Degradation of suspended proteins in an Anaerobic Rotating Bed Contactor

Summary A protein rich suspension, obtained from spent grains, was digested in an Anaerobic Rotating Bed Contactor (AnRBC) at 37°C, pH=7 and a hydrolic retention time of 1 hr. The hydrolysis and acidification of the suspension was about 50% on organic carbon basis at reactor loads of 2.5 kg TOC/m3.d (7.5 kg COD/m3.d). A further increase of the conversion rate seems quite possible with an improved reactor configuration.     

含易降解有机质时对苯二甲酸(TA)厌氧降解动力学

含易降解有机物的对苯二甲酸有机废水在厌氧处理时,其中的易降解有机质通过甲烷发酵被优先利用,其中间代谢物对对苯二甲酸的降解有抑制作用,同时,对苯二甲酸自身的降解也存在底物抑制现象。本文建立了有易降解有机质存在时对苯二甲酸厌氧降解的抑制动力学模型方程：q=q_max SS+Ks[1+(I-0.86)/K_I,s,并利用非线性回归,确定了模型参数q_max=1972.0mgTA/gVSS·d;K_s=20.2844gTA/L;K_I,I=2.041gCOD/L;K_I,s=0.0108 gTA/L,实验数据对该动力学方程能较好拟合。在此基础上提出两段厌氧处理新工艺。     

Enrichment and Detection of Microorganisms Involved in Direct and Indirect Methanogenesis from Methanol in an Anaerobic Thermophilic Bioreactor

To gain insight into the microorganisms involved in direct and indirect methane formation from methanol in a laboratory-scale thermophilic (55°C) methanogenic bioreactor, reactor sludge was disrupted and serial dilutions were incubated in specific growth media containing methanol and possible intermediates of methanol degradation as substrates. With methanol, growth was observed up to a dilution of 10⁸. However, when Methanothermobacter thermoautotrophicus strain Z245 was added for H₂ removal, growth was observed up to a 10¹⁰-fold dilution. With H₂/CO₂ and acetate, growth was observed up to dilutions of 10⁹ and 10⁴, respectively. Dominant microorganisms in the different dilutions were identified by 16S rRNA-gene diversity and sequence analysis. Furthermore, dilution polymerase chain reaction (PCR) revealed a similar relative abundance of Archaea and Bacteria in all investigated samples, except in enrichment with acetate, which contained 100 times less archaeal DNA than bacterial DNA. The most abundant bacteria in the culture with methanol and strain Z245 were most closely related to Moorella glycerini. Thermodesulfovibrio relatives were found with high sequence similarity in the H₂/CO₂ enrichment, but also in the original laboratory-scale bioreactor sludge. Methanothermobacter thermoautotrophicus strains were the most abundant hydrogenotrophic archaea in the H₂/CO₂ enrichment. The dominant methanol-utilizing methanogen, which was present in the 10⁸-dilution, was most closely related to Methanomethylovorans hollandica. Compared to direct methanogenesis, results of this study indicate that syntrophic, interspecies hydrogen transfer-dependent methanol conversion is equally important in the thermophilic bioreactor, confirming previous findings with labeled substrates and specific inhibitors.     

Organic Matter Mineralization with Reduction of Ferric Iron in Anaerobic Sediments

The potential for ferric iron reduction with fermentable substrates, fermentation products, and complex organic matter as electron donors was investigated with sediments from freshwater and brackish water sites in the Potomac River Estuary. In enrichments with glucose and hematite, iron reduction was a minor pathway for electron flow, and fermentation products accumulated. The substitution of amorphous ferric oxyhydroxide for hematite in glucose enrichments increased iron reduction 50-fold because the fermentation products could also be metabolized with concomitant iron reduction. Acetate, hydrogen, propionate, butyrate, ethanol, methanol, and trimethylamine stimulated the reduction of amorphous ferric oxyhydroxide in enrichments inoculated with sediments but not in uninoculated or heat-killed controls. The addition of ferric iron inhibited methane production in sediments. The degree of inhibition of methane production by various forms of ferric iron was related to the effectiveness of these ferric compounds as electron acceptors for the metabolism of acetate. The addition of acetate or hydrogen relieved the inhibition of methane production by ferric iron. The decrease of electron equivalents proceeding to methane in sediments supplemented with amorphous ferric oxyhydroxides was compensated for by a corresponding increase of electron equivalents in ferrous iron. These results indicate that iron reduction can outcompete methanogenic food chains for sediment organic matter. Thus, when amorphous ferric oxyhydroxides are available in anaerobic sediments, the transfer of electrons from organic matter to ferric iron can be a major pathway for organic matter decomposition.     

