Die-off of enteric bacterial pathogens during mesophilic anaerobic digestion

Conventionally treated sewage sludge may contain high concentrations of potentially pathogenic microorganisms and additional treatment is required to minimise the risks to health if it is to be recycled to agricultural land. Mesophilic anaerobic digestion (MAD) is the most widely used process in the UK for stabilising sludge prior to agricultural recycling, but little is known about the fate of a number of enteric pathogens as the sludge passes through the treatment processes. The aim of this study was to determine the efficiency of MAD in removing the bacterial enteric pathogens, Salmonella senftenberg, Listeria monocytogenes and Campylobacter jejuni which were added as a spike to the digester feedstock, together with the die-off of indigenous Escherichia coli already present in the sludge. The primary sludge digestion stage of MAD was found to achieve a log removal of 1.66 for E. coli, 2.23 for L. monocytogenes and 2.23 for S. senftenberg. However, the extent of die-off was a function of the numbers of pathogens in the feed and as these increased the log removal also increased. The numbers of C. jejuni were not affected by primary sludge digestion. Additional die-off was provided by secondary sludge digestion with log removals of 1.70 for E. coli, 2.10 for S. senftenberg and 0.36 for C. jejuni.     

Influence of high gas production during thermophilic anaerobic digestion in pilot-scale and lab-scale reactors on survival of the thermotolerant pathogens Clostridium perfringens and Campylobacter jejuni in piggery wastewater

Safe reuse of animal wastes to capture energy and nutrients, through anaerobic digestion processes, is becoming an increasingly desirable solution to environmental pollution. Pathogen decay is the most important safety consideration and is in general, improved at elevated temperatures and longer hydraulic residence times. During routine sampling to assess pathogen decay in thermophilic digestion, an inversely proportional relationship between levels of Clostridium perfringens and gas production was observed. Further samples were collected from pilot-scale, bench-scale thermophilic reactors and batch scale vials to assess whether gas production (predominantly methane) could be a useful indicator of decay of the thermotolerant pathogens C. perfringens and Campylobacter jejuni. Pathogen levels did appear to be lower where gas production and levels of methanogens were higher. This was evident at each operating temperature (50, 57, 65 °C) in the pilot-scale thermophilic digesters, although higher temperatures also reduced the numbers of pathogens detected. When methane production was higher, either when feed rate was increased, or pH was lowered from 8.2 (piggery wastewater) to 6.5, lower numbers of pathogens were detected. Although a number of related factors are known to influence the amount and rate of methane production, it may be a useful indicator of the removal of the pathogens C. perfringens and C. jejuni.     

Involvement of the TCA cycle in the anaerobic metabolism of polyphosphate accumulating organisms (PAOs)

For decades, glycolysis has been generally accepted to supply the reducing power for the anaerobic conversion of volatile fatty acids (VFAs) to polyhydroxyalkanoates (PHAs) by polyphosphate accumulating organisms (PAOs). However, the importance of the tricarboxylic acid (TCA) cycle has also been raised since 1980s. The aim of this study is to demonstrate the involvement of the TCA cycle in the anaerobic metabolism of PAOs. To achieve this goal, the glycogen pool of an activated sludge highly enriched in Candidatus Accumulibacter Phosphatis (hereafter referred to as Accumulibacter), a putative PAO was reduced substantially through starving the sludge under intermittent anaerobic and aerobic conditions. After the starvation, acetate added was still taken up anaerobically and stored as PHA, with negligible glycogen degradation. The metabolic models proposed by Pereira, Hesselmann and Yagci, which predict the formation of reducing power through glycolysis and the full or partial TCA cycle, were used to estimate the carbon fluxes. The results demonstrate that Accumulibacter can use both glycogen and acetate to generate reducing power anaerobically. The anaerobic production of reducing power from acetate is likely through the full TCA cycle. The proportion of TCA cycle involvement depends on the availability of degradable glycogen.     

