Adaption of microbial community during the start-up stage of a thermophilic anaerobic digester treating food waste

A successful start-up enables acceleration of anaerobic digestion (AD) into steady state. The microbial community influences the AD performance during the start-up. To investigate how microbial communities changed during the start-up, microbial dynamics was analyzed via high-throughput sequencing in this study. The results confirmed that the AD was started up within 25 d. Thermophilic methanogens and bacterial members functioning in hydrolysis, acidogenesis, and syntrophic oxidation became predominant during the start-up stage, reflecting a quick adaption of microorganisms to operating conditions. Such predominance also indicated the great contribution of these members to the fast start-up of AD. Redundancy analysis confirmed that the bacterial abundance significantly correlated with AD conditions. The stable ratio of hydrogenotrophic methanogens to aceticlastic methanogens is also important to maintain the stability of the AD process. This work will be helpful to understand the contribution of microbial community to the start-up of AD.     

Concurrent microscopic observations and activity measurements of cellulose hydrolyzing and methanogenic populations during the batch anaerobic digestion of crystalline cellulose

This study compares process data with microscopic observations from an anaerobic digestion of organic particles. As the first part of the study, this article presents detailed observations of microbial biofilm architecture and structure in a 1.25-L batch digester where all particles are of an equal age. Microcrystalline cellulose was used as the sole carbon and energy source. The digestions were inoculated with either leachate from a 220-L anaerobic municipal solid waste digester or strained rumen contents from a fistulated cow. The hydrolysis rate, when normalized by the amount of cellulose remaining in the reactor, was found to reach a constant value 1 day after inoculation with rumen fluid, and 3 days after inoculating with digester leachate. A constant value of a mass specific hydrolysis rate is argued to represent full colonization of the cellulose surface and first-order kinetics only apply after this point. Additionally, the first-order hydrolysis rate constant, once surfaces were saturated with biofilm, was found to be two times higher with a rumen inoculum, compared to a digester leachate inoculum. Images generated by fluorescence in situ hybridization (FISH) probing and confocal laser scanning microscopy show that the microbial communities involved in the anaerobic biodegradation process exist entirely within the biofilm. For the reactor conditions used in these experiments, the predominant methanogens exist in ball-shaped colonies within the biofilm.     

Effects of acetate and propionate on the performance of a photosynthetic biofilm reactor for sulfide removal

The effects of acetate and propionate on the performance of a recently proposed and characterized photosynthetic biological sulfide removal system have been investigated with a view to predicting this concept's suitability for removing sulfide from wastewater undergoing or having undergone anaerobic treatment. The concept relies on substratum-irradiated biofilms dominated by green sulfur bacteria (GSB), which are supplied with radiant energy in the band 720-780 nm. A model reactor was fed for 7 months with a synthetic wastewater free of volatile fatty acids (VFAs), after which time intermittent dosing of the wastewater with acetate or propionate was begun. Such dosing suppressed the areal net sulfide removal rate by approximately 50%, and caused the principal net product of sulfide removal to switch from sulfate to elemental-S. Similarly suppressed values of this rate were observed when the wastewater was dosed continuously with acetate, and this rate was not significantly affected by changes in the concentration of ammonia-N in the feed. The main net product of sulfide removal was again elemental-S, which was scarcely released into the liquid, however. Sulfate reduction and sulfur reduction were observed when the light supply was interrupted and were inferred to be occurring within the irradiated biofilm. A preexisting conceptual model of the biofilm was augmented with both of these reductive processes, and this augmented model was shown to account for most of the observed effects of VFA dosing. The implications of these findings for the practicality of the technology are considered.     

Effect of operating variables on biofilm formation and performance of an anaerobic fluidized-bed bioreactor

The effect of various operating variables such as initial inoculum circulation, dilution rate, chemical oxygen demand (COD) loading rate, and quantity and quality of inoculum on the process of film formation on sand surface and reactor performance were studied using synthetic glucose based wastewater. It was found that the film formation process is favored by a high dilution rate, a large quantity of inoculum, and an inoculum having high methane producing capacity. Experimental observations indicate that the biofilm formation process is initiated by methanogenic bacteria.     

