Sulfate reduction with methanol by a thermophilic consortium obtained from a methanogenic reactor

 An enrichment culture obtained from anaerobic granular sludge of a bench-scale anaerobic reactor degraded methanol at 65°C via sulfate reduction and acetogenesis. Sulfate reduction was the dominant process (S^2-/acetate=2.5). No methane formation was observed. Approximately 30% of the methanol was converted by acetogenic bacteria to acetate, while the remainder was degraded by these bacteria to H₂ and CO₂ in syntrophy with hydrogen-consuming sulfate-reducing bacteria. Pure cultures of sulfate-reducing and acetogenic bacteria were isolated and characterized. Received: 4 December 1995 / Received revision: 15 April 1996 / Accepted: 22 April 1996     

Microbiology and performance of a methanogenic biofilm reactor during the start-up period

Aims:  To understand the interactions between anaerobic biofilm development and process performances during the start-up period of methanogenic biofilm reactor.
Methods and Results:  Two methanogenic inverse turbulent bed reactors have been started and monitored for 81 days. Biofilm development (adhesion, growth, population dynamic) and characteristics (biodiversity, structure) were investigated using molecular tools (PCR–SSCP, FISH-CSLM). Identification of the dominant populations, in relation to process performances and to the present knowledge of their metabolic activities, was used to propose a global scheme of the degradation routes involved. The inoculum, which determines the microbial species present in the biofilm influences bioreactor performances during the start-up period. FISH observations revealed a homogeneous distribution of the Archaea and bacterial populations inside the biofilm.
Conclusion:  This study points out the link between biodiversity, functional stability and methanogenic process performances during start-up of anaerobic biofilm reactor. It shows that inoculum and substrate composition greatly influence biodiversity, physiology and structure of the biofilm.
Significance and Impact of the Study:  The combination of molecular techniques associated to a biochemical engineering approach is useful to get relevant information on the microbiology of a methanogenic growing biofilm, in relation with the start-up of the process.     

Desulfitobacterium hafniense is present in a high proportion within the biofilms of a high-performance pentachlorophenol-degrading, methanogenic fixed-film reactor

We developed a pentachlorophenol (PCP)-degrading, methanogenic fixed-film reactor by using broken granular sludge from an upflow anaerobic sludge blanket reactor. This methanogenic consortium was acclimated with increasing concentrations of PCP. After 225 days of acclimation, the reactor was performing at a high level, with a PCP removal rate of 1,173 muM day(-1), a PCP removal efficiency of up to 99%, a degradation efficiency of approximately 60%, and 3-chlorophenol as the main chlorophenol residual intermediate. Analyses by PCR-denaturing gradient gel electrophoresis (DGGE) showed that Bacteria and Archaea in the reactor stabilized in the biofilms after 56 days of operation. Important modifications in the profiles of Bacteria between the original granular sludge and the reactor occurred, as less than one-third of the sludge DGGE bands were still present in the reactor. Fluorescence in situ hybridization experiments with probes for Archaea or Bacteria revealed that the biofilms were composed mostly of Bacteria, which accounted for 70% of the cells. With PCR species-specific primers, the presence of the halorespiring bacterium Desulfitobacterium hafniense in the biofilm was detected very early during the reactor acclimation period. D. hafniense cells were scattered in the biofilm and accounted for 19% of the community. These results suggest that the presence of PCP-dehalogenating D. hafniense in the biofilm was crucial for the performance of the reactor.     

Reduction of nitrate and nitrite in a cyclically operated continuous biological reactor

Biological reduction of nitrate and nitrite was studied with a continuously operated cyclic reactor. The medium was fed to the reactor during the first phase of the cycle, and the effluent was drawn from the reactor during the third phase of the cycle; reaction occurred throughout the cycle. The process was described mathematically based on kinetic expressions revealed in an independent study. The model equations were subjected to detailed analysis with numerical codes based on the bifurcation theory for forced systems. The analysis has shown that in the operating parameter space there are extensive regions where the system can reach up to three different periodic states. The results of this analysis are shown in the form of two-dimensional operating diagrams. Numerical results have also shown that under certain operating conditions nitrate can be completely eliminated, while nitrite remains practically untreated. An experimental unit was designed, constructed, and used in experiments with a strain of Pseudomonas denitrificans [American Type Culture Collection (ATCC) 13867] under different operating conditions. The experimental results confirmed the theoretical predictions both qualitatively and quantitatively. Conditions under which complete reduction of both nitrate and nitrite is achieved, were found and experimentally verified. The results of this study suggest a methodology for analysis and design of cyclically operated bioreactors employed in denitrification of wastewaters. (c) 1995 John Wiley & Sons, Inc.     

