Influence of co-substrates on structure of microbial aggregates in long-chain fatty acid-fed anaerobic digesters

AIMS: The purpose of this study was to investigate the influence of co-substrates, such as glucose and cysteine, on the structure of microbial aggregates in anaerobic digesters treating oleate, a long-chain fatty acid (LCFA). METHODS AND RESULTS: Transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) were used to examine the structure of microbial aggregates. Fluorescence in situ hybridization (FISH) techniques were also used to characterize and localize the different trophic groups present in the aggregates. Oleate was found to inhibit the methanogenic activity and formation of granular biomass in digesters. The addition of co-substrates, such as glucose and cysteine either singly or in combination, increased the methanogenic activity and formation of granular biomass. Glucose was more effective than cysteine in reducing the inhibition by oleate on the methanogenic bacteria and in enhancing the formation of granules. CONCLUSIONS: The addition of nutrient substrate, such as glucose and cysteine could decrease the toxicity of LCFA on anaerobic granulation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the addition of other substrates might decrease the toxicity of LCFA on the granulation of biomass in anaerobic digesters and enhance methanogenic activity. A combination of TEM, CLSM and FISH techniques provides a better tool for visualizing microbial aggregates and for differentiating and localizing different microbial groups within these aggregates.     

Control of interspecies electron transfer flow during anaerobic digestion: dynamic diffusion reaction models for hydrogen gas transfer in microbial flocs

Dynamic reaction diffusion models were used to analyze the consequences of aggregation for syntrophic reactions in methanogenic ecosystems. Flocs from a whey digestor were used to measure all model parameters under the in situ conditions of a particular defined biological system. Fermentation simulations without adjustable parameters could precisely predict the kinetics of H(2) gas production of digestor flocs during syntrophic methanogenesis from ethanol. The results demonstrated a kinetic compartmentalization of H(2) metabolism inside the flocs. The interspecies electron transfer reaction was mildly diffusion controlled. The H(2) gas profiles across the flocs showed high H (2) concentrations inside the flocs at any time. Simulations of the syntrophic metabolism at low substrate concentrations such as in digestors or sediments showed that it is impossible to achieve high H(2) gas turnovers at simultaneously low steady-state H(2) concentrations. This showed a mechanistic contradiction in the concept of postulated low H(2) microenvironments for the anaerobic digestion process. The results of the computer experiments support the conclusion that syntrophic H(2) production may only be a side reaction of H(2) independent interspecies electron transfer in methanogenic ecosystems.     

An experimental study of mass diffusion and reaction rate in an anaerobic biofilm

An experimental reactor consisting of two chambers, separated by a porous ceramic immobilization matrix, was constructed to measure the effective diffusivity of different compounds and the consumption rates of acetate in developing biofilms. In initial experiments, effective diffusivities for acetate, propionate, isopropanol, and lithium salt through the ceramic immobilization matrix in the absence of biofilm were determined to be 40% to 50% less than in water at infinite dilution. The effective diffusivity of the lithium salt was similar to that of acetate. The effective diffusivity of the lithium salt through biofilms of thickness in the range of 200 to 1200 mum was essentially constant with a value of approximately 7% of that in water at infinite dilution. Acetate consumption in the biofilm was linearly proportional to biofilm thickness up to a biofilm depth of 800 mum. Deviation from linearity appeared in biofilm thicknesses greater than 800 mum. Results of these experiments support previous reports that immobilized cell reactors have significantly higher bioconversion rates than suspended cell systems.     

Substrate controlled development of anaerobic acidifying aggregates at different shear rates in a gas lift reactor.

The influence of liquid shear rates on the development of acidifying mixed-culture aggregates was studied in a gas-lift reactor. The glucose concentration was kept at a constant and relatively high level by operating the reactor in pH-auxostat mode. Size, strength, and wet density of aggregates cultivated at different superficial gas velocities (Ug) were investigated. Image analysis showed that the Sauter mean diameter (Ds) decreased with increasing Ug. A stirred tank was used to characterize the surface detachment rate (Rd) under non-growth conditions. An exponential decrease was observed in Rd with the applied Ug during cultivation, i.e., aggregates became stronger. The increased strength coincided with an increase in aggregate wet density. Size classified aggregates showed an increase in Rd with the square of the aggregate diameter (Dp), however, this contribution was much smaller than the effect of adaptation. Experiments in a similar gas-lift reactor under dynamic conditions without adaptation, showed that Rd increased exponentially with increasing Ug. So, two important contributions to Rd can be distinguished: adaptation, which induces stronger aggregates, and aggregate size, which makes them less susceptible to hydrodynamic shear. A general expression for Rd was derived, which depends on Dp and Ug. Combining this equation with the surface biomass growth rate (Rg) allowed for the estimation of the maximal diameter (Dmax) aggregates can reach at any Ug, and it was found that the estimated and measured Dmax were in good agreement.     

