Organic substrate diffusibility governs microbial community composition, nutrient removal performance and kinetics of granulation of aerobic granular sludge.

Basic understanding of formation of aerobic granular sludge (AGS) has mainly been derived from lab-scale systems with simple influents containing only highly diffusible volatile fatty acids (VFA) as organic substrate. This study compares start-up of AGS systems fed by different synthetic and municipal wastewaters (WW), characterised by increasing complexity in terms of non-diffusible organic substrate. Four AGS reactors were started with the same inoculum activated sludge and operated for one year. The development of AGS, settling characteristics, nutrient and substrate removal performance as well as microbial community composition were monitored. Our results indicate that the higher the content of diffusible organic substrate in the WW, the faster the formation of AGS. The presence of non-diffusible organic substrate in the influent WW led to the formation of small granules and to the presence of 20-40% (% of total suspended solids) of flocs in the AGS. When AGS was fed with complex influent WW, the classical phosphorus and glycogen accumulating organisms (PAO, GAO) were outcompeted by their fermentative equivalents. Substrate and nutrient removal was observed in all reactors, despite the difference in physical and settling properties of the AGS, but the levels of P and N removal depended on the influent carbon composition. Mechanistically, our results indicate that increased levels of non-diffusible organic substrate in the influent lower the potential for microbial growth deep inside the granules. Additionally, non-diffusible organic substrates give a competitive advantage to the main opponents of AGS formation - ordinary heterotrophic organisms (OHO). Both of these mechanisms are suspected to limit AGS formation. The presented study has relevant implications for both practice and research. Start-up duration of AGS systems treating high complexity WW were one order of magnitude higher than a typical lab-scale system treating VFA-rich synthetic WW, and biomass as flocs persisted as a significant fraction. Finally, the complex synthetic influent WW - composed of VFA, soluble fermentable and particulate substrate - tested here seems to be a more adequate surrogate of real municipal WW for laboratory studies than 100%-VFA WW.

Temporal evolution of bacterial communities associated with the in situ wetland-based remediation of a marine shore porphyry copper tailings deposit.

Mine tailings are a serious threat to the environment and public health. Remediation of these residues can be carried out effectively by the activation of specific microbial processes. This article presents detailed information about temporal changes in bacterial community composition during the remediation of a section of porphyry copper tailings deposited on the Bahía de Ite shoreline (Peru). An experimental remediation cell was flooded and transformed into a wetland in order to prevent oxidation processes, immobilizing metals. Initially, the top oxidation zone of the tailings deposit displayed a low pH (3.1) and high concentrations of metals, sulfate, and chloride, in a sandy grain size geological matrix. This habitat was dominated by sulfur- and iron-oxidizing bacteria, such as Leptospirillum spp., Acidithiobacillus spp., and Sulfobacillus spp., in a microbial community which structure resembled acid mine drainage environments. After wetland implementation, the cell was water-saturated, the acidity was consumed and metals dropped to a fraction of their initial respective concentrations. Bacterial communities analyzed by massive sequencing showed time-dependent changes both in composition and cell numbers. The final remediation stage was characterized by the highest bacterial diversity and evenness. Aside from classical sulfate reducers from the phyla δ-Proteobacteria and Firmicutes, community structure comprised taxa derived from very diverse habitats. The community was also characterized by an elevated proportion of rare phyla and unaffiliated sequences. Numerical ecology analysis confirmed that the temporal population evolution was driven by pH, redox, and K. Results of this study demonstrated the usefulness of a detailed follow-up of the remediation process, not only for the elucidation of the communities gradually switching from autotrophic, oxidizing to heterotrophic and reducing living conditions, but also for the long term management of the remediation wetlands.

Nitrogen removal from digested manure in a simple one-stage process.