Characterization of the methanogenic Archaea within two-phase biogas reactor systems operated with plant biomass

The two-phase leach-bed system is a biogas reactor system optimized for the utilization of energy crop silages at maximized loading rates under maintenance of an optimal microbial activity. In this study, a characterization of the methanogenic microbial community within this reactor system was conducted for the first time. Accordingly, effluent samples from the anaerobic filter and the silage digesting leach-bed reactors of both a laboratory-scale two-phase biogas reactor system and a scaled-up commercial on-farm pilot plant were investigated. In total, five Archaea-specific 16S rDNA libraries were constructed and analyzed by amplified rDNA restriction analysis (ARDRA), with subsequent phylogenetic analysis of nucleotide sequences for individual ARDRA patterns. A quantification of major methanogenic Archaea groups was conducted by real-time PCR. A total of 663 clones were analyzed and 45 operational taxonomic units (OTUs) related to methanogenic Archaea were detected. These OTUs were related to the orders Methanosarcinales, Methanomicrobiales and Methanobacteriales, as well as the hitherto uncultured CA-11 and ARC-I groups, and most of them occurred throughout all the compartments of both two-phase biogas reactors. The proportion of acetotrophic to hydrogenotrophic methanogens differed between the laboratory and the pilot scale system. A total of 56% of the clones from the 16S rDNA library derived from the laboratory biogas system were assigned to presumably acetotrophic members of Methanosarcinales. In contrast, these OTUs were less abundant in the 16S rDNA library derived from samples of the pilot plant. Therein, the most dominant OTUs were Methanoculleus-related OTUs, which presumably indicated the predominant presence of hydrogenotrophic methanogens. These findings were confirmed by group-specific quantitative real-time PCR assays. The results indicated that the fraction of acetotrophic and hydrogenotrophic methanogens within a biogas reactor caused certain variations, which may reflect varying substrate utilization during methanogenesis.     

Influence of hydrodynamic conditions on biofilm behavior in a methanogenic inverse turbulent bed reactor

This paper presents a study about the influence of gas velocity on a methanogenic biofilm in an inverse turbulent bed reactor. Experimental results indicate a dynamic response of the growing attached biomass to the changes of hydrodynamic conditions, mainly attrition constraints. Short but intensive increases of gas velocity (U(g)) are shown to induce more detachment than a high but constant gas flow rate. Hydrodynamic conditions control the composition of the growing biofilm in terms of cells and exocellular polymeric substances (EPS). The cell fraction within the biofilm (R(cell)) was found to be inversely proportional to the gas velocity. The specific activity expressed in methane production rate or COD removal rate is higher in biofilms formed under high hydrodynamic constraints. The control of the hydrodynamic conditions in a biofilm reactor should make it possible to obtain a resistant and active biofilm.     

Magnetic resonance microscopy analysis of advective transport in a biofilm reactor

In this article we present magnetic resonance microscopy (MRM) characterization of the advective transport in a biofilm capillary reactor. The biofilm generates non-axial flows that are up to 20% of the maximum axial velocity. The presence of secondary velocities of this magnitude alters the mass transport in the bioreactor relative to non-biofilm fouled reactors and questions the applicability of empirical mass transfer coefficient approaches. The data are discussed in the context of simulations and models of biofilm transport and conceptual aspects of transport modeling in complex flows are also discussed. The variation in the residence time distribution due to biofilm growth is calculated from the measured propagator of the motion. Dynamical systems methods applied to model fluid mixing in complex flows are indicated as a template for extending mass transport theory to quantitatively incorporate microscale data on the advection field into macroscale mass transfer models.     

