Effect of temperature on microbial community of a glucose-degrading methanogenic consortium under hyperthermophilic chemostat cultivation

We continuously fed an anaerobic chemostat with synthetic wastewater containing glucose as the sole source of carbon and energy to study the effects of temperature on the microbial community under hyperthermophilic (65-80 degrees C) conditions. Methane was produced normally up to 77.5 degrees C at a dilution rate of 0.025 d(-1). However, the concentration of microorganisms and the rate of gas production decreased with increasing operation temperature. The microbial community in the chemostat at various temperatures was analyzed based on the 16S rRNA gene using molecular biological techniques including clone library analysis and denaturing gradient gel electrophoresis (DGGE). Aceticlastic methanogens related to Methanosarcina thermophila were detected at 65 degrees C and hydrogenotrophilic methanogens related to Methanothermobacter thermautotrophicus were the dominant methanogens between 70 degrees C to 77.5 degrees C. Bacteria related to Clostridium stercorarium and Thermoanaerobacter subterraneus comprised the dominant glucose-fermenting bacteria at temperatures of 65 degrees C and above, respectively. Bacteria related to Thermacetogenium phaeum and to Tepidiphilus margaritifer and Petrobacter succinatimandens were the dominant acetate-oxidizing bacteria at 70 degrees C and at 75-77.5 degrees C, respectively. The results suggested that, at temperatures of 70 degrees C and above, methane production via the aceticlastic pathway was negligible and indirect methanogenesis from acetate was dominant. Since acetate oxidation is a rate limiting step and a higher temperature favors the hydrolysis and acid formation, a two stage fermentation process, acidogenic and methanogenic fermentation stages operated under different temperatures, should be more suitable for the thermophilic anaerobic treatment at temperatures above 65 degrees C.     

Microbial community analysis of mesophilic anaerobic protein degradation process using bovine serum albumin (BSA)-fed continuous cultivation

Two mesophilic anaerobic chemostats, one without added Ni2+ and Co2+ (chemostat 1) and the other with added Ni2+ and Co2+ (chemostat 2), were supplied with synthetic wastewater containing bovine serum albumin (BSA) as the sole carbon and energy source in order to study the capacity of protein degradation, microbial community structure and the effects of the addition of trace metals. Volatile fatty acids and ammonia were the main products of chemostat 1, while methane, CO2 and ammonia were the main products of chemostat 2, and critical dilution rates of 0.15 d-1 and 0.08 d-1 were obtained, respectively. Fluorescence in situ hybridization (FISH) with archaeal and bacterial domain-specific probes showed that archaeal cells were very limited in chemostat 1 while large populations of several types of archaeal cells were present in chemostat 2. Phylogenetic analyses based on 16S rRNA gene clonal sequences, DGGE, and quantitative real-time polymerase chain reaction (PCR) showed that, within the domain Archaea, methanogens affiliated with the genera Methanosaeta and Methanoculleus were predominant in chemostat 2. Within the domain Bacteria, rRNA genes obtained from chemostat 1 were affiliated with the three phyla; Firmicutes (43%), Bacteroidetes (50%) and Proteobacteria (7%). A total of 56% of rRNA genes obtained from chemostat 2 was affiliated with the three phyla, Firmicutes (32%), Bacteroidetes (11%) and Proteobacteria (13%) while 44% of rRNA genes remained unclassified. Phylogenetically distinct clones were obtained in these two chemostats, suggesting that different protein degradation pathways were dominant in the two chemostats: coupled degradation of amino acids via the Stickland reaction in chemostat 1 and uncoupled degradation of amino acids via syntrophic association of amino acid degraders and hydrogenotrophic methanogens in chemostat 2.     

Formaldehyde-limited cultivation of a newly isolated methylotrophic bacterium, Methylobacterium sp. MF1: enzymatic analysis related to C1 metabolism