The evolution of slow dispersal rates: a reaction diffusion model

 We consider n phenotypes of a species in a continuous but heterogeneous environment. It is assumed that the phenotypes differ only in their diffusion rates. With haploid genetics and a small rate of mutation, it is shown that the only nontrivial equilibrium is a population dominated by the slowest diffusing phenotype. We also prove that if there are only two possible phenotypes, then this equilibrium is a global attractor and conjecture that this is true in general. Numerical simulations supporting this conjecture and suggesting that this is a robust phenomenon are also discussed. Received: 29 January 1997 / Revised version: 23 September 1997     

Comparison of long-term performances and final microbial compositions of anaerobic reactors treating landfill leachate

Laboratory scale anaerobic upflow filter, sludge blanket and hybrid bed reactors were operated for 860 days in the treatment of high ammonia landfill leachate. Organic loading was gradually increased from 1.3 to 23.5 kg COD/m3 day in the start-up period and then fluctuated according to the COD concentration of raw leachate. To prevent free ammonia inhibition, influent pH was reduced to 4.5 after Day 181 and consequently COD removal efficiencies above 80% were achieved in all reactors. However, the anaerobic filter and hybrid bed reactor were generally found slightly more efficient and stable than the UASB reactor. In addition to conventional anaerobic reactor control parameters, the complementary techniques of denaturing gradient gel electrophoresis (DGGE), cloning and fluorescent in situ hybridization (FISH) were used to identify and compare the microbial profiles in the reactors at Day 830. Molecular analyses revealed that acetoclastic Methanosaeta species were prevalent in all reactors and configuration did not have an impact on microbial diversity in the long-term.     

Growth and metabolism characteristics of anaerobic ammonium-oxidizing bacteria aggregates

The present study reported the growth and metabolism characteristics of anaerobic ammonium-oxidizing (anammox) bacteria aggregates in an expanded granular sludge bed (EGSB). The results showed that the anammox bacteria aggregates presented starvation, growth, and inhibition phase along with the increase of substrate supply. The substrate conversion rates for survival were 0.05 kgNH ₄ ⁺ –N/(kgVSS·day), 0.07 kgNO ₂ ^? –N/(kgVSS·day), and 0.12 kgN/(kgVSS·day); the substrate conversion rates for maximum growth were 0.21 kgNH ₄ ⁺ –N/(kgVSS·day), 0.24 kgNH ₄ ⁺ –N/(kgVSS·day), and 0.45 kgNH ₄ ⁺ –N/(kgVSS·day), respectively. In the growth phase, the yield of anammox bacteria aggregates was 0.14 gVSS/(gNH ₄ ⁺ –N), 0.12 gVSS/(gNO ₂ ^? –N), and 0.70 gVSS/(gNO ₃ ^? –N); the yield of extracellular polymeric substances (EPS) was 0.11 gEPS/(gNH ₄ ⁺ –N), 0.09 gEPS/(gNO ₂ ^? –N), and 0.55 gEPS/(gNO ₃ ^? –N), respectively. The EPS contents in anammox bacteria aggregates were high compared to that in anaerobic granular sludge. Speculated from the cell yield, the energy for anammox bacteria growth was not only from nitrite oxidation, but also from anammox reaction.     

Immobilized enzymes: Electrokinetic effects on reaction rates under external diffusion

The rates of reactions catalyzed by enzymes immobilized on a nonporous solid surface have been computed employing a Nernst film model. The Nernst-Planck equations for the transport of the charged substrate and product species in the film and the Poisson equation for the distribution of electrical potential are solved numerically with the appropriate boundary conditions. The electrical charge at the surface is assumed to arise from the dissociation equilibria of the acidic and basic surface groups of the enzyme. The pH at the surface affects both the surface charge as well as the intrinsic kinetics of the enzyme-catalyzed reaction. Factors which determine the pH at the surface include the pH in the bulk solution and the release of H(+) ions in the enzyme-catalyzed reaction. The latter causes a lowering of pH at the surface, causing the reaction rate to differ from that computed assuming an equilibrium distribution of electrical potential. Another kind of nonequilibrium contribution is caused by unequal charges or diffusivities of the substrate and products, which results in a diffusion potential being set up. Two moduli are introduced to evaluate the significance of the reaction-generated lowering of pH and the diffusion potential effect. The effect of changing various parameters, e.g., reaction rate constant, substrate concentration, enzyme concentration, pH, etc., on the overall reaction rate are studied.     