Inhibition of the anaerobic digestion process by linear alkylbenzene sulfonates

Linear Alkylbenzene Sulfonates (LAS) are the most widely used synthetic anionicsurfactants. They are anthropogenic, toxic compounds and are found in the primarysludge generated in municipal wastewater treatment plants. Primary sludge is usuallystabilized anaerobically and therefore it is important to investigate the effect of thesexenobiotic compounds on an anaerobic environment. The inhibitory effect of LinearAlkylbenzene Sulfonates (LAS) on the acetogenic and methanogenic step of theanaerobic digestion process was studied. LAS inhibit both acetogenesis from propionate and methanogenesis from acetate and hydrogen and it is shown that the propionate-utilising bacteria are more sensitive to the presence of LAS than the acetoclastic methanogens. It has been proven that the inhibition intensity depends on the solids concentration and thus the term ``biomass specific LAS concentration' has been introduced in order to describe the phenomenon better. Conclusively, it is believed that the inhibitory effect of LAS is the main reason that anaerobic microbial enrichments on LAS have not been succeeded yet. Also, the inhibition caused by LAS on the acetogenic and methanogenic step of the anaerobic digestion process should be seriously taken into consideration when wastewater from a surfactant producing industry is to be treated biologically or enter a municipal wastewater treatment plant that employs anaerobic technology. The upper allowable biomass specific LAS concentration should be 14 mg LAS (gVSS)^-1.     

Microbial population dynamics during start-up and overload conditions of anaerobic digesters treating municipal solid waste and sewage sludge

Microbial population dynamics were investigated during start-up and during periods of overload conditions in anaerobic co-digesters treating municipal solid waste and sewage sludge. Changes in community structure were monitored using ribosomal RNA-based oligonucleotide probe hybridization to measure the abundance of syntrophic propionate-oxidizing bacteria (SPOB), saturated fatty acid-beta-oxidizing syntrophs (SFAS), and methanogens. These changes were linked to traditional performance parameters such as biogas production and volatile fatty acid (VFA) concentrations. Digesters with high levels of Archaea started up successfully. Methanosaeta concilii was the dominant aceticlastic methanogen in these systems. In contrast, digesters that experienced a difficult start-up period had lower levels of Archaea with proportionally more abundant Methanosarcina spp. Syntrophic propionate-oxidizing bacteria and saturated fatty acid-beta-oxidizing syntrophs were present at low levels in all digesters, and SPOB appeared to play a role in stabilizing propionate levels during start-up of one digester. Digesters with a history of poor performance tolerated a severe organic overload event better than digesters that had previously performed well. It is hypothesized that higher levels of SPOB and SFAS and their methanogenic partners in previously unstable digesters are responsible for this behavior.     

Microbial community structure in a biofilm anode fed with a fermentable substrate: The significance of hydrogen scavengers

We compared the microbial community structures that developed in the biofilm anode of two microbial electrolysis cells fed with ethanol, a fermentable substrate—one where methanogenesis was allowed and another in which it was completely inhibited with 2‐bromoethane sulfonate. We observed a three‐way syntrophy among ethanol fermenters, acetate‐oxidizing anode‐respiring bacteria (ARB), and a H₂ scavenger. When methanogenesis was allowed, H₂‐oxidizing methanogens were the H₂ scavengers, but when methanogenesis was inhibited, homo‐acetogens became a channel for electron flow from H₂ to current through acetate. We established the presence of homo‐acetogens by two independent molecular techniques: 16S rRNA gene based pyrosequencing and a clone library from a highly conserved region in the functional gene encoding formyltetrahydrofolate synthetase in homo‐acetogens. Both methods documented the presence of the homo‐acetogenic genus, Acetobacterium, only with methanogenic inhibition. Pyrosequencing also showed a predominance of ethanol‐fermenting bacteria, primarily represented by the genus Pelobacter. The next most abundant group was a diverse community of ARB, and they were followed by H₂‐scavenging syntrophic partners that were either H₂‐oxidizing methanogens or homo‐acetogens when methanogenesis was suppressed. Thus, the community structure in the biofilm anode and suspension reflected the electron‐flow distribution and H₂‐scavenging mechanism. Biotechnol. Bioeng. 2010;105: 69–78. © 2009 Wiley Periodicals, Inc.     