Monitoring the denitrification of wastewater containing high concentrations of nitrate with methanol in a sulfur-packed reactor

Biological denitrification of high nitrate-containing wastewater was examined in a sulfur-packed column using a smaller amount of methanol than required stoichiometrically for heterotrophic denitrification. In the absence of methanol, the observed nitrate removal efficiency was only about 40%, and remained at 400 mg NO(3)(-)-N/l, which was due to an alkalinity deficiency of the pH buffer and of CO(2) as a carbon source. Complete denitrification was achieved by adding approximately 1.4 g methanol/g nitrate-nitrogen (NO(3)(-)-N) to a sulfur-packed reactor. As the methanol concentration increased, the overall nitrate removal efficiency increased. As influent methanol concentrations increased from 285 to 570, 855, and 1,140 mg/l, the value of Delta mg alkalinity as CaCO(3) consumed/Delta mg NO(3)(-)-N removed increased from -1.94 to -0.84, 0.24, and 0.96, and Delta mg SO(4)(2-) produced/Delta mg NO(3)(-)-N removed decreased from 4.42 to 3.57, 2.58, and 1.26, respectively. These results imply the co-occurrence of simultaneous autotrophic and heterotrophic denitrification. Sulfur-utilizing autotrophic denitrification in the presence of a small amount of methanol is very effective at decreasing both sulfate production and alkalinity consumption. Most of methanol added was removed completely in the effluent. A small amount of nitrite accumulated in the mixotrophic column, which was less than 20 mg NO(2)(-) -N/l, while under heterotrophic denitrification conditions, nitrite accumulated steadily and increased to 60 mg NO(2)(-) -N/l with increasing column height.     

Biodegradation of the herbicide propanil, and its 3,4-dichloroaniline by-product in a continuously operated biofilm reactor

The persistence of propanil in soil and aquatic environments along with the possible accumulation of toxic degradation products, such as chloroanilines, is of environmental concern. In this work, a continuous small-scale bioprocess to degrade the herbicide propanil, its main catabolic by-product, 3,4-dichloroaniline (3,4-DCA), and the herbicide adjuvants is carried out. A microbial consortium, constituted by nine bacterial genera, was selected. The isolated strains, identified by amplification and sequencing of their 16S rDNA, were: Acidovorax sp., Luteibacter (rhizovicinus), Xanthomonas sp., Flavobacterium sp., Variovorax sp., Acinetobacter (calcoaceticus), Pseudomonas sp., Rhodococcus sp., and Kocuria sp. The ability of the microbial consortium to degrade the herbicide was evaluated in a biofilm reactor at propanil loading rates ranging from 1.9 to 36.8 mg L^?1 h^?1. Complete removal of propanil, 3,4-DCA, chemical oxygen demand and total organic carbon was obtained at propanil loading rates up to 24.9 mg L^?1 h^?1. At higher loading rates, the removal efficiencies decayed. Four of the identified strains could grow individually in propanil, and 3,4-DCA: Pseudomonas sp., Acinetobacter calcoaceticus, Rhodococcus sp., and Xanthomonas sp. The Kokuria strain grew on 3,4-DCA, but not on propanil. The first three bacteria have been related to biodegradation of phenyl urea herbicides or chlorinated anilines. Although some strains of the genera Xanthomonas and Kocuria have a role in the biodegradation of several xenobiotic compounds, as far as we know, there are no reports about degradation of propanil by Xanthomonas or 3,4-DCA by Kocuria species.     

Biodegradation of acetonitrile by adapted biofilm in a membrane-aerated biofilm reactor

A membrane-aerated biofilm reactor (MABR) was developed to degrade acetonitrile (ACN) in aqueous solutions. The reactor was seeded with an adapted activated sludge consortium as the inoculum and operated under step increases in ACN loading rate through increasing ACN concentrations in the influent. Initially, the MABR started at a moderate selection pressure, with a hydraulic retention time of 16 h, a recirculation rate of 8 cm/s and a starting ACN concentration of 250 mg/l to boost the growth of the biofilm mass on the membrane and to avoid its loss by hydraulic washout. The step increase in the influent ACN concentration was implemented once ACN concentration in the effluent showed almost complete removal in each stage. The specific ACN degradation rate achieved the highest at the loading rate of 101.1 mg ACN/g-VSS h (VSS, volatile suspended solids) and then declined with the further increases in the influent ACN concentration, attributed to the substrate inhibition effect. The adapted membrane-aerated biofilm was capable of completely removing ACN at the removal capacity of up to 21.1 g ACN/m² day, and generated negligible amount of suspended sludge in the effluent. Batch incubation experiments also demonstrated that the ACN-degrading biofilm can degrade other organonitriles, such as acrylonitrile and benzonitrile as well. Denaturing gradient gel electrophoresis studies showed that the ACN-degrading biofilms contained a stable microbial population with a low diversity of sequence of community 16S rRNA gene fragments. Specific oxygen utilization rates were found to increase with the increases in the biofilm thickness, suggesting that the biofilm formation process can enhance the metabolic degradation efficiency towards ACN in the MABR. The study contributes to a better understanding in microbial adaptation in a MABR for biodegradation of ACN. It also highlights the potential benefits in using MABRs for biodegradation of organonitrile contaminants in industrial wastewater.     