Common key acidogen populations in anaerobic reactors treating different wastewaters: molecular identification and quantitative monitoring

Bacterial population dynamics during the start-up of three lab-scale anaerobic reactors treating different wastewaters, i.e., synthetic glucose wastewater, whey permeate, and liquefied sewage sludge, were assessed using a combination of denaturing gradient gel electrophoresis (DGGE) and real-time PCR techniques. The DGGE results showed that bacterial populations related to Aeromonas spp. and Clostridium sticklandii emerged as common and prominent acidogens in all reactors. Two real-time PCR primer/probe sets targeting Aeromonas or C. sticklandii were developed, and successfully applied to quantitatively investigate their dynamics in relation to changes in reactor performance. Quantitative analysis demonstrated that both Aeromonas- and C. sticklandii-related populations were highly abundant for acidogenic period in all reactors. Aeromonas populations accounted for up to 86.6-95.3% of total bacterial 16S rRNA genes during start-up, suggesting that, given its capability of utilizing carbohydrate, Aeromonas is likely the major acidogen group responsible for the rapid initial fermentation of carbohydrate. C. sticklandii, able to utilize specific amino acids only, occupied up to 8.5-55.2% of total bacterial 16S rRNA genes in the reactors tested. Growth of this population is inferred to be supported, at least in part, by non-substrate amino acid sources like cell debris or extracellular excretions, particularly in the reactor fed on synthetic glucose wastewater with no amino acid source. The quantitative dynamics of the two acidogen groups of interest, together with their putative functions, suggest that Aeromonas and C. sticklandii populations were numerically as well as functionally important in all reactors tested, regardless of the differences in substrate composition. Particularly, the members of Aeromonas supposedly play vital roles in anaerobic digesters treating various substrates under acidogenic, fermentative start-up conditions.     

Temporal flux and spatial dynamics of nutrients, fecal indicators, and zoonotic pathogens in anaerobic swine manure lagoon water

Confined animal feeding operations (CAFOs) often use anaerobic lagoons for manure treatment. In the USA, swine CAFO lagoon water is used for crop irrigation that is regulated by farm-specific nutrient management plans (NMPs). Implementation of stricter US environmental regulations in 2013 will set soil P limits; impacting land applications of manure and requiring revision of NMPs. Precise knowledge of lagoon water quality is needed for formulating NMPs, for understanding losses of N and C in ammonia and greenhouse gas emissions, and for understanding risks of environmental contamination by fecal bacteria, including zoonotic pathogens. In this study we determined year-round levels of nutrients and bacteria from swine CAFO lagoon water. Statistical analysis of data for pH, electrical conductivity (EC), inorganic and organic C, total N, water-soluble and total minerals (Ca, Cu, Fe, K, Mg, Mn, P, and Zn) and bacteria (Escherichia coli, enterococci, Clostridium perfringens, Campylobacter spp., Listeria spp., Salmonella spp., and staphylococci) showed that all differed significantly by dates of collection. During the irrigation season, levels of total N decreased by half and the N:P ratio changed from 9.7 to 2.8. Some seasonal differences were correlated with temperature. Total N and inorganic C increased below 19 °C, and decreased above 19 °C, consistent with summer increases in ammonia and greenhouse gas emissions. Water-soluble Cu, Fe, and Zn increased with higher summer temperatures while enterococci and zoonotic pathogens (Campylobacter, Listeria, and Salmonella) decreased. Although their populations changed seasonally, the zoonotic pathogens were present year-round. Increasing levels of E. coli were statistically correlated with increasing pH. Differences between depths were also found. Organic C, total nutrients (C, Ca, Cu, Fe, Mg, Mn, N, P, and Zn) and C. perfringens were higher in deeper samples, indicating stratification of these parameters. No statistical interactions were found between collection dates and depths.     

Chromium bioaccumulation: comparison of the capacity of two floating aquatic macrophytes

The capacity of Salvinia herzogii and Pistia stratiotes to remove Cr (III) from water and their behaviour at different Cr (III) concentrations were studied in outdoor experiments. Cr distribution in aerial parts and roots with time and the possible mechanisms of Cr uptake were analyzed. Both macrophytes efficiently removed Cr from water at concentrations of 1, 2, 4 and 6 mgCrL(-1). S. herzogii was the best adapted species. At a greater initial concentration, greater bioaccumulation rates were observed. Root Cr uptake was a rapid process that was completed within the first 24h. Cr uptake through direct contact between the leaves and the solution is the main cause of the increase of Cr in the aerial parts, Cr being poorly translocated from the roots to the aerial parts. Both mechanisms were fast processes. The Cr uptake mechanism involves two components: a fast component and a slow one. The former occurs mainly due to the roots and leaves adsorption and is similar for both species. The slow component is different for each species probably because in P. stratiotes a Cr precipitation occurs induced by the roots.     