Effective biofilm control in a membrane biofilm reactor using a quenching bacterium (Rhodococcus sp. BH4)

The biofilm thickness in membrane biofilm reactors (MBfRs) is an important factor affecting system performance because excessive biofilm formation on the membrane surface inhibits gas diffusion to the interior of the biofilm, resulting in a significant reduction in the performance of contaminant removal. This study provides innovative insights into the control of biofilm thickness in O₂-based MBfRs by using the quorum quenching (QQ) method. The study was carried out in MBfRs operated at different gas pressures and hydraulic retention times (HRTs) using QQ beads containing Rhodococcus sp. BH4 at different amounts. The highest performance was observed in reactors operated with 0.21 ml QQ bead/cm² membrane surface area, 12 HRTs and 1.40 atm. Over this period, the performance increase in chemical oxygen demand (COD) removal was 25%, while the biofilm thickness on the membrane surface was determined to be 250 μm. Moreover, acetate and equivalent oxygen flux results reached 6080 and 10 640 mg·m⁻²·d⁻¹ maximum values, respectively. The extracellular polymeric substances of the biofilm decreased significantly with the increase of gas pressure and QQ beads amount. Polymerase chain reaction denaturing gradient gel electrophoresis results showed that the microbial community in the MBfR system changed depending on operating conditions and bead amount. The results showed that the QQ method was an effective method to control the biofilm thickness in MBfR and provide insights for future research.     

Characterization of a methanogenic sludge to be used as inoculum for a high-rate reactor

The sludge of an anaerobic lagoon treating the wastewater from a factory producing baker's yeast was evaluated as inoculum for anaerobic digestion. Specific methanogenic activity tests failed to give a good estimation of the trophic groups that were evidenced by enumerations involving Most Probable Number estimations. This failure was ascribed to the toxic effects of either the acetate concentrations used or to the ammonia content of the sludge.     

Modelling MSW decomposition under landfill conditions considering hydrolytic and methanogenic inhibition

A landfill typically progresses through a series of microbial degradation phases, in which hydrolysis, production and consumption of fermentation products, such as fatty acids, and methane formation play important roles. For ultimate degradation of the waste, stable methanogenic conditions have to be attained, and maintained for sufficient time. Using experimental data from 100-L landfill simulation reactors containing municipal solid waste from a residential area, a distributed model, which accounts for vertical water flow, was developed. As a first step, the waste was divided into two fractions: readily degradable and recalcitrant waste. Secondly, the general hydrolysis of the recalcitrant waste was accounted for by including a specific, well-defined chemical substance in the model that generally occurs in Municipal Solid Waste (MSW) and is hydrolysed before its further degradation to methane. For this purpose we chose diethyl phthalate and its hydrolysis product monoethyl phthalate, for which leachate data are available from the reactors. The model indicated that inhibition of the hydrolytic and methanogenic processes occurred during␣the acidogenic phase and that it could be overcome either by improving the chemical environment or by the complete oxidation of the inhibiting, i.e. the easily degraded, fraction of the waste. The generality of the model was confirmed by the patterns of the phthalate di- and monoester transformations obtained. The validity of the model was further confirmed using experimental data from parallel reactors, which were subjected to either leachate exchange with an already methanogenic reactor or to initial aeration to force the reactor into stable methanogenic conditions.     

Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor

Microbial methanogenesis in subseafloor sediments is a key process in the carbon cycle on the Earth. However, the cultivation-dependent evidences have been poorly demonstrated. Here we report the cultivation of a methanogenic microbial consortium from subseafloor sediments using a continuous-flow-type bioreactor with polyurethane sponges as microbial habitats, called down-flow hanging sponge (DHS) reactor. We anaerobically incubated methane-rich core sediments collected from off Shimokita Peninsula, Japan, for 826 days in the reactor at 10 °C. Synthetic seawater supplemented with glucose, yeast extract, acetate and propionate as potential energy sources was provided into the reactor. After 289 days of operation, microbiological methane production became evident. Fluorescence in situ hybridization analysis revealed the presence of metabolically active microbial cells with various morphologies in the reactor. DNA- and RNA-based phylogenetic analyses targeting 16S rRNA indicated the successful growth of phylogenetically diverse microbial components during cultivation in the reactor. Most of the phylotypes in the reactor, once it made methane, were more closely related to culture sequences than to the subsurface environmental sequence. Potentially methanogenic phylotypes related to the genera Methanobacterium, Methanococcoides and Methanosarcina were predominantly detected concomitantly with methane production, while uncultured archaeal phylotypes were also detected. Using the methanogenic community enrichment as subsequent inocula, traditional batch-type cultivations led to the successful isolation of several anaerobic microbes including those methanogens. Our results substantiate that the DHS bioreactor is a useful system for the enrichment of numerous fastidious microbes from subseafloor sediments and will enable the physiological and ecological characterization of pure cultures of previously uncultivated subseafloor microbial life.     