A process based on partial nitrification and recirculation into the anaerobic digester was studied to remove nitrogen from digested manure and thus reduce enhanced gaseous ammonia emissions due to on-farm biogas production. An anaerobic reactor representing an anaerobic manure digester was fed with a nitrite solution and digested manure liquor. Nitrite was efficiently removed from the influent and ammonium formation was observed first. Ammonium was subsequently eliminated up to a maximum of 90% of the influent concentration, indicating anaerobic ammonium oxidation activity. This activity, however, decreased again and was lost at the end of the 4-month operation period. In a 1.5 L aerobic CSTR that was fed with digested manure liquor, ammonium was efficiently removed from the influent. Nitrite and nitrate formation was observed but mass balances indicated significant N-removal. Accumulation of suspended solids was observed at the end of the experiment suggesting presence of oxygen-free environments. In a second test in a 15 L CSTR where suspended solids sedimentation could be avoided, low N-removal rates were observed in the absence of biofilm carrier elements whereas high N-removal rates were achieved in their presence. A simple one-stage process based on immobilized biomass could therefore be installed downstream of agricultural anaerobic digesters in order to mitigate undesirable gaseous ammonia emissions.

Proposal of biostimulation for hexachlorocyclohexane (HCH)-decontamination and characterization of culturable bacterial community from high-dose point HCH-contaminated soils.

AIMS: To locate a high-dose point hexachlorocyclohexane (HCH)-contaminated site, to identify HCH-degrading bacteria in it and assay HCH-decontamination by biostimulation. METHODS AND RESULTS: Bacteria were isolated by serial dilution method from HCH-contaminated soil samples collected from areas near an HCH-manufacturing unit and its dumpsite in North India. After confirming the presence of indigenous HCH-degraders (seven of 24 strains), an ex situ biostimulation experiment was conducted. For this, residue levels in soil were diluted by mixing with pristine garden soil and aeration, moisture and nutrients were provided intermittently. This soil was monitored for reduction in Sigma-HCH (sum of alpha-, beta-, gamma- and delta-HCH) levels and stimulation of HCH-degraders. Experiments were conducted twice, in March-April (c. 75 microg Sigma-HCH g(-1) soil) and October-November 2006 (c. 280 microg Sigma-HCH g(-1) soil) at 26-30 degrees C. Sigma-HCH levels were reduced to <30% of the original in 24 days and <3% in 240 days in the experimental pits. Terminal restriction fragment length polymorphism analysis reflected changes in microbial community structure during the course of experiment. CONCLUSIONS: Our results show presence of HCH-degrading sphingomonads at a high-dose point HCH-contaminated site and presents biostimulation as an effective approach for its decontamination via aeration, addition of nutrients and moisture, of the indigenous population. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates that biostimulation of indigenous HCH-degrading microbial population can be used for decontamination of chronically HCH-contaminated sites.

Steady-state and transient-state performance of a biotrickling filter treating chlorobenzene-containing waste gas.

Biotrickling filter (BTF) technology was applied for the treatment of waste gas containing a mixture of chlorobenzene and 1,2-dichlorobenzene. An adapted microbial community was immobilised on a structured packing material. The strategy followed was to reach high removal efficiencies at initially low mass loading rates followed by an increase of the latter. This procedure was successful and resulted in a short start-up period of only 2 weeks. A 3-month operation under steady-state conditions showed good performance, with >95% removal efficiency at a mass loading rate of 1,800 g m(-3) day(-1). Dimensionless concentration profiles showed that the chlorobenzenes were simultaneously degraded. Low dissolved organic carbon of 15 mg l(-1) and stoichiometric chloride concentrations in the trickling liquid indicated complete mineralisation of the pollutant. Transient-state experiments with five times higher mass loading rates caused a decrease in the removal efficiency that recovered rapidly once the mass loading rate returned to its original steady-state level. A progressive increase of the mass loading rate in a long-term performance experiment showed that the removal efficiency could be kept stable between 95 and 99% at loads of up to 5,200 g m(-3) day(-1) over several days. Above this mass loading rate, the elimination capacity did not increase any further. These results demonstrated that with a well-adapted inoculum and optimal operation parameters, a BTF system with excellent performance and stability that efficiently removes a mixture of chlorobenzene vapours from air can be obtained.

Evaluation of kinetic coefficients using integrated monod and haldane models for low-temperature acetoclastic methanogenesis.