Mesophilic and thermophilic anaerobic digestion of faecal sludge in a pilot plant digester

In many developing countries, the sewage consisting of faecal sludge is discharged untreated into rivers, lakes and coastal areas. This poses a health hazard and a risk to the ecosystem, and wastes a resource which could produce sustainable energy. This paper reports results from an anaerobic digester of 1000L used for digestion of faecal waste at mesophilic and thermophilic conditions. The specific biogas production rate from faecal sludge was in the range of 0.06–0.12 m³/(kg DM.d) at mesophilic conditions at NTP (Normal Temperature & Pressure i.e. 25 °C and 1 atm. Pressure) and 0.1–0.21 m³/(kg DM.d) at thermophilic conditions calculated at NTP. The number of toilet users affects the biogas production with changes in the organic loading rate. The results showed 97% reductionin chemical oxygen demand and 90% reduction in biological oxygen demand of anaerobic digester discharge water as compared to inlet substrate values.     

Identification of microorganisms involved in reductive dehalogenation of chlorinated ethenes in an anaerobic microbial community

In this study, we report on phylogenetic and physiological characterization of an anaerobic culture capable of reductive dehalogenation of tetrachloroethene (PCE) obtained from a PCE-contaminated site. The culture was enriched using different combinations of electron donors (hydrogen and acetate) and electron acceptors (PCE, cis-1,2-dichloroethene (cDCE) and controls without chlorinated ethenes). The resulting subcultures were analyzed using three different approaches: chemical analysis to document conversion of chlorinated ethenes; polymerase chain reaction (PCR) of 16S rRNA gene fragments and denaturing gradient gel electrophoresis (DGGE) to compare community compositions; fluorescence in situ hybridization (FISH) to quantify specific groups of microorganisms using oligonucleotide probes previously designed or newly designed based on the sequences retrieved from sequence analysis of specific DGGE bands. Members of two genera which contain bacteria capable of reductive dehalogenation were detected in the culture: Dehalococcoides and Desulfitobacterium. The combined analyses suggested that Dehalococcoides-like bacteria are associated with complete dehalogenation of chlorinated ethenes to ethene with hydrogen as electron donor; and Desulfitobacterium-like bacteria, in contrast, are associated with incomplete PCE dehalogenation to cDCE and appear to be able to use acetate as electron donor. In addition, Sporomusa-like bacteria were identified, which most likely act as homoacetogens. The results demonstrated that combination of culture enrichment with different substrates, DGGE, and FISH allowed a detailed qualitative and quantitative characterization of the dominant microorganisms associated with reductive dehalogenation.     

Monitoring the temporal variations of PM2.5 and gases close to the highway in Sohar,Oman

The aim of this study was to identify the relationship between the concentrations of PM_2.5 (particulate matter less than 2.5 μm) and the temporal variation of the monitored gases at Sohar highway, Oman, from November 2014 to February 2015. The hourly concentrations of surface ozone (O₃), nitric dioxide (NO₂) and sulphur dioxide (SO₂) were measured by an open-path differential optical absorption spectroscopy instrument installed across Sohar highway. Additionally, the same gases and the meteorological parameters were measured in the same location of the PM_2.5 analyser. The findings of this study show that on the hourly time scale, PM_2.5 and O₃ were very weakly and negatively correlated. In contrast, on the daily time scale, PM_2.5 and O₃ were positively rather weakly correlated. Stronger correlation coefficient was found between 24 h averages of PM_2.5 and daily maximum O₃ concentrations. A policy implication of these findings could be that reducing the emissions of O₃ precursors reduces the levels of PM_2.5 as well.     