Exocellular electron transfer in anaerobic microbial communities

Exocellular electron transfer plays an important role in anaerobic microbial communities that degrade organic matter. Interspecies hydrogen transfer between microorganisms is the driving force for complete biodegradation in methanogenic environments. Many organic compounds are degraded by obligatory syntrophic consortia of proton-reducing acetogenic bacteria and hydrogen-consuming methanogenic archaea. Anaerobic microorganisms that use insoluble electron acceptors for growth, such as iron- and manganese-oxide as well as inert graphite electrodes in microbial fuel cells, also transfer electrons exocellularly. Soluble compounds, like humic substances, quinones, phenazines and riboflavin, can function as exocellular electron mediators enhancing this type of anaerobic respiration. However, direct electron transfer by cell-cell contact is important as well. This review addresses the mechanisms of exocellular electron transfer in anaerobic microbial communities. There are fundamental differences but also similarities between electron transfer to another microorganism or to an insoluble electron acceptor. The physical separation of the electron donor and electron acceptor metabolism allows energy conservation in compounds as methane and hydrogen or as electricity. Furthermore, this separation is essential in the donation or acceptance of electrons in some environmental technological processes, e.g. soil remediation, wastewater purification and corrosion.     

Relationship between microbial activity and microbial community structure in six full-scale anaerobic digesters

High activity levels and balanced anaerobic microbial communities are necessary to attain proper anaerobic digestion performance. Therefore, this work was focused on the kinetic performance and the microbial community structure of six full-scale anaerobic digesters and one lab-scale co-digester. Hydrolytic (0.6–3.5 g COD g^?1 VSS d^?1) and methanogenic (0.01–0.84 g COD g^?1 VSS d^?1) activities depended on the type of biomass, whereas no significant differences were observed among the acidogenic activities (1.5–2.2 g COD g^?1 VSS d^?1). In most cases, the higher the hydrolytic and the methanogenic activity, the higher the Bacteroidetes and Archaea percentages, respectively, in the biomasses. Hydrogenotrophic methanogenic activity was always higher than acetoclastic methanogenic activity, and the highest values were achieved in those biomasses with lower percentages of Methanosaeta. In sum, the combination of molecular tools with activity tests seems to be essential for a better characterization of anaerobic biomasses.     

Changes in the microbial community during repeated anaerobic microbial dechlorination of pentachlorophenol

Pentachlorophenol (PCP) has been widely used as a pesticide in paddy fields and has imposed negative ecological effect on agricultural soil systems, which are in typically anaerobic conditions. In this study, we investigated the effect of repeated additions of PCP to paddy soil on the microbial communities under anoxic conditions. Acetate was added as the carbon source to induce and accelerate cycles of the PCP degradation. A maximum degradation rate occurred at the 11th cycle, which completely transformed 32.3 μM (8.6 mg L^?1) PCP in 5 days. Illumina high throughput sequencing of 16S rRNA gene was used to profile the diversity and abundance of microbial communities at each interval and the results showed that the phyla of Bacteroidates, Firmicutes, Proteobacteria, and Euryarchaeota had a dominant presence in the PCP-dechlorinating cultures. Methanosarcina, Syntrophobotulus, Anaeromusa, Zoogloea, Treponema, W22 (family of Cloacamonaceae), and unclassified Cloacamonales were found to be the dominant genera during PCP dechlorination with acetate. The microbial community structure became relatively stable as cycles increased. Treponema, W22, and unclassified Cloacamonales were firstly observed to be associated with PCP dechlorination in the present study. Methanosarcina that have been isolated or identified in PCP dechlorination cultures previously was apparently enriched in the PCP dechlorination cultures. Additionally, the iron-cycling bacteria Syntrophobotulus, Anaeromusa, and Zoogloea were enriched in the PCP dechlorination cultures indicated they were likely to play an important role in PCP dechlorination. These findings increase our understanding for the microbial and geochemical interactions inherent in the transformation of organic contaminants from iron rich soil, and further extend our knowledge of the PCP-transforming microbial communities in anaerobic soil conditions.     