The integrated Monod and Haldane models were used to evaluate the kinetic coefficients and their standard deviations using the methane accumulation curves of low-temperature acetoclastic methanogenesis. The linear and exponential approximations and the limitations of their applicability were deduced from the integrated models. The samples of lake sediments and biomass taken from a low-temperature upflow anaerobic sludge blanket (UASB) reactor were used as inoculum in batch assays for acetate methanation. In comparison, the Monod and Haldane models were applied to evaluate the kinetic coefficients for mesophilic acetoclastic methanogenesis accomplished by the pure culture of Methanosarcina barkeri strain MS. The Monod and Haldane models and their approximations were fitted by using non-linear regression. For the wide range of initial acetateconcentrations (4.2-84 mM: 5-100 mM) applied to the UASB biomass at 11 and 22 degrees C and for the lake sediment samples at 6 and 15 degrees C, a better fit was obtained with the Haldane models and their exponential approximations, respectively. For the lake sediments the values of inhibition coefficients decreased at decreasing temperatures. At the highest temperature of 30 degrees C no difference was found between the Haldane and Monod models and the simpler Monod model should be preferred. The values of the maximum growth rate of biomass were highest at 30 degrees C (lake sediment) and 22 degrees C (the UASB biomass) being in a range presented in the literature for mesophilic acetoclastic methanogenesis.

Vertical distribution of methanogens in the anoxic sediment of Rotsee (Switzerland).

Anoxic sediments from Rotsee (Switzerland) were analyzed for the presence and diversity of methanogens by using molecular tools and for methanogenic activity by using radiotracer techniques, in addition to the measurement of chemical profiles. After PCR-assisted sequence retrieval of the 16S rRNA genes (16S rDNA) from the anoxic sediment of Rotsee, cloning, and sequencing, a phylogenetic analysis identified two clusters of sequences and four separated clones. The sequences in cluster 1 grouped with those of Methanosaeta spp., whereas the sequences in cluster 2 comprised the methanogenic endosymbiont of Plagiopyla nasuta. Discriminative oligonucleotide probes were constructed against both clusters and two of the separated clones. These probes were used subsequently for the analysis of indigenous methanogens in a core of the sediment, in addition to domain-specific probes against members of the domains Bacteria and Archaea and the fluorescent stain 4', 6-diamidino-2-phenylindole (DAPI), by fluorescent in situ hybridization. After DAPI staining, the highest microbial density was obtained in the upper sediment layer; this density decreased with depth from (1.01 +/- 0.25) x 10(10) to (2.62 +/- 0.58) x 10(10) cells per g of sediment (dry weight). This zone corresponded to that of highest metabolic activity, as indicated by the ammonia, alkalinity, and pH profiles, whereas the methane profile was constant. Probes Eub338 and Arch915 detected on average 16 and 6% of the DAPI-stained cells as members of the domains Bacteria and Archaea, respectively. Probe Rotcl1 identified on average 4% of the DAPI-stained cells as Methanosaeta spp., which were present throughout the whole core. In contrast, probe Rotcl2 identified only 0.7% of the DAPI-stained cells as relatives of the methanogenic endosymbiont of P. nasuta, which was present exclusively in the upper 2 cm of the sediment. Probes Rotp13 and Rotp17 did not detect any cells. The spatial distribution of the two methanogenic populations corresponded well to the methane production rates determined by incubation with either [14C]acetate or [14C]bicarbonate. Methanogenesis from acetate accounted for almost all of the total methane production, which concurs with the predominance of acetoclastic Methanosaeta spp. that represented on average 91% of the archaeal population. Significant hydrogenotrophic methanogenesis was found only in the organically enriched upper 2 cm of the sediment, where the probably hydrogenotrophic relatives of the methanogenic endosymbiont of P. nasuta, accounting on average for 7% of the archaeal population, were also detected.

Pelagic Bacteria-Particle Interactions and Community-Specific Growth Rates in Four Lakes along a Trophic Gradient.