Potential acidifying capacity of deposition experiences from regions with high NH4+ and dry deposition in China

Acid rain may cause soil acidification possibly leading to indirect forest damage. Assessment of acidification potential of atmospheric deposition is problematic where dry and occult deposition is significant. Furthermore, uncertainty is enhanced where a substantial part of the potential acidity is represented by deposition of ammonium (NH(4)(+)) since the degree of assimilation and nitrification is not readily available. Estimates of dry deposition based on deposition velocity are highly uncertain and the models need to be verified or calibrated by field measurements of total deposition. Total deposition may be monitored under the forest canopy. The main problem with this approach is the unknown influence of internal bio-cycling. Moreover, bio-cycling may neutralize much of the acidity by leaching of mainly K(+). When the water percolates down into the rooting zone this K(+) is assimilated again and acidity is regenerated. Most monitoring stations only measure deposition. Lacking measurements of output flux of both NH(4)(+) and NO(3)(-) from the soil one cannot assess current net N transformation rates. Assumptions regarding the fate of ammonium in the soil have strong influence on the estimated acid load. Assuming that all the NH(4)(+) is nitrified may lead to an overestimation of the acidifying potential. In parts of the world where dry deposition and ammonium are important special consideration of these factors must be made when assessing the acidification potential of total atmospheric loading. In China dry and occult deposition is considerable and often greater than wet deposition. Furthermore, the main part of the deposited N is in its reduced state (NH(4)(+)). The IMPACTS project has monitored the water chemistry as it moves through watersheds at 5 sites in China. This paper dwells at two important findings in this study. 1) Potassium leached from the canopy by acid rain is assimilated again upon entering the mineral soil. 2) Nitrification apparently mainly takes place in forest floor (H- and O-) horizon as NH(4)(+) that escapes this horizon is efficiently assimilated in the A-horizon. This suggests that the potential acidification capacity of the deposition may be found in the throughfall and forest floor solution by treating K(+) and NH(4)(+), respectively, as acid cations in a base neutralization capacity (BNC) calculation.     

Monitoring of Cryptosporidium and Giardia river contamination in Paris area

This study evaluates the protozoan contamination of river waters, which are used for drinking water in Paris and its surrounding area (about 615,000 m(3) per day in total, including 300,000 m(3) for Paris area). Twenty litre samples of Seine and Marne Rivers were collected over 30 months and analyzed for Cryptosporidium oocysts and Giardia cysts detection according to standard national or international methods. Cryptosporidium oocysts and Giardia cysts were found, respectively, in 45.7% and 93.8% of a total of 162 river samples, with occasional high concentration peaks. A significant seasonal pattern was observed, with positive samples for Cryptosporidium more frequent in autumn than spring, summer and winter, and positive samples for Giardia less frequent in summer. Counts of enterococci and rainfalls were significantly associated with Giardia concentration but not Cryptosporidium. Other faecal bacteria were not correlated with monitored protozoan. Marne seems to contribute mainly to the parasitic contamination observed in Seine. Based on seasonal pattern and rainfall correlation, we hypothesize that the origin of contamination is agricultural practices and possible dysfunction of sewage treatment plants during periods of heavy rainfalls. High concentrations of protozoa found at the entry of drinking water plants justify the use of efficient water treatment methods. Treatment performances must be regularly monitored to ensure efficient disinfection according to the French regulations.     

Relevance of side reactions in anaerobic reductive dechlorination microcosms amended with different electron donors