Formaldehyde is a highly toxic compound to most living organisms. We have isolated a bacterial strain that is able to efficiently degrade formaldehyde and use it as a sole carbon source. The isolated strain was identified as Methylobacterium sp. MF1, which could grow on formaldehyde and methanol. Methylobacterium sp. MF1 was grown in batch culture using 1.2 g/l formaldehyde as a sole carbon source, which was all consumed within 200 h. In order to decompose formaldehyde more efficiently, formaldehyde-limited chemostat cultivation of Methylobacterium sp. MF1 was investigated. Formaldehyde was consumed at 1.7 g/l/d when the dilution rate was 0.012 h(-1). Under these conditions, the cell turbidity (OD610) reached 2.0. Furthermore, when the initial turbidity was adjusted to 3.0 using methanol-grown cells, continuous cultivation could be started at an initial dilution rate of 0.008 h(-1). Using these conditions, consumption of formaldehyde could be continued for at least 600 h. The enzyme activities of cells growing as a chemostat culture, using methanol or formaldehyde as a sole carbon source, were compared to that of C1 metabolism. No difference was detected in the enzyme activities for the oxidation and assimilation of C1 compounds between the two cell-free extracts. Furthermore, methanol dehydrogenase activity was detected at the same level when formaldehyde was used as a sole carbon source. These results suggest that the resistance to the toxic effects of formaldehyde exhibited by Methylobacterium sp. MF1 is related to factors other than C1 metabolism.     

驼、牛、羊乳中脂肪酸含量的比较分析

采用气相色谱法对驼、牛、羊乳3 种乳中脂肪酸（fatty acids,FA）种类及含量进行比较。结果表明,3 种乳脂肪酸种类和含量差异显著。驼乳检测出33 种脂肪酸,棕榈酸相对含量最高（（23.73±2.17）%）;驼乳的多不饱和脂肪酸（polyunsaturated fatty acids,PUFA）相对含量（（4.09±0.54）%）显著高于牛乳和羊乳;牛乳中检测出29 种脂肪酸,棕榈酸相对含量最高（（27.44±2.45）%）;羊乳检测出34 种脂肪酸,且油酸相对含量最高（（30.89±3.57）%）。3 种乳中羊乳单不饱和脂肪酸（monounsaturated fatty acids,MUFA）含量最高（（35.26±3.86）%）。此外,结合主成分分析和相关性分析方法,对驼乳的脂肪酸含量进行进一步分析,结果显示,驼乳的饱和脂肪酸（saturated fatty acids,SFA）∶MUFA∶PUFA为1∶0.60∶0.08,驼乳更接近联合国粮食及农业组织/世界卫生组织推荐SFA∶MUFA∶PUFA为1∶1∶1。PUFA与短链脂肪酸（short-chain fatty acids,SCFA）、长链脂肪酸（long-chain fatty acids,LCFA）呈负相关,与n-6 FA相关性最大（0.978）;SCFA与LCFA呈正相关,n-6 FA与n-3 FA呈正相关。     

Effect of dilution rate on structure of a mesophilic acetate-degrading methanogenic community during continuous cultivation

The community structures of two mesophilic acetate-degrading methanogenic consortia enriched at dilution rates of 0.025 and 0.6 d(-1) were analyzed by fluorescence in situ hybridization (FISH) and phylogenetic analyses based on 16S rDNA clonal sequences and quantitative real-time polymerase chain reaction (PCR). FISH experiments with archaeal and bacterial domain-specific probes showed that archaeal cells were predominant and only a small number of bacterial cells were detected at both dilution rates. In the domain Archaea, the number of cells closely related to Methanosarcina barkeri was shown to be greater at the high dilution rate using FISH with species-specific probes. Taxonomic analyses based on rDNA clonal sequences obtained at the low and high dilution rates showed that 43% of 100 clones and 72% of 92 clones, respectively, were affiliated with the domain Archaea and the remainders at each dilution rate were affiliated with the domain Bacteria. Within the domain Archaea, all rDNA clones at both dilution rates were affiliated with the genera Methanosaeta or Methanosarcina of the aceticlastic methanogens. Within the domain Bacteria, the rDNA clones obtained at the low dilution rate were affiliated with four phyla, Firmicutes (36%), Bacteroidetes (9%), Chloroflexi (6%) and candidate division OP12 (5%). The rDNA clones obtained at the high dilution rate were affiliated with four phyla, Firmicutes (16%), Bacteroidetes (8%), Proteobacteria (1%) and candidate division OP12 (3%). Real-time quantitative PCR experiments showed that the number of rDNA sequences affiliated with the genus Methanosarcina was greater at the high dilution rate. In addition, a significant number of rDNA sequences affiliated with the genus Methanoculleus were detected only at the low dilution rate. Detection of a hydrogenotrophic methanogen at the low dilution rate suggests that the syntrophic acetate oxidation by hydrogenotrophic methanogens and acetate-oxidizing bacteria could occur at the low dilution rate.     