Abstract The relationships between bacterial concentration, bacterial production, and cell-specific activity of both free and attached bacteria and environmental factors such as suspended solids, nutrients, and temperature were examined in four lakes, two in New Zealand and two in Switzerland. Estimates of cell-specific production were obtained by microautoradiographic counts of [3H]thymidine-labeled cells. Bacteria attached to particles accounted for only 1.3 to 11.6% of the total bacterial abundance, but showed overall 20-fold higher specific growth rates and were relatively more active than their free counterparts. On average, 80 to 100% of epibacteria were attached to organic particles. The abundance and production of free and attached bacteria were positively correlated; however, relationships between these fractions and some environmental variables differed. Cell-specific activities of active bacteria were not equivalent to mean cellular activities of the entire bacterial community and differed in their relationship to trophic state. [3H]Thymidine-positive bacteria were more tightly linked to chlorophyll a than were total bacteria. Our findings indicate that production by attached bacteria, fueled by phytoplankton carbon, supplies "new" free bacteria to the bacterial community. Our results support the idea that particulate organic matter acts as a source of dissolved nutrients to free bacteria. Bottom-up control of bacterial biomass, as shown by regressions of biomass vs production, appeared to be stronger in two ultraoligotrophic lakes than in two more eutrophic ones.

Characterization of the pectin methylesterase-like gene AtPME3: a new member of a gene family comprising at least 12 genes in Arabidopsis thaliana.

Pectin demethylesterification appears to be catalysed by a number of pectin methylesterase (PME) isoenzymes in higher plant species. In order to better define the biological role of these isoenzymes in plant cell growth and differentiation, we undertook molecular studies on the PME-encoding genes in Arabidopsis thaliana. In this paper, we report the characterization of AtPME3, a new PME-related gene of 4kb in length that we have mapped on Chromosome III. AtPME3 encodes a putative mature PME-related isoenzyme of 34kDa with a basic isoelectric point. Since the extent of the gene family encoding PME in higher plant species is still unknown, we resorted to the use of degenerate primers designed from several well-known consensus regions to identify new PME-related genes in the genome of Arabidopsis. Our results, in combination with several known expressed sequences tags (ESTs), indicate that the Arabidopsis genome contains at least 12 PME-related genes. Consequently, a method of systematic gene expression analysis has been applied in order to discern the expression pattern of these 12 genes throughout the plant at the floral stage. Whereas most of these genes appeared to be more or less ubiquitously expressed throughout the plant, several genes are distinguishable by their strikingly specific expression in certain organs. The present data bring a new insight into the role of specific PME-related genes in flower and root development.

Localization and solubilization of the Iron(III) reductase of geobacter sulfurreducens

The iron(III) reductase activity of Geobacter sulfurreducens was determined with the electron donor NADH and the artificial electron donor horse heart cytochrome c. The highest reduction rates were obtained with Fe(III) complexed by nitrilotriacetic acid as an electron acceptor. Fractionation experiments indicated that no iron(III) reductase activity was present in the cytoplasm, that approximately one-third was found in the periplasmic fraction, and that two-thirds were associated with the membrane fraction. Sucrose gradient separation of the outer and cytoplasmic membranes showed that about 80% of the iron(III) reductase was present in the outer membrane. The iron(III) reductase could be solubilized from the membrane fraction with 0.5 M KCl showing that the iron(III) reductase was weakly bound to the membranes. In addition, solubilization of the iron(III) reductase from whole cells with 0.5 M KCl, without disruption of cells, indicated that the iron(III) reductase is a peripheral protein on the outside of the outer membrane. Redox difference spectra of KCl extracts showed the presence of c-type cytochromes which could be oxidized by ferrihydrite. Only one activity band was observed in native polyacrylamide gels stained for the iron(III) reductase activity. Excision of the active band from a preparative gel followed by extraction of the proteins and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of high-molecular-mass, cytochrome-containing proteins in this iron(III) reductase activity band. From these experimental data it can be hypothesized that the iron(III) reductase of G. sulfurreducens is a peripheral outer membrane protein that might contain a c-type cytochrome.

Dehalobacter restrictus gen. nov. and sp. nov., a strictly anaerobic bacterium that reductively dechlorinates tetra- and trichloroethene in an anaerobic respiration.