In this study we examined the relative importance of side reactions, i.e. the formation of volatile fatty acids (VFA), and the reduction of alternative electron acceptors (nitrate, sulfate, Fe(III)), in enhanced dechlorination microcosms, amended with different electron donors, namely lactate, butyrate, and H(2)+acetate mixture. Dechlorination reactions proceeded at low rates and consequently, nearly all of the reducing equivalents (over 99%) available from electron donor consumption were channeled to (side) reactions other than dechlorination. The relevance of these side reactions was more evident with lactate which was consumed at higher rate than other electron donors. Correspondingly, high levels of VFA and soluble Fe(II) accumulated in the supernatant of lactate-amended microcosms. Ecotoxicity experiments (Lepidium sativum germination tests) also indicated that the supernatant was much more toxic/inhibitory than that of other microcosms. Among the electron donors tested, the H(2)+acetate mixture, yielded the most promising results in terms of extent of dechlorination, negligible accumulation of by-products, and residual groundwater toxicity. Fluorescent In situ Hybridization analysis (FISH) confirmed that H(2)+acetate-amended microcosms were dominated by Dehalococcoides spp., while a higher biodiversity was observed in the cultures fed with lactate or butyrate. Overall, the average amount of donor that was required for the removal of 1micromol of chloride from the contaminant differed greatly among the donors, namely 2.13meq/micromolCl(-) for lactate, 1.01meq/micromolCl(-) for butyrate, and 0.39meq/micromolCl(-) for H(2)+acetate mixture. Interestingly, dechlorinating activity was observed under sulfate-reducing conditions; this suggests that it may not be necessary to deplete the sulfate from the groundwater, for instance by supplying an excess electron donor, in order to achieve substantial dechlorination. Finally, in this study we found that the pathway of anaerobic lactate degradation shifted from the production of acetate and H(2) during active sulfate reduction to acetate and propionate upon sulfate depletion. Energetic considerations that support this finding were presented.     

Model selection, identification and validation in anaerobic digestion: a review

Anaerobic digestion enables waste (water) treatment and energy production in the form of biogas. The successful implementation of this process has lead to an increasing interest worldwide. However, anaerobic digestion is a complex biological process, where hundreds of microbial populations are involved, and whose start-up and operation are delicate issues. In order to better understand the process dynamics and to optimize the operating conditions, the availability of dynamic models is of paramount importance. Such models have to be inferred from prior knowledge and experimental data collected from real plants. Modeling and parameter identification are vast subjects, offering a realm of approaches and methods, which can be difficult to fully understand by scientists and engineers dedicated to the plant operation and improvements. This review article discusses existing modeling frameworks and methodologies for parameter estimation and model validation in the field of anaerobic digestion processes. The point of view is pragmatic, intentionally focusing on simple but efficient methods.     

A comparison of the role of two blue-green algae in THM and HAA formation

The contribution of two blue-green algae species, Anabaena flos-aquae and Microcystis aeruginosa, to the formation of trihalomethanes (THMs) and haloacetic acids (HAAs) was investigated. The experiments examined the formation potential of these disinfection by-products (DBPs) from both algae cells and extracellular organic matter (EOM) during four algal growth phases. Algal cells and EOM of Anabaena and Microcystis exhibited a high potential for DBP formation. Yields of total THMs (TTHM) and total HAAs (THAA) were closely related to the growth phase. Reactivity of EOM from Anabaena was slightly higher than corresponding cells, while the opposite result was found for Microcystis. Specific DBP yields (yield/unit C) of Anabaena were in the range of 2-11 μmol/mmol C for TTHM and 2-17 μmol/mmol C for THAA, while those of Microcystis were slightly higher. With regard to the distributions of individual THM and HAA compounds, differences were observed between the algae species and also between cells and EOM. The presence of bromide shifted the dominant compounds from HAAs to THMs.     

Focused-Pulsed sludge pre-treatment increases the bacterial diversity and relative abundance of acetoclastic methanogens in a full-scale anaerobic digester

The low yield of methane in anaerobic digestion systems represents a loss of energy that can be captured as renewable energy when the input sludge is pre-treated to make it more bioavailable. We investigated Focused-Pulsed (FP) pre-treatment, which make complex biological solids more bioavailable by exposing them to rapid pulses of a very strong electric field. We investigated how the microbial ecology in full-scale anaerobic digesters was altered when the digesters' methane production rate was significantly increased by FP pre-treatment. Using clone libraries and quantitative PCR, we demonstrated a shift in methanogenic genera to the acetate-cleaving Methanosaeta and away from the H₂-oxidizing Methanoculleus. In addition, the acetate concentration in the effluent was very low, probably due to the dominance of Methanosaeta, which are capable of scavenging low acetate concentrations. By analyzing 36,797 pyrosequencing tags from the V6 region of the bacterial 16S rRNA gene, along with archaeal and bacterial clone libraries and quantitative PCR, we compared the microbial community composition before and after FP treatment. The bacterial community became more diverse after FP pre-treatment and was populated more by phylotypes associated with cellulose fermentation (Ruminococcus), scavenging of biomass-derived organic carbon (Chloroflexi), and homo-acetogenesis (Treponema). We interpret that, as the overall activity of the community was stimulated by addition of more bioavailable organic matter, the bacterial community became more phylogenetically diverse to take advantage of the added input of biodegradable material and in response to the more efficient utilization of acetate by Methanosaeta.     