Keratin Degradation by Fervidobacterium pennavorans, a Novel Thermophilic Anaerobic Species of the Order Thermotogales

From a hot spring of the Azores islands a novel thermophilic bacterium belonging to the Thermotogales order was isolated. This strain, which grows optimally at 70(deg)C and pH 6.5, is the first known extreme thermophile that is able to degrade native feathers at high temperatures. The enzyme system converts feather meal to amino acids and peptides. On the basis of physiological, morphological, and 16S rDNA studies the new isolate was found to be a member of the Thermotogales order and was identified as Fervidobacterium pennavorans. The strain was highly related to Fervidobacterium islandicum and Fervidobacterium pullulanolyticum. The cell-bound keratinolytic enzyme system was purified 32-fold by detergent treatment with CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was characterized as a serine protease with a molecular mass of 130 kDa and an isoelectric point of 3.8. Optimal activity was measured at 80(deg)C and pH 10.0. Furthermore, 19 anaerobic thermophilic archaea and bacteria belonging to the orders Thermococcales, Thermoproteales, Thermotogales, and Clostridiales (growth temperatures between 60 and 105(deg)C) were tested for their abilities to grow on feathers and produce heat-stable keratinolytic enzymes. None of the tested extremophilic microorganisms was able to attack the substrate in a native form.     

Physiological and Molecular Characterisation of an Anaerobic Thermophilic Oleate-degrading Enrichment Culture

Thermophilic oleate-degrading bacteria were enriched in mineral medium in association withMethanobacterium thermoautotrophicum . The enrichment culture also produced methane from linear saturated fatty acids (two to 18 carbon atoms), and fermented crotonate to acetate and butyrate, with a stoichiometry similar to that reported forSyntrophomonaswolfei . Two thermophilic, anaerobic members of the domain Bacteria were isolated and phylogenetically characterised as Coprothermobacter proteolyticus and Anaerobaculum thermoterrenum. Amplified ribosomal DNA restriction analysis of the members of the domain Bacteria in the enrichment culture showed that there were three dominant restriction patterns, two of which corresponded to those of the isolated non-syntrophic organisms. The sequence of the third pattern clustered with a group of bacteria that degrade C4 and higher fatty acids in syntrophic association. However, it diverged considerably from the closest thermophilic relatives, Syntrophothermus lipocalidus and Thermosyntropha lipolytica, indicating the presence of a new thermophilic long-chain fatty acid-degrading bacterium in the enrichment culture.     

Kinetics of Butyrate, Acetate, and Hydrogen Metabolism in a Thermophilic, Anaerobic, Butyrate-Degrading Triculture

Kinetics of butyrate, acetate, and hydrogen metabolism were determined with butyrate-limited, chemostat-grown tricultures of a thermophilic butyrate-utilizing bacterium together with Methanobacterium thermoautotrophicum and the TAM organism, a thermophilic acetate-utilizing methanogenic rod. Kinetic parameters were determined from progress curves fitted to the integrated form of the Michaelis-Menten equation. The apparent half-saturation constants, K_m, for butyrate, acetate, and dissolved hydrogen were 76 μM, 0.4 mM, and 8.5 μM, respectively. Butyrate and hydrogen were metabolized to a concentration of less than 1 μM, whereas acetate uptake usually ceased at a concentration of 25 to 75 μM, indicating a threshold level for acetate uptake. No significant differences in K_m values for butyrate degradation were found between chemostat- and batch-grown tricultures, although the maximum growth rate was somewhat higher in the batch cultures in which the medium was supplemented with yeast extract. Acetate utilization was found to be the rate-limiting reaction for complete degradation of butyrate to methane and carbon dioxide in continuous culture. Increasing the dilution rate resulted in a gradual accumulation of acetate. The results explain the low concentrations of butyrate and hydrogen normally found during anaerobic digestion and the observation that acetate is the first volatile fatty acid to accumulate upon a decrease in retention time or increase in organic loading of a digestor.     