日粮添加不同来源脂肪酸对奶牛脂质代谢及激素水平的影响

研究日粮中添加不同来源脂肪酸对泌乳中期奶牛脂质代谢及激素水平的影响。选择36头泌乳中期中国荷斯坦奶牛,采用随机区组设计,按照产奶量、胎次及泌乳日龄将奶牛分为对照组、中短链脂肪酸(SMC-FA)组和长链脂肪酸(LCFA)组。对照组饲喂基础日粮,SMCFA组和LCFA组分别在基础日粮的基础上添加400g/d SMCFA和400 g/d LCFA,正试期为56 d。结果显示,LCFA组奶牛血清中非酯化脂肪酸的含量显著高于对照组(P<0.05),其余组间差异不显著(P>0.05);血清中胆固醇的含量在SMCFA和LCFA组均有增加的趋势,但未达到显著水平(P>0.05);胰岛素样生长因子的含量在SMCFA组显著降低(P<0.05),其余组间没有显著差异(P>0.05);添加LCFA或SMCFA对奶牛血清中三酰甘油、高密度脂蛋白、低密度脂蛋白、生长激素及胰岛素均没有显著影响(P>0.05)。结果表明日粮中添加SMCFA对泌乳中期奶牛脂质代谢有一定的负面影响。     

Efficiency of energy utilization by mixed rumen bacteria in continuous culture.

Mixed bacterial cultures derived from the rumen were grown in a remen fluid medium in a chemostat at three dilution rates (.02, .06, and .12 per h), each at four growth-limiting glucose concentrations (5.8, 9.9, 12.7, and 25.0 mM). Microscopic observations indicated that a relatively complex mixture of bacterial species was maintained and proportions of fermentations products were similar to those of the rumen except for elevated proportions of methane and acetate. Cell concentration increased linearly with increases in glucose concentration. The range of glucose concentrations had little effect on yields of cells or products produced per mole of glucose fermented. With increases in dilution rates, the amount of butyrate and methane produced per mole of glucose fermented decreased and the amount of propionate increased. Yield glucose (grams cells produced per mole of glucose fermented) increased from 42 at a dilution rate of .02 to 84 at a dilution rate of .12. These large increases are discussed in relationship to the energy requirements for maintenance of bacteria. A theoretical maximum yield glucose of 89.3 and a maintenance requirement of .26 mmol glucose per g cells per h were calculated. Moles of adenosine triphosphate produced per mole of glucose fermented and yield of cells produced per mole of adenosine triphosphate are discussed.     

Effects of Long‐Chain and Medium‐Chain Fatty Acids on Apoptosis and Oxidative Stress in Human Liver Cells with Steatosis

Nonalcoholic fatty liver disease (NAFLD) is closely associated with obesity‐related metabolic complications, which caused by excess energy intake and physical inactivity apart from genetic defects. The mechanisms that promote disease progression from NAFLD to further liver injury are still unclear. We hypothesize that the progression involved “2nd hit” is strongly influenced by the type of fatty acids in diets. Flow cytometric analysis showed that medium‐chain fatty acid (MCFA) markedly decreased the percentage of late apoptotic and necrotic cells compared with long‐chain fatty acid (LCFA), and MCFA inhibited the activities of caspase‐3 and ‐9 in human liver cells with steatosis. Western blot analysis found that the levels of inflammatory markers (interleukin [IL]‐6, IL‐1‐β, and tumor necrosis factor‐α) were substantially reduced by MCFA compared with LCFA. Proteomic analysis further showed that LCFA inhibited the expression of antioxidant enzymes, and increased the expression of proteins associated with oxidative stress. It was found that LCFA (palmitate), not MCFA induced apoptosis, oxidative stress and chronic inflammatory responses in the hepatic cells with steatosis. In conclusion, reasonable selection of dietary fats has potential to translate therapeutically by ameliorating disease progression in patients with NAFLD.     

Development of a strain of Hansenula polymorpha for the efficient expression of guar alpha-galactosidase.

A strain of the methylotrophic yeast Hansenula polymorpha, A16, has been developed that expresses the guar alpha-galactosidase gene to 22.4 mg/g dry cell weight in chemostat cultures at a dilution rate of 0.1 h(-1). This corresponds to more than 13.1% of soluble cell protein, of which 56-62% is secreted into the medium. The alpha-galactosidase gene was flanked by the promoter and terminator sequences of the H.polymorpha mox gene, which can direct expression of the mox gene itself more than 30% of total cell protein under methanol growth. The expression cassette (pUR3510) based on the Saccharomyces cerevisiae plasmid, YEp13, was integrated into the genome. Such transformants were stable in chemostat cultures and exhibited 100% stability for both alpha-galactosidase+ and leu+ phenotypes. Chemostat cultures produced higher levels of alpha-galactosidase with higher specific productivities expressed as mg alpha-galactosidase g(-1) h(-1) compared to batch cultures.     