Natural and enhanced anaerobic degradation of 1,1,1-trichloroethane and its degradation products in the subsurface--a critical review

1,1,1-Trichloroethane (TCA) in groundwater is susceptible to a variety of natural degradation mechanisms. Evidence of intrinsic decay of TCA in aquifers is commonly observed; however, TCA remains a persistent pollutant at many sites and some of the daughter products that accumulate from intrinsic decay of TCA have been determined to be more toxic than the parent compound. Research advances from the past decade indicate that in situ enhanced reductive dechlorination (ERD) offers promise as a cost-effective solution toward the cleanup of groundwater contaminated with TCA and its transformation daughter products. Laboratory studies have demonstrated that pure or mixed cultures containing certain Dehalobacter (Dhb) bacteria can catalyze respiratory dechlorination of TCA and 1,1-dichloroethane (1,1-DCA) to monochloroethane (CA) in groundwater systems. 16S rRNA Dhb gene probes have been used as biomarkers in groundwater samples to both assess ERD potential and quantify growth of Dhb in ERD applications at TCA sites. Laboratory findings suggest that iron-bearing minerals and methanogenic bacteria that co-occur in reduced aquifers may synergistically affect dechlorination of TCA. Despite these advances, a number of significant challenges remain, including an inability of any known cultures to completely dechlorinate TCA to ethane. CA is commonly observed as a terminal product of the biological reductive dechlorination of TCA and 1,1-DCA. Also important is the lack of rigorous field studies demonstrating the utility of bioaugmentation with Dhb cultures for remediation of TCA in the field. In this paper we review the state-of-the-science of TCA degradation in aquifers, examining results from both laboratory experiments and twenty-two field case studies, focusing on the capabilities and limits of ERD technology, and identifying aspects of the technology that warrant further development.     

Temperature dependence of anaerobic TCE-dechlorination in a highly enriched Dehalococcoides-containing culture

Temperature dependence of reductive trichloroethene (TCE) dechlorination was investigated in an enrichment culture (KB-1), using lactate or propionate as electron donors at a temperature interval from 4 to 60 degrees C. Dechlorination was complete to ethene at temperatures between 10 and 30 degrees C (lactate-amended) and between 15 and 30 degrees C (propionate-amended). Dechlorination stalled at cis-1,2-dichloroethene (cDCE) at 4 degrees C (lactate-amended), at and below 10 degrees C (propionate-amended), and at 40 degrees C with both electron donors. No dechlorination of TCE was observed at 50 and 60 degrees C. Concentrations of Dehalococcoides had increased or remained constant after 15 days of incubation at temperatures involving complete dechlorination to ethene. Temperature dependence of dechlorination rates was compared using zero order degradation kinetics and a Monod growth-rate model for multiple electron acceptor usage with competition. Maximum growth rates (mu) and zero order degradation rates were highest for TCE dechlorination at 30 degrees C with lactate as substrate (mu(TCE) of 7.00+/-0.14 days(-1)). In general, maximum growth rates and dechlorination rates of TCE were up to an order of magnitude higher than rates for utilization of cis-dichloroethene (cDCE, mu(c)(DCE) up to 0.17+/-0.02 days(-1)) and vinyl chloride (VC, mu(VC) up to 0.52+/-0.09 days(-1)). Temperature dependence of maximum growth rates and degradation rates of cDCE and VC were similar and highest at 15-30 degrees C, with growth rates on cDCE being lower than on VC. This study demonstrates that bioaugmentation of chlorinated ethene sites may be more efficient at elevated temperatures.     