Bacterial Degradation of Dissolved Organic Matter from Two Northern Michigan Streams

This study used high-pressure size exclusion chromatography (HPSEC) to measure the changes in molecular weight distributions of dissolved organic matter (DOM) of two Northern Michigan streams following inoculation with bacterial concentrates from the same locations. During the initial 12 h of the experiment, weight average molecular weight (M _w ) of DOM decreased, as high molecular weight components were lost from solution. After 12 h, the M _w of DOM increased, primarily because of a loss of intermediate to lower molecular weight components. Leucine incorporation showed little or no bacterial metabolism during the first 12 h, but metabolism increased substantially after 12 h. The initial loss of high molecular weight components during the period of little or no bacterial metabolism suggests preferential adsorption of these components to the bacterial surfaces, perhaps followed by metabolism. This suggested interpretation is consistent with previous observations of preferential adsorption of higher molecular weight components to viable but non-metabolizing Bacillus subtilis and to mineral surfaces. The latter loss of lower molecular weight components was most likely due to bacterial metabolism of the DOM, which is consistent with previous observations that lower molecular weight components are more biodegradable. The HPSEC technique uses 254 nm wavelength for detection and focuses primarily on humic- and fulvic-type components rather than low molecular weight organic molecules, such as carbohydrates. Thus, results confirmed that humic/fulvic components are biodegradable, but did not address other DOM components.     

A Stochastic Model for the Synthesis and Degradation of Natural Organic Matter. Part I. Data Structures and Reaction Kinetics

Here we present a stochastic biogeochemical model for the formation, transformation and mineralization of natural organic matter (NOM). The model is agent-based, with each software agent representing a single molecule of defined composition. Molecular properties and reactivities are estimated from composition and environmental parameters. Environmental parameters including temperature, pH, light intensity, dissolved O₂, moisture and enzyme activities are user controlled. Time is treated in discrete steps, and during each step potential reaction probabilities are evaluated for each molecule based on its structure and the environmental parameters. When reactions occur, the molecular composition is modified accordingly. The model uses small natural products and biopolymers for inputs, and the composition of the molecules produced is constrained only by the inputs and reaction stoichiometries, not by pre-defined structures. Example simulations using the program AlphaStep are presented, in which the breakdown of biopolymers and the condensation of small molecules both lead to molecular assemblages with elemental composition and average properties similar to those of aquatic NOM. This batch-reactor model can be expanded to include spatial information and environmental feedback.     

Growth Kinetics of Microbacterium lacticum and Nitrate-Dependent Degradation of Ethylbenzene under Anaerobic Conditions

A pure strain of Microbacterium lacticum DJ-1 capable of anaer-obic biodegradation of ethylbenzene was isolated from soil contaminated with gasoline. Growth of the strain and biodegradation of ethylbenzene in batch cultures led to stoichiometric reduction of nitrate. M. lacticum DJ-1 could degrade 100 mg L^?1 of ethylbenzene completely, with a maximum degradation rate of 15.02 ± 1.14 mg L^?1 day^?1. Increasing the initial concentration of ethy-lbenzene resulted in decreased degradative ability. The cell-specific growth rates on ethylbenzene conformed to the Haldane–Andrew model in the substrate level range of 10–150 mg L^?1. Kinetic parameters were determined by nonlinear regression on specific growth rates and various initial substrate concentrat-ions, and the values of the maximum specific growth rate, half saturation constant, and inhibition constant were 0.71 day^?1, 34.3 mg L^?1, and 183.5 mg L^?1, respectively. This is the first report of ethylbenzene biodegradation by a bacterium of Microbacterium lacticum under nitrate-reducing conditions.     

Organic Matter Degradation Drives Benthic Cyanobacterial Mat Abundance on Caribbean Coral Reefs

Benthic cyanobacterial mats (BCMs) are impacting coral reefs worldwide. However, the factors and mechanisms driving their proliferation are unclear. We conducted a multi-year survey around the Caribbean island of Curaçao, which revealed highest BCM abundance on sheltered reefs close to urbanised areas. Reefs with high BCM abundance were also characterised by high benthic cover of macroalgae and low cover of corals. Nutrient concentrations in the water-column were consistently low, but markedly increased just above substrata (both sandy and hard) covered with BCMs. This was true for sites with both high and low BCM coverage, suggesting that BCM growth is stimulated by a localised, substrate-linked release of nutrients from the microbial degradation of organic matter. This hypothesis was supported by a higher organic content in sediments on reefs with high BCM coverage, and by an in situ experiment which showed that BCMs grew within days on sediments enriched with organic matter (Spirulina). We propose that nutrient runoff from urbanised areas stimulates phototrophic blooms and enhances organic matter concentrations on the reef. This organic matter is transported by currents and settles on the seabed at sites with low hydrodynamics. Subsequently, nutrients released from the organic matter degradation fuel the growth of BCMs. Improved management of nutrients generated on land should lower organic loading of sediments and other benthos (e.g. turf and macroalgae) to reduce BCM proliferation on coral reefs.     