Characterization of functional microbial community in a membrane-aerated biofilm reactor operated for completely autotrophic nitrogen removal

The 16S rDNA-based molecular technique was applied to investigate the functional microbial community of a membrane-aerated biofilm (MAB) that was used for completely autotrophic nitrogen removal over nitrite (CANON). The relationships among two kinds of key bacteria responsible for CANON: aerobic ammonia-oxidizing bacteria (AOB) and Anammox bacteria, and their possible distributions in the MAB were discussed based on the microbial community analysis. FISH analysis showed the existence of two visible active layers in experimental MAB. One is the partial nitrifying layer located in the region of oxygen-rich membrane-biofilm interface, dominated by NSO190-positive AOB. The other is the Anammox active layer located in the region of anoxic liquid-biofilm interface, dominated by PLA46 and AMX820-positive Anammox microorganisms. As a result of this study, the AOB as well as Anammox bacteria were present and active in experimental MABR, and the cooperation between AOB and Anammox bacteria was considered to be responsible for CANON.     

Light/electricity conversion by a self-organized photosynthetic biofilm in a single-chamber reactor

Biological energy-conversion systems are attractive in terms of their self-sustaining and self-organizing nature and are expected to be applied to low-cost and environment-friendly processes. Here we show a biofilm-based light/electricity-conversion system that was self-organized from a natural microbial community. A bioreactor equipped with an air cathode and graphite-felt anode was inoculated with a green hot-spring microbial mat. When the reactor was irradiated with light, electric current was generated between the anode and cathode in accordance with the formation of green biofilm on the anode. Fluorescence microscopy of the green biofilm revealed the presence of chlorophyll-containing microbes of ∼10 μm in size, and these cells were abundant close to the surface of the biofilm. The biofilm community was also analyzed by sequencing of polymerase chain reaction-amplified small-subunit rRNA gene fragments, showing that sequence types affiliated with Chlorophyta, Betaproteobacteria, and Bacteroidetes were abundantly detected. These results suggest that green algae and heterotrophic bacteria cooperatively converted light energy into electricity.     

Bioreduction of para-chloronitrobenzene in a hydrogen-based hollow-fiber membrane biofilm reactor: effects of nitrate and sulfate

A continuous-stirred, hydrogen-based, hollow-fiber membrane biofilm reactor (HFMBfR) that was active in nitrate and sulfate reductions was shown to be effective for degradation or detoxification of para-chloronitrobenzene (p-CNB) in water by biotransforming it first to para-chloroaniline (nitro-reduction) and then to aniline (reductive dechlorination) with hydrogen (H₂) as an electron donor. A series of short-term experiments examined the effects of nitrate and sulfate on p-CNB bioreduction. The results obtained showed both higher nitrate and sulfate concentration declined the p-CNB bioreduction in the biofilm, and this suggests the competition for H₂ caused less H₂ available for the p-CNB bioreduction when the H₂ demand for the reductions was larger. Denitrification and sulfate reduction intermediates were thought to be potential factors inhibiting the p-CNB bioreduction. Analysis of electron-equivalent fluxes and reaction orders in the biofilm further demonstrated both denitrification and sulfate reduction competed more strongly for H₂ availability than p-CNB bioreduction. These findings have significant implications for the HFMBfR used for degrading p-CNB under denitrifying and/or sulfate reducing conditions.     

Catechol and phenol degradation by a methanogenic population of bacteria

An anaerobic population of bacteria became acclimated to catechol and phenol in 32 and 18 days, respectively. Evidence from carbon balance measurements indicates that the aromatic ring is cleaved and that the products are stoichiometrically fermentable to methane and carbon dioxide.     