Mixed aerobic and anaerobic microbial communities in benzene-contaminated groundwater

Aims:  To investigate the factors affecting benzene biodegradation and microbial community composition in a contaminated aquifer.
Methods and Results:  We identified the microbial community in groundwater samples from a benzene-contaminated aquifer situated below a petrochemical plant. Eleven out of twelve groundwater samples with in situ dissolved oxygen concentrations between 0 and 2·57 mg l⁻¹ showed benzene degradation in aerobic microcosm experiments, whereas no degradation in anaerobic microcosms was observed. The lack of aerobic degradation in the remaining microcosm could be attributed to a pH of 12·1. Three groundwaters, examined by 16S rRNA gene clone libraries, with low in situ oxygen concentrations and high benzene levels, each had a different dominant aerobic (or denitrifying) population, either Pseudomonas , Polaromonas or Acidovorax species. These groundwaters also had syntrophic organisms, and aceticlastic methanogens were detected in two samples. The alkaline groundwater was dominated by organisms closely related to Hydrogenophaga .
Conclusions:  Results show that pH 12·1 is inimical to benzene biodegradation, and that oxygen concentrations below 0·03 mg l⁻¹ can support aerobic benzene-degrading communities.
Significance and Impact of the Study:  These findings will help to guide the treatment of contaminated groundwaters, and raise questions about the extent to which aerobes and anaerobes may interact to effect benzene degradation.     

Mechanisms and rates of bacterial colonization of sinking aggregates

Quantifying the rate at which bacteria colonize aggregates is a key to understanding microbial turnover of aggregates. We used encounter models based on random walk and advection-diffusion considerations to predict colonization rates from the bacteria's motility patterns (swimming speed, tumbling frequency, and turn angles) and the hydrodynamic environment (stationary versus sinking aggregates). We then experimentally tested the models with 10 strains of bacteria isolated from marine particles: two strains were nonmotile; the rest were swimming at 20 to 60 microm s(-1) with different tumbling frequency (0 to 2 s(-1)). The rates at which these bacteria colonized artificial aggregates (stationary and sinking) largely agreed with model predictions. We report several findings. (i) Motile bacteria rapidly colonize aggregates, whereas nonmotile bacteria do not. (ii) Flow enhances colonization rates. (iii) Tumbling strains colonize aggregates enriched with organic substrates faster than unenriched aggregates, while a nontumbling strain did not. (iv) Once on the aggregates, the bacteria may detach and typical residence time is about 3 h. Thus, there is a rapid exchange between attached and free bacteria. (v) With the motility patterns observed, freely swimming bacteria will encounter an aggregate in <1 day at typical upper-ocean aggregate concentrations. This is faster than even starving bacteria burn up their reserves, and bacteria may therefore rely solely on aggregates for food. (vi) The net result of colonization and detachment leads to a predicted equilibrium abundance of attached bacteria as a function of aggregate size, which is markedly different from field observations. This discrepancy suggests that inter- and intraspecific interactions among bacteria and between bacteria and their predators may be more important than colonization in governing the population dynamics of bacteria on natural aggregates.     

Changes in microbial ecology in an anaerobic reactor

This study examined the behaviour of the microbial population in an anaerobic reactor, in terms of changes in numbers of total bacterial community, autofluorescent methanogens, non-methanogens and morphology of the autofluorescent methanogens, using epifluorescence microscopy and microbiological enumeration techniques. A laboratory-scale, continuous flow-completely mixed anaerobic reactor, coupled with a conventional gravity settling tank and a continuous recycling system, was operated at an HRT range between 24 and 12 h, using dairy wastewater as the substrate. The numbers of the total bacterial community and autofluorescent methanogens both decreased during start-up. Also, the proportion of the number of autofluorescent methanogens in the total bacterial community varied from 5% to 16% during operation. In particular, the activity of the methane-forming bacteria decreased significantly at HRTs of 16 and 12 h. A membrane module, instead of a conventional settling tank, would obviously have been a more effective method if recycling were required in the anaerobic treatment system.     

Stay connected: Electrical conductivity of microbial aggregates

The discovery of direct extracellular electron transfer offers an alternative to the traditional understanding of diffusional electron exchange via small molecules. The establishment of electronic connections between electron donors and acceptors in microbial communities is critical to electron transfer via electrical currents. These connections are facilitated through conductivity associated with various microbial aggregates. However, examination of conductivity in microbial samples is still in its relative infancy and conceptual models in terms of conductive mechanisms are still being developed and debated. The present review summarizes the fundamental understanding of electrical conductivity in microbial aggregates (e.g. biofilms, granules, consortia, and multicellular filaments) highlighting recent findings and key discoveries. A greater understanding of electrical conductivity in microbial aggregates could facilitate the survey for additional microbial communities that rely on direct extracellular electron transfer for survival, inform rational design towards the aggregates-based production of bioenergy/bioproducts, and inspire the construction of new synthetic conductive polymers.