Nitrogen removal via nitrite from municipal wastewater at low temperatures using real-time control to optimize nitrifying communities

Although many studies regarding nitrogen removal via nitrite have been carried out, very limited research has been undertaken on nitrogen removal via nitrite at low temperatures. In this study, to improve the nitrogen removal efficiency from municipal wastewater, a pilot-plant of sequencing batch reactor with a working volume of 54 m3 was used to investigate nitrogen removal via nitrite from municipal wastewater at normal and low water temperature. The obtained results showed that high nitrogen removal efficiency with effluent total nitrogen below 3 mg/L could be achieved. Using real-time control with temperature ranging from 11.9 to 26.5 degrees C under normal dissolved oxygen condition (> or =2.5 mg/L), nitrogen removal via nitrite was successfully and stably achieved for a long period (180 days) with average nitrite accumulation rate above 95%. Fluorescence in situ hybridization was carried out to investigate the quantitative changes of nitrifying microbial community in the activated sludge. Fluorescence in situ hybridization results approved that the nitrifying microbial communities were optimized; ammonia oxidizing bacteria became the dominant nitrifying bacteria and nitrite oxidizing bacteria had been washed out of the activated sludge.     

Comparison of Growth Response by Chemostat Cultured and Batch Cultured Clostridium perfringens Cells in Various Food Substrates

A constant source of exponential phase cells of Clostridium perfringens ATCC 3624 could reduce the time required to carry out the protein quality evaluation test of Solberg et al. (1979) to 4 hr. Clostridium perfringens ATCC 3624 was grown in an anaerobic chemostat using the chemically defined medium, R&S, with glucose as the growth limiting nutrient. The dilution rate was set at 0.06 hr^?1, the pH at 7.2, and the temperature at 43°C. In the transition from a batch culture to a continuous culture an initial oscillatory cell density response was observed. In the steady state, which was continued for as long as 50 days, the cells were typical Gram positive rods, occurring singly or in chains as long as 15 rods, which were occasionally without septa. The fermentative and biochemical responses of the cells did not change. No sporulation occurred when the cells were growing in the chemostat, but spores were observed in the glucose free culture effluent after incubation at 37°C for 24 hr. When cells produced in the chemostat, were cultured in complex media they demonstrated a growth response similar to cells which had been grown in a batch culture. In defined medium the generation time for the chemostat cultured cells was decreased approximately 17%. The chemostat cultured cells can be used as inoculum for the C. perfringens protein quality assay.     

Microbial community in methanogenic packed-bed reactor successfully operating at short hydraulic retention time

The microbial community in a thermophilic anaerobic packed-bed reactor, which had been successfully operated to convert acetic and butyric acids to methane at a short hydraulic retention time (from 24 h to 1.9 h), was investigated. Archaea closely related to known methanogens were detected by 16S rRNA gene analyses of the effluents, together with diverse types of unidentified bacteria.     

Lipid and fatty acid composition and lipid peroxidation of sublimated mare's milk after its long term preservation using antioxidants]

Sublimated mare's milk was stored with and without antioxidants during one year. The antioxidants quercetin and bisalcophen were introduced before drying in a dose of 0.02% relative to milk fat. Lipid, fatty acid composition and lipid peroxidation (LPO) parameters (diene and triene conjugates, total lipid oxidation) were studied immediately after drying and after 3, 6, 9 and 12 months of storage. No significant changes were detected in lipid, fatty-acid composition and LPO rate after 3 months of dried milk storage without additives. Significant deterioration of lipid, fatty-acid composition, increase of LPO and oxidation of milk were recorded after 6 months. Addition of quercetin and bisalcophen promoted stabilization of fatty-acid composition and prolonged the term of sublimated mare's milk storage.     