The highly enriched anaerobic bacterium that couples the reductive dechlorination of tetrachloroethene to growth, previously referred to as PER-K23, was obtained in pure culture and characterized. The bacterium, which does not form spores, is a small, gram-negative rod with one lateral flagellum. It utilized only H2 as an electron donor and tetrachloroethene and trichloroethene as electron acceptors in an anaerobic respiration process; it could not grow fermentatively. Acetate served as a carbon source in a defined medium containing iron as the sole trace element, the two vitamins thiamine and cyanocobalamin, and the three amino acids arginine, histidine, and threonine. The cells contained menaquinones and b-type cytochromes. The G+C content of the DNA was 45.3 +/- 0.3 mol%. The cell wall consisted of type-A3gamma peptidoglycan with ll-diaminopimelic acid and one glycine as an interpeptide bridge. The cells are surrounded by an S-layer; an outer membrane was absent. Comparative sequence analysis of the 16S rRNA sequence showed that PER-K23 is related to gram-positive bacteria with a low G+C content of the DNA. Based on the cytological, physiological, and phylogenetic characterization, it is proposed to affiliate the isolate to a new genus, Dehalobacter, with PER-K23 as the type strain of the new species Dehalobacter restrictus.

Contaminated environments in the subsurface and bioremediation: organic contaminants.

Due to leakages, spills, improper disposal and accidents during transport, organic compounds have become subsurface contaminants that threaten important drinking water resources. One strategy to remediate such polluted subsurface environments is to make use of the degradative capacity of bacteria. It is often sufficient to supply the subsurface with nutrients such as nitrogen and phosphorus, and aerobic treatments are still dominating. However, anaerobic processes have advantages such as low biomass production and good electron acceptor availability, and they are sometimes the only possible solution. This review will focus on three important groups of environmental organic contaminants: hydrocarbons, chlorinated and nitroaromatic compounds. Whereas hydrocarbons are oxidized and completely mineralized under anaerobic conditions in the presence of electron acceptors such as nitrate, iron, sulfate and carbon dioxide, chlorinated and nitroaromatic compounds are reductively transformed. For the aerobic often persistent polychlorinated compounds, reductive dechlorination leads to harmless products or to compounds that are aerobically degradable. The nitroaromatic compounds are first reductively transformed to the corresponding amines and can subsequently be bound to the humic fraction in an aerobic process. Such new findings and developments give hope that in the near future contaminated aquifers can efficiently be remediated, a prerequisite for a sustainable use of the precious-subsurface drinking water resources.

Redox chemistry of cobalamin and iron-sulfur cofactors in the tetrachloroethene reductase of Dehalobacter restrictus.

Respiration of Dehalobacter restrictus is based on reductive dechlorination of tetrachloroethene. The terminal component of the respiratory chain is the membrane-bound tetrachloroethene reductase. The metal prosthetic groups of the purified enzyme have been studied by optical and EPR spectroscopy. The 60-kDa monomer contains one cobalamin with Em(Co[1+/2+]) = -350 mV and Em(Co[2+/3+]) > 150 mV and two electron-transferring [4Fe-4S](2+;1+) clusters with rather low redox potentials of Em approximately -480 mV. The cob(II)alamin is present in the base-off configuration. A completely reduced enzyme sample reacted very rapidly with tetrachloroethene yielding base-off cob(II)alamin rather than trichlorovinyl-cob(III)alamin.

Anaerobic biodegradation of hydrocarbons.

Anaerobic biodegradation of aliphatic and aromatic hydrocarbons is a promising alternative to aerobic biodegradation treatments in bioremediation processes. It is now proven that, besides toluene, benzene and ethylbenzene can be oxidized under anaerobic redox conditions. Anaerobic bacteria have also been shown capable of utilizing substrates not only in the pure form, but also in complex hydrocarbon mixtures, such as crude oil. In addition, crucial steps in anaerobic treatment processes have been studied in vitro to better understand the enzymes involved in monoaromatic hydrocarbon degradation. Knowledge remains incomplete, however, about the anaerobic degradation of aliphatic and polycyclic aromatic hydrocarbons.

The proton/electron ration of the menaquinone-dependent electron transport from dihydrogen to tetrachloroethene in "Dehalobacter restrictus".