Spatial distribution of major bacterial species and different volatile fatty acids in a two-phase anaerobic biofilm reactor with PVA gel beads as bio-carrier

Abstract

Conventional completely mixed anaerobic treatment systems limit the chances of the different species of bacteria to spatially group together according to their mutual cooperation and as a result, show a lower efficiency and vulnerability towards shock situations. It is interesting to know about the stratification of the different bacterial species participating in the degradation process and the intermediates that they produce. In this study, we established and optimized a two-phase anaerobic packed bed biofilm reactor system (AnPBR) with porous PVA gel beads used as bio-carriers and ran the reactor system in a steady state to observe the VFAs produced along with the microbial diversity of the predominant species at different stages of the reactor system. We observed that acetate and butyrate were the predominant intermediate VFAs while concentrations of other VFAs such that propionic acid were low. Acetobacterium and Clostridium were found to be the most abundant bacterial species in acidogenic reactor while methanogenic reactor was highly enriched with Methanobacterium and Methanosarcina. Apart from the above, syntrophic populations such as Syntrophobactor wolinii were also observed to be dominant in both the reactors – especially towards the end of acidogenic reactor and the initial part of the methanogenic reactor.     

Rapid start-up of thermophilic anaerobic digestion with the turf fraction of MSW as inoculum

This study aims to determine suitable start-up conditions and inoculum sources for thermophilic anaerobic digestion. Within days of incubation MSW at 55 °C, methane was produced at a high rate. In an attempt to narrow down which components of typical MSW contained the thermophilic methanogens, vacuum cleaner dust, banana peel, kitchen waste, and garden waste were tested as inoculum for thermophilic methanogenesis with acetate as the substrate. Results singled out grass turf as the key source of thermophilic acetate degrading methanogenic consortia. Within 4 days of anaerobic incubation (55 °C), anaerobically incubated grass turf samples produced methane accompanied by acetate degradation enabling successful start-up of thermophilic anaerobic digestion. Other essential start-up conditions are specified. Stirring of the culture was not conducive for successful start-up as it resulted specifically in propionate accumulation.     

Kinetics of anaerobic methane oxidation coupled to denitrification in the membrane biofilm reactor

Anaerobic oxidation of methane coupled to denitrification (AOM-D) in a membrane biofilm reactor (MBfR), a platform used for efficiently coupling gas delivery and biofilm development, has attracted attention in recent years due to the low cost and high availability of methane. However, experimental studies have shown that the nitrate-removal flux in the CH₄-based MBfR (<1.0 g N/m²-day) is about one order of magnitude smaller than that in the H₂-based MBfR (1.1–6.7 g N/m²-day). A one-dimensional multispecies biofilm model predicts that the nitrate-removal flux in the CH₄-based MBfR is limited to <1.7 g N/m²-day, consistent with the experimental studies reported in the literature. The model also determines the two major limiting factors for the nitrate-removal flux: The methane half-maximum-rate concentration (K₂) and the specific maximum methane utilization rate of the AOM-D syntrophic consortium (k_max2), with k_max2 being more important. Model simulations show that increasing k_max2 to >3 g chemical oxygen demand (COD)/g cell-day (from its current 1.8 g COD/g cell-day) and developing a new membrane with doubled methane-delivery capacity (D_m) could bring the nitrate-removal flux to ≥4.0 g N/m²-day, which is close to the nitrate-removal flux for the H₂-based MBfR. Further increase of the maximum nitrate-removal flux can be achieved when D_m and k_max2 increase together.     