螺旋纤维床固定化生物反应器同时产酶降解壳聚糖的研究

采用多孔聚酯泡沫固定里氏木霉,在鼓泡柱固定化反应器中同时产酶降解壳聚糖。结果表明通过控制降解时间可以得到不同平均聚合度的降解产物。在28℃,pH4.8,通气量3vvm条件下,利用固定化反应器,在30d内连续进行10批同时产酶降解试验,结果发现壳聚糖酶活力和壳聚糖降解率能保持稳定。每批产生的壳聚糖酶活力平均达到0.15u/mL以上,壳聚糖平均降解率为73%。     

Impact of an Aerobic Thermophilic Sequencing Batch Reactor on Antibiotic-Resistant Anaerobic Bacteria in Swine Waste

The introduction of antibiotics to animal feed has contributed to the selection of antibiotic-resistant bacteria in concentrated animal feeding operations. The aim of this work was to characterize the impact of an aerobic thermophilic biotreatment on anaerobic antibiotic-resistant bacteria in swine waste. Despite 162- to 6,166-fold reduction in antibiotic-resistant populations enumerated in the swine waste at 25°C and 37°C, resistant populations remained significant (10⁴ to 10⁵ most probable number per milliliter) in the treated swine waste. Five resistance genes were detected before [tet(LMOS) erm(B)], and six resistance genes were detected after [tet(LMOSY) erm(B)] biotreatment. However, the biotreatment decreased the frequency of detection of resistance genes by 57%. Analysis by denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16 S ribosomal DNA (rDNA) fragments showed that the biotreatment reduced the bacterial diversity of resistant populations enumerated at 37°C. Cloning and sequencing of the 16 S rDNA of these populations revealed that most clones in the treated swine waste were closely similar to some of the clones retrieved from the untreated swine waste. This study revealed that the aerobic thermophilic biotreatment developed in our laboratory does not prevent the introduction of facultatively anaerobic antibiotic-resistant bacteria and their resistance genes into agricultural ecosystems. Horizontal transfer of ecologically advantageous genes within microbial communities are likely to prevent thermophilic biotreatments from completely eliminating antibiotic-resistant bacteria and their resistance genes in animal wastes.     

The Intracellular pH of Clostridium paradoxum, an Anaerobic, Alkaliphilic, and Thermophilic Bacterium

When the extracellular pH was increased from 7.6 to 9.8, Clostridium paradoxum, a novel alkalithermophile, increased its pH gradient across the cell membrane ((Delta)pH, pH(infin) - pH(infout)) by as much as 1.3 U. At higher pH values (>10.0), the (Delta)pH and membrane potential ((Delta)(psi)) eventually declined, and the intracellular pH increased significantly. Growth ceased when the extracellular pH was greater than 10.2 and the intracellular pH increased to above 9.8. The membrane potential increased to 110 (plusmn) 8.6 mV at pH 9.1, but the total proton motive force ((Delta)p) declined from about 65 mV at pH 7.6 to 25 mV at pH 9.8. Between the extracellular pH of 8.0 and 10.3, the intracellular ATP concentration was around 1 mM and decreased at lower and higher pH values concomitantly with a decrease in growth rate.     

Detection of Putatively Thermophilic Anaerobic Methanotrophs in Diffuse Hydrothermal Vent Fluids

The anaerobic oxidation of methane (AOM) is carried out by a globally distributed group of uncultivated Euryarchaeota, the anaerobic methanotrophic arachaea (ANME). In this work, we used G+C analysis of 16S rRNA genes to identify a putatively thermophilic ANME group and applied newly designed primers to study its distribution in low-temperature diffuse vent fluids from deep-sea hydrothermal vents. We found that the G+C content of the 16S rRNA genes (P_GC) is significantly higher in the ANME-1GBa group than in other ANME groups. Based on the positive correlation between the P_GC and optimal growth temperatures (T_opt) of archaea, we hypothesize that the ANME-1GBa group is adapted to thrive at high temperatures. We designed specific 16S rRNA gene-targeted primers for the ANME-1 cluster to detect all phylogenetic groups within this cluster, including the deeply branching ANME-1GBa group. The primers were successfully tested both in silico and in experiments with sediment samples where ANME-1 phylotypes had previously been detected. The primers were further used to screen for the ANME-1 microorganisms in diffuse vent fluid samples from deep-sea hydrothermal vents in the Pacific Ocean, and sequences belonging to the ANME-1 cluster were detected in four individual vents. Phylotypes belonging to the ANME-1GBa group dominated in clone libraries from three of these vents. Our findings provide evidence of existence of a putatively extremely thermophilic group of methanotrophic archaea that occur in geographically and geologically distinct marine hydrothermal habitats.     

