococcus, Lactobacillus and Megasphaera species and showed a product spectrum of acetate, ethanol, propionate, butyrate and valerate. An intermediate peak of lactate was observed, showing the simultaneous production and consumption of lactate, which is of concern for lactic acid production purposes. This study underlines that the competitive advantage for lactic acid-producing bacteria primarily lies in their ability to attain a high biomass specific uptake rate of glucose, which was two times higher for the complex medium enrichment when compared to the mineral medium enrichment. The competitive advantage of lactic acid production in rich media can be explained using a resource allocation theory for microbial growth processes.     

Variability of hydrocarbon and fatty acid components in cultures of the filamentous cyanobacterium Scytonema sp. isolated from microbial community "black cover" of limestone walls in Jerusalem

The hydrocarbon and lipid components of four strains of the filamentous cyanobacterium Scytonema sp. isolated from microbial community Black Cover of limestone walls in Jerusalem were identified by gas chromatography–mass spectrometry using serially coupled capillary columns. The dominant compounds were: 1-heptadecyne (1.5-8%), hexadecanoic acid (14-36%), (Z,Z)-9,12-octadecadienoic acid (12-30%), (Z,Z,Z)-9,12,15-octadecatrienoic acid (6-12%), n-heptadecane (4-16%), and 1-heptadecene (1.5-8%). In addition to unsaturated alkanes and fatty acids, the very long-chain (C₃₀-C₃₂) hydrocarbons, squalene (2.4-3.0%), and branched 4,8,12-trimethyl-C_13:0 acid were also isolated. Two major hydrocarbons were detected in the cyanobacteria species using GC-MS and ¹³C-NMR.     

Common vetch varietal differences in hay nutritive value,ruminal fermentation,nutrient digestibility and performance of fattening lambs

In some lower rainfall regions of the world (300–750 mm), common vetch (Vicia sativa L., hereafter referred to as vetch) has been shown to have yields competitive with alternate crops and provide high-quality hay for ruminant diets, but there are few studies of vetch performance as a livestock feed, or of vetch varietal differences in livestock feeding value. This study evaluated vetch varietal differences in hay nutritive value, ruminal fermentation properties, nutrient digestibility, nitrogen retention and animal performance in fattening lambs consuming a diet comprising 20% vetch. Fifty male Hu lambs with an initial BW of 17.7 ± 0.27 kg and 2–3 months of age were assigned randomly into five groups of 10 lambs, and each allocated one of five dietary treatments for 67 days (10 days of adaptation and a 57 day experimental period). All diets contained 30% maize stover, 50% concentrate, and with a different forage source (on an as-fed basis): 20% alfalfa hay (Control), 20% vetch 333A (C333A) hay, 20% vetch Lanjian No. 1 (CLJ1) hay, 20% vetch Lanjian No. 2 (CLJ2) hay, or 20% vetch Lanjian No. 3 (CLJ3) hay. Hay CLJ3 had greater contents of ash, CP, ether extract, in vitro organic matter digestibility and metabolizable energy, and lower cell wall contents (P < 0.05) than those of C333A and CLJ1 hays, but similar to the CLJ2 hay (P > 0.05). Compared to the Control diet, the CLJ2 and CLJ3 diets resulted in greater (P < 0.05) final BW, average daily gain, total tract apparent digestibility of CP and NDF, and nitrogen balance, both when expressed as g/day and relative to nitrogen intake, while animal performance when fed diets with C333A or CLJ1 diet did not differ (P > 0.05) from the Control diet. No differences (P > 0.05) were observed between dietary treatments with respect to average daily feed intake, ruminal pH, total volatile fatty acid contents and molar proportions of acetate, butyrate, valerate, isobutyrate, and isovalerate, or total tract apparent digestibility of DM, organic matter, and ADF. The feed efficiency of tested feeds ranked CLJ3 = CLJ2 > CLJ1 > Control with C333A intermediate between CLJ1 and Control. In summary, considering hay quality, nutrient digestibility and animal weight gain, performance as a ruminant feed of hays from recently released vetch cultivars Lanjian No. 2 and Lanjian No. 3 was superior to the older cultivar C333A and the alfalfa control.