In the anaerobic respiration chain of "Dehalobacter restrictus," dihydrogen functioned as the electron donor and tetrachloroethene (PCE) functioned as the electron acceptor. The hydrogenase faced the periplasm, and the PCE reductase faced the cytoplasmic side of the membrane. Both activities were associated with the cytoplasmic membrane. UV spectroscopy showed that membrane-bound menaquinone (MQ) was reduced by oxidation of H2 and reoxidized by reduction of PCE, indicating that MQ functions as an electron mediator. Fast proton liberation (t1/2 = 6 +/- 2 s) during electron transport from H2 to PCE and to trichloroethene (TCE) after addition of either PCE or TCE to H2-saturated cells resulted in an extrapolated H+/e- ratio of 1.25 +/- 0.2. This ratio indicated that besides the formation of protons upon oxidation of H2, vectorial translocation of protons from the inside to the outside could also occur. Proton liberation was inhibited by carbonylcyanide m-chlorophenylhydrazone (CCCP), 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO), and CuCl2. Fast proton liberation with an H+/e- ratio of 0.65 +/- 0.1 was obtained after addition of the MQ analog 2,3-dimethyl-1,4-naphthoquinone (DMN) as an oxidant pulse. This acidification was also inhibited by CCCP, HOQNO, and CuCl2. Oxidation of reduced DMN by PCE was not associated with fast acidification. The results with DMN indicate that the consumption and release of protons associated with redox reactions of MQ during electron transfer from H2 to PCE both occurred at the cytoplasmic side of the membrane. The PCE reductase was photoreversibly inactivated by 1-iodopropane, indicating that a corrinoid was involved in the PCE reduction.

Physiological meaning and potential for application of reductive dechlorination by anaerobic bacteria.

The physiological meaning of reductive dechlorination reactions catalyzed by anaerobic bacteria can be explained as a co-metabolic activity or as a novel type of respiration. Co-metabolic activities have been found mainly with alkyl halides. They are non-specific reactions catalyzed by various enzyme systems of facultative as well as obligate anaerobic bacteria. In contrast, the reductive dechlorinations involved in metabolic respiration processes are very specific reactions. Only a limited number of alkyl and aryl chlorinated compounds is presently known to function as a terminal electron acceptor in a few, recently isolated bacteria. Metabolic dechlorination rates are in general several orders of magnitude higher than co-metabolic ones. Both reaction types are suitable for the anaerobic treatment of waste streams.

Reductive dehalogenation as a respiratory process.

Anaerobic bacteria can reductively dehalogenate aliphatic and aromatic halogenated compounds in a respiratory process. Only a few of these bacteria have been isolated in pure cultures. However, long acclimation periods, substrate specificity, high dehalogenation rates, and the possibility to enrich for the dehalogenation activity by subcultivation in media containing an electron donor indicate that many of the reductive dehalogenations in the environment are catalyzed by specific bacteria. Molecular hydrogen or formate appear to be good electron donors for the enrichment of such organisms. Furthermore, systems have to be employed which supply the cultures with the halogenated compounds beyond their toxicity level. All bacteria that are presently available in pure culture and grow with a halogenated compound as electron acceptor are members of new genera. Based on experimental results with the membrane-impermeable electron mediator methyl viologen, a model of the respiration system of Dehalobacter restrictus, a tetrachloroethene-dechlorinating bacterium, is presented. Further studies of the biochemistry and energetics of respiratory-dehalogenating strains will help to understand the mechanisms involved and perhaps reveal the evolutionary origin of the dehalogenating enzyme systems.

Abiotic reduction of 4-chloronitrobenzene to 4-chloroaniline in a dissimilatory iron-reducing enrichment culture.

4-chloronitrobenzene (4-Cl-NB) was rapidly reduced to 4-chloroaniline with half-lives of minutes in a dissimilatory Fe(III)-reducing enrichment culture. The initial pseudo-first-order rate constants at 25 degrees C ranged from 0.11 to 0.19 per minute. The linear Arrhenius correlation in a temperature range of 6 to 85 degrees C and the unchanged reactivity after pasteurization indicated that the nitroreduction occurred abiotically. A fine-grained black solid which was identified as poorly crystalline magnetite (Fe(3)O(4)) by X-ray diffraction accumulated in the enrichments. Magnetite produced by the Fe(III)-reducing bacterium Geobacter metallireducens GS-15 and synthetic magnetite also reduced 4-Cl-NB. These results suggest that the reduction of 4-Cl-NB by the enrichment material was a surface-mediated reaction by dissimilatory formed Fe(II) associated with magnetite.