Bacterial production of organic acids enhances H2O2-dependent iodide oxidation

To develop an understanding of the role that microorganisms play in the transport of (129)I in soil-water systems, bacteria isolated from subsurface sediments were assessed for iodide oxidizing activity. Spent liquid medium from 27/84 bacterial cultures enhanced iodide oxidation 2-10 fold in the presence of H(2)O(2). Organic acids secreted by the bacteria were found to enhance iodide oxidation by (1) lowering the pH of the spent medium, and (2) reacting with H(2)O(2) to form peroxy carboxylic acids, which are extremely strong oxidizing agents. H(2)O(2)-dependent iodide oxidation increased exponentially from 8.4 to 825.9 μM with decreasing pH from 9 to 4. Organic acids with ≥2 carboxy groups enhanced H(2)O(2)-dependent iodide oxidation (1.5-15-fold) as a function of increasing pH above pH 6.0, but had no effect at pH ≤ 5.0. The results indicate that as pH decreases (≤5.0), increasing H(2)O(2) hydrolysis is the driving force behind iodide oxidation. However, at pH ≥ 6.0, spontaneous decomposition of peroxy carboxylic acids, generated from H(2)O(2) and organic acids, contributes significantly to iodide oxidation. The results reveal an indirect microbial mechanism, organic acid secretion coupled to H(2)O(2) production, that could enhance iodide oxidation and organo-iodine formation in soils and sediments.     

Kinetics of growth and glucose transport in glucose-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066

The glucose transport capacity of Saccharomyces cerevisiae CBS 8066 was studied in aerobic glucose-limited chemostat cultures. Two different transport systems were encountered with affinity constants of 1 and 20 mM, respectively. The capacity of these carriers (Vmax) was dependent on the dilution rate and the residual glucose concentration in the culture. From the residual glucose concentration in the fermenter and the kinetic constants of glucose transport, their in situ contribution to glucose consumption was determined. The sum of these calculated in situ transport rates correlated well with the observed rate of glucose consumption of the culture. The growth kinetics of S. cerevisiae CBS 8066 in glucose-limited cultures were rather peculiar. At low dilution rates, at which glucose was completely respired, the glucose concentration in the fermenter was constant at 110 microM, independent of the glucose concentration in the reservoir. At higher dilution rates, characterized by the occurrence of both respiration and alcoholic fermentation, the residual substrate concentration followed Monod kinetics. In this case, however, the overall affinity constant was dependent on the reservoir glucose concentration.     

Selective production of lactic acid in continuous anaerobic acidogenesis by extremely low pH operation

The selective production of lactic acid by anaerobic acidogenesis with low pH control was examined using a chemostat culture. By decreasing culture pH to 3.5 in a chemostat culture containing mixed microbial populations for anaerobic acidogenesis, heterolactic fermentation became dominant, resulting in the selective production of lactic acid and ethanol. This phenomenon was reversible between the acidic and neutral conditions, and was not affected by the dilution rate. The extremely low pH operation was effective for selective lactic acid production in anaerobic acidogenesis.     

Fatty acid composition of the lipids of whitefish

The fatty-acid composition of lipids was studied in the muscular tissue and internal organs of whitefish living in Siberian rivers, that had been moved to lakes where it had not lived before. Changes in the lipid composition were recorded in mature fish moved into the lakes and in this year's brood. It was found that lipids of mature fish were resistant to oxidizing damage.     

Segregation of biomass in cyclic anaerobic/aerobic granular sludge allows the enrichment of anaerobic ammonium oxidizing bacteria at low temperatures

A cyclic anaerobic/aerobic bubble column reactor was run for 420 days to study the competition for nitrite between nitrite oxidizing bacteria (NOB) and anaerobic ammonium oxidizing bacteria (Anammox) at low temperatures. An anaerobic feeding period with nitrite and ammonium in the influent followed by an aerated period was applied resulting in a biomass specific conversion rate of 0.18 ± 0.02 [gN(2) - N · gVSS(-1)· day(-1)] when the dissolved oxygen concentration was maintained at 1.0 mgO(2) · L(-1). An increase in white granules was observed in the reactor which were mainly located at the top of the settled sludge bed, whereas red granules were located at the bottom. FISH, activity tests, and qPCR techniques revealed that red biomass was dominated by Anammox bacteria and white granules by NOB. Granules from the top of the sludge bed were smaller and therefore had a higher aerobic volume fraction, a lower density, and consequently a slower settling rate. Sludge was manually removed from the top of the settled sludge bed to selectively remove NOB which resulted in an increased overall biomass specific N-conversion rate of 0.32 ± 0.02 [gN(2) - N · gVSS(-1) · day(-1)]. Biomass segregation in granular sludge reactors gives an extra opportunity to select for specific microbial groups by applying a different SRT for different microbial groups.