Managing methanogens and homoacetogens to promote reductive dechlorination of trichloroethene with direct delivery of H2 in a membrane biofilm reactor

A study with H(2)-based membrane biofilm reactors (MBfRs) was undertaken to examine the effectiveness of direct H(2) delivery in ex-situ reductive dechlorination of chlorinated ethenes. Trichloroethene (TCE) could be reductively dechlorinated to ethene with up to 95% efficiency as long as the pH-increase effects of methanogens and homoacetogens were managed and dechlorinators were selected for during start-up by creating H(2) limitation. Based on quantitative PCR, the dominant bacterial groups in the biofilm at the end of reactor operation were Dehalococcoides, Geobacter, and homoacetogens. Pyrosequencing confirmed the dominance of the dechlorinators and identified Acetobacterium as the key homoacetogen. Homoacetogens outcompeted methanogens for bicarbonate, based on the effluent concentration of acetate, by suppressing methanogens during batch start-up. This was corroborated by the methanogenesis functional gene mcrA, which was 1-2 orders of magnitude lower than the FTHFS functional gene for homoacetogens. Imaging of the MBfR fibers using scanning electron microscopy showed a distinct Dehalococcoides-like morphology in the fiber biofilm. These results support that direct addition of H(2) can allow for efficient and complete reductive dechlorination, and they shed light into how H(2)-fed biofilms, when operated to manage methanogenic and homoacetogenic activity, can be used for ex-situ bioremediation of chlorinated ethenes.     

Population dynamics of biofilm development during start-up of a butyrate-degrading fluidized-bed reactor

Summary Population dynamics during start-up of a fluidized-bed reactor with butyrate or butyrate plus acetate as sole substrates as well as biofilm development on the sand substratum were studied microbiologically, immunologically and by scanning electron microscopy. An adapted syntrophic consortium consisting of Syntrophospora sp., Methanothrix soehngenii, Methanosarcina mazei and Methanobrevibacter arboriphilus or Methanogenium sp. achieved high-rate butyrate degradation to methane and carbon dioxide. Desulfovibrio sp., Methanocorpusculum sp., and Methanobacterium sp. were also present in lower numbers. Immunological analysis demonstrated methanogens antigenically related to Methanobrevibacter ruminantium M1, Methanosarcina mazei S6, M. thermophila TM1, Methanobrevibacter arboriphilus AZ and Methanothrix soehngenii Opfikon in the biofilm. Immunological analysis also showed that the organisms isolated from the butyrate-degrading culture used as a source of inoculum were related to M. soehngenii Opfikon, Methanobacterium formicicum MF and Methanospirillum hungatei JF1. Offprint requests to: G. Zellner     

High-rate acidophilic ferrous iron oxidation in a biofilm airlift reactor and the role of the carrier material

In this study, the feasibility and engineering aspects of acidophilic ferrous iron oxidation in a continuous biofilm airlift reactor inoculated with a mixed culture of Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans bacteria were investigated. Specific attention was paid to biofilm formation, competition between both types of bacteria, ferrous iron oxidation rate, and gas liquid mass transfer limitations. The reactor was operated at a constant temperature of 30 degrees C and at pH values of 0-1.8. Startup of the reactor was performed with basalt carrier material. During the experiments the basalt was slowly removed and the ferric iron precipitates formed served as a biofilm carrier. These precipitates have highly suitable characteristics as a carrier material for the immobilization of ferrous iron-oxidizing bacteria and dense conglomerates were observed. Lowering the pH (0.6-1) resulted in dissolution of the ferric precipitates and induced granular sludge formation. The maximum ferrous iron oxidation rate achieved in this study was about 145 molFe(2+)/m(3).h at a hydraulic residence time of 0.25 h. Optimal treatment performance was obtained at a loading rate of 100 mol/m(3).h at a conversion efficiency as high as 98%. Fluorescent in situ hybridization (FISH) studies showed that when the reactor was operated at high ferrous iron conversion (>85%) for 1 month, the desirable L. ferrooxidans species could out-compete A. ferrooxidans due to the low Fe(2+) and high Fe(3+) concentrations.