Cluster Structure of Anaerobic Aggregates of an Expanded Granular Sludge Bed Reactor

The metabolic properties and ultrastructure of mesophilic aggregates from a full-scale expanded granular sludge bed reactor treating brewery wastewater are described. The aggregates had a very high methanogenic activity on acetate (17.19 mmol of CH₄/g of volatile suspended solids [VSS]·day or 1.1 g of CH₄ chemical oxygen demand/g of VSS·day). Fluorescent in situ hybridization using 16S rRNA probes of crushed granules showed that 70 and 30% of the cells belonged to the archaebacterial and eubacterial domains, respectively. The spherical aggregates were black but contained numerous whitish spots on their surfaces. Cross-sectioning these aggregates revealed that the white spots appeared to be white clusters embedded in a black matrix. The white clusters were found to develop simultaneously with the increase in diameter. Energy-dispersed X-ray analysis and back-scattered electron microscopy showed that the whitish clusters contained mainly organic matter and no inorganic calcium precipitates. The white clusters had a higher density than the black matrix, as evidenced by the denser cell arrangement observed by high-magnification electron microscopy and the significantly higher effective diffusion coefficient determined by nuclear magnetic resonance imaging. High-magnification electron microscopy indicated a segregation of acetate-utilizing methanogens (Methanosaeta spp.) in the white clusters from syntrophic species and hydrogenotrophic methanogens (Methanobacterium-like and Methanospirillum-like organisms) in the black matrix. A number of physical and microbial ecology reasons for the observed structure are proposed, including the advantage of segregation for high-rate degradation of syntrophic substrates.     

Effect of Biowaste Sludge Maturation on the Diversity of Thermophilic Bacteria and Archaea in an Anaerobic Reactor

Prokaryotic diversity was investigated near the inlet and outlet of a plug-flow reactor. After analyzing 800 clones, 50 bacterial and 3 archaeal phylogenetic groups were defined. Clostridia (>92%) dominated among bacteria and Methanoculleus (>90%) among archaea. Significant changes in pH and volatile fatty acids did not invoke a major shift in the phylogenetic groups. We suggest that the environmental filter imposed by the saline conditions (20 g liter⁻¹) selected a stable community of halotolerant and halophilic prokaryotes.The anaerobic digestion of organic wastes constitutes a major research focus due to the global needs for waste recycling and renewable energy production. Currently, the linkage between digester performance and the diversity and dynamics of anaerobic prokaryotes is still not fully understood (2). Bacterial diversity in anaerobic reactors has always been judged to be greater than archaeal diversity (9, 13, 30). This probably reflects the metabolic flexibility of bacteria and the range of available substrates in complex input materials. However, several recent discoveries pose the question as to whether archaeal diversity and physiological versatility are greater than currently thought: that is, the huge diversity of yet-to-be cultured archaea (4, 6), the detection of energy metabolisms not known previously in archaea (e.g., chemoorganotrophy [1]), and the unexpected predominance of archaeal groups among prokaryotes in unstressed environments, such as ammonia oxidizers in soils (19).Several surveys have investigated the shifts in prokaryotic diversity occurring with waste maturation or under different reactor operating conditions. Some evidence demonstrates bacterial phylogenetic stability under constant operation conditions (18). Generally, however, the dominant bacterial communities are very dynamic, showing chaotic shifts even with stable reactor performance (9, 32). Hypothetically, this is due to the functional redundancy among diverse phylogenetic groups allowing oscillations of their populations with no effects on the reactor function (2). Archaeal communities are less dynamic than bacterial communities (32), their shifts being related to changes in reactor performance (6) and correlated with important process parameters such as volatile fatty acids (VFAs) (13, 16).We aimed to analyze the change in prokaryotic diversity in a plug-flow reactor associated with the maturation of biowastes. In a previous study, stable bacterial and archaeal denaturing gradient gel electrophoresis patterns were found in the sludge collected close to the outlet over a year of unstable reactor performance (23). This temporal pattern contradicts the general idea of extremely dynamic bacterial communities proliferating in bioreactors. Here, we investigated the phylogenetic identity of the organisms in sludge samples collected near the inlet and outlet pipes after a period of stable operation and performance in terms of pH and biogas production.     

螺旋纤维床固定化反应器两步法生产壳聚糖酶

采用了菌体生长与产酶分步的新工艺利用里氏木霉（Trichoderma reesei） ATCC56764生产壳聚糖酶,酶活力比在相同条件下进行的一步法产酶提高了1.7倍。采用此工艺在螺旋纤维床生物反应器中进行产酶试验,酶活比采用游离细胞培养又提高了39%,达到0.246U/mL。固定化菌丝还能够长期保持活性,在重复分批操作中,经过10批共15天的产酶实验,平均每批的酶活保持在0.235U/mL左右。