Coexistence of nitrifiers, denitrifiers and Anammox bacteria in a sequencing batch biofilm reactor as revealed by PCR-DGGE

Aims:  The bacterial diversity in a sequencing batch biofilm reactor (SBBR) treating landfill leachate was studied to explain the mechanism of nitrogen removal.
Methods and Results:  The total microbial DNA was extracted from samples collected from landfill leachate and biofilm of the reactor with the removal efficiencies of NH₄⁺-N higher than 97% and that of chemical oxygen demand (determined by K₂Cr₂O₇, COD_Cr) higher than 86%. Denaturing gradient gel electrophoresis (DGGE) fingerprints based on total community 16S rRNA genes were analyzed with statistical methods, and excised DNA bands were sequenced. The results of phylogenetic analyses revealed high diversity within the SBBR biofilm community, and DGGE banding patterns showed that the community structure in the biofilm remained stable during the running period.
Conclusions:  A coexistence of nitrifiers, including ammonia-oxidizing bacteria and nitrite-oxidizing bacteria, denitrifiers, including aerobic or anaerobic denitrifying bacteria and Anammox bacteria were detected, which might be the real matter of high removal efficiencies of NH₄⁺-N and COD_Cr in the reactor.
Significance and Impact of the Study:  The findings in this study indicated that PCR-DGGE analysis could be used for microbial community detection as prior method, and the SBBR technique could provide preferable growing environment for bacteria with N removal function.     

Simultaneous removal of phosphorus and nitrogen in a sequencing batch biofilm reactor with transgenic bacteria expressing polyphosphate kinase

To improve phosphorus removal from wastewater, we constructed a high-phosphate-accumulating microorganism, KTPPK, using Pseudomonas putida KT2440 as a host. The expression plasmid was constructed by inserting and expressing polyphosphate kinase gene (ppk) from Microcystis aeruginosa NIES-843 into broad-host-range plasmid, pBBR1MCS-2. KTPPK was then added to a sequencing batch biofilm reactor (SBBFR) using lava as a biological carrier. The results showed that SBBFR with KTPPK not only efficiently removed COD, NH(3)-N, and NO(3)(-)-N but also had a high removal capacity for PO(4)(3-)-P, resulting in a low phosphorus concentration remaining in the outflow of the SBBFR. The biofilm increased by 30-53% on the lava in the SBBFR that contained KTPPK after 11 days when compared with the reactor that contained P. putida KT2440. Real-time quantitative polymerase chain reaction confirmed that the copy of ppk was maintained at about 3.5 × 10(10) copies per μL general DNA in the biofilm after 20 days. Thus, the transgenic bacteria KTPPK could maintain a high density and promote phosphorus removal in the SBBFR. In summary, this study indicates that the use of SBBFR with transgenic bacteria has the potential to remove phosphorus and nitrogen from wastewater.     

Catechol and phenol degradation by a methanogenic population of bacteria.

An anaerobic population of bacteria became acclimated to catechol and phenol in 32 and 18 days, respectively. Evidence from carbon balance measurements indicates that the aromatic ring is cleaved and that the products are stoichiometrically fermentable to methane and carbon dioxide.     

Enzymatic cleaning of biofouled thin-film composite reverse osmosis (RO) membrane operated in a biofilm membrane reactor

Application of environmentally friendly enzymes to remove thin-film composite (TFC) reverse osmosis (RO) membrane biofoulants without changing the physico-chemical properties of the RO surface is a challenging and new concept. Eight enzymes from Novozyme A/S were tested using a commercially available biofouling-resistant TFC polyamide RO membrane (BW30, FilmTech Corporation, Dow Chemical Co.) without filtration in a rotating disk reactor system operated for 58 days. At the end of the operation, the accumulated biofoulants on the TFC RO surfaces were treated with the three best enzymes, Subtilisin protease and lipase; dextranase; and polygalacturonase (PG) based enzymes, at neutral pH (~7) and doses of 50, 100, and 150 ppm. Contact times were 18 and 36 h. Live/dead staining, epifluorescence microscopy measurements, and 5 μm thick cryo-sections of enzyme and physically treated biofouled membranes revealed that Subtilisin protease- and lipase-based enzymes at 100 ppm and 18 h contact time were optimal for removing most of the cells and proteins from the RO surface. Culturable cells inside the biofilm declined by more than five logs even at the lower dose (50 ppm) and shorter incubation period (18 h). Subtilisin protease- and lipase-based enzyme cleaning at 100 ppm and for 18 h contact time restored the hydrophobicity of the TFC RO surface to its virgin condition while physical cleaning alone resulted in a 50° increase in hydrophobicity. Moreover, at this optimum working condition, the Subtilisin protease- and lipase-based enzyme treatment of biofouled RO surface also restored the surface roughness measured with atomic force microscopy and the mass percentage of the chemical compositions on the TFC surface estimated with X-ray photoelectron spectroscopy to its virgin condition. This novel study will encourage the further development and application of enzymes to remove biofoulants on the RO surface without changing its surface properties.