Functional bacterial and archaeal community structures of major trophic groups in a full-scale anaerobic sludge digester

Functional Bacteria and Archaea community structures of a full-scale anaerobic sludge digester were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescent in situ hybridization (FISH) technique and micromanipulation. FISH analysis with a comprehensive set of 16S and 23S rRNA-targeted oligonucleotide probes based on 16S rRNA clone libraries revealed that the Gram-positive bacteria represented by probe HGC69A-hybridized Actinobacteria (8.5+/-1.4% of total 4', 6-diamidino-2-phenylindole (DAPI)-stained cells) and probe LGC354-hybridized Firmicutes (3.8+/-0.8%) were the major phylogenetic bacterial phyla, followed by Bacteroidetes (4.0+/-1.2%) and Chloroflexi (3.7+/-0.8%). The probe MX825-hybridized Methanosaeta (7.6+/-0.8%) was the most abundant archaeal group, followed by Methanomicrobiales (2.8+/-0.6%) and Methanobacteriaceae (2.7+/-0.4%). The functional community structures (diversity and relative abundance) of major trophic groups were quantitatively analyzed by MAR-FISH. The results revealed that glucose-degrading microbial community had higher abundance (ca. 10.6+/-4.9% of total DAPI-stained cells) and diversity (at least seven phylogenetic groups) as compared with fatty acid-utilizing microbial communities, which were more specialized to a few phylogenetic groups. Despite the dominance of Betaproteobacteria, members of Chloroflexi, Smithella, Syntrophomonas and Methanosaeta groups dominated the [(14)C]glucose-, [(14)C]propionate-, [(14)C]butyrate- and [(14)C]acetate-utilizing microorganism community, and accounted for 27.7+/-4.3%, 29.6+/-7.0%, 34.5+/-7.6% and 18.2+/-9.5%, respectively. In spite of low abundance (ca. 1%), the hitherto unknown metabolic functions of Spirochaeta and candidate phylum of TM7 as well as Synergistes were found to be glucose and acetate utilization, respectively.     

Effects of wood ash fertilization on forest floor greenhouse gas emissions and tree growth in nutrient poor drained peatland forests

Wood ash (3.1, 3.3 or 6.6 tonnes dry weight ha^− 1) was used to fertilize two drained and forested peatland sites in southern Sweden. The sites were chosen to represent the Swedish peatlands that are most suitable for ash fertilization, with respect to stand growth response. The fluxes of carbon dioxide (CO₂), methane (CH₄) and nitrous oxide (N₂O) from the forest floor, measured using opaque static chambers, were monitored at both sites during 2004 and 2005 and at one of the sites during the period 1 October 2007-1 October 2008. No significant (p > 0.05) changes in forest floor greenhouse gas exchange were detected. The annual emissions of CO₂ from the sites varied between 6.4 and 15.4 tonnes ha^− 1, while the CH₄ fluxes varied between 1.9 and 12.5 kg ha^− 1. The emissions of N₂O were negligible. Ash fertilization increased soil pH at a depth of 0-0.05 m by up to 0.9 units (p < 0.01) at one site, 5 years after application, and by 0.4 units (p < 0.05) at the other site, 4 years after application. Over the first 5 years after fertilization, the mean annual tree stand basal area increment was significantly larger (p < 0.05) at the highest ash dose plots compared with control plots (0.64 m² ha^− 1 year^− 1 and 0.52 m² ha^− 1 year^− 1, respectively). The stand biomass, which was calculated using tree biomass functions, was not significantly affected by the ash treatment. The groundwater levels during the 2008 growing season were lower in the high ash dose plots than in the corresponding control plots (p < 0.05), indicating increased evapotranspiration as a result of increased tree growth. The larger basal area increment and the lowered groundwater levels in the high ash dose plots suggest that fertilization promoted tree growth, while not affecting greenhouse gas emissions.     

Impact of nano zero valent iron (NZVI) on methanogenic activity and population dynamics in anaerobic digestion

Nano zero valent iron (NZVI), although being increasingly used for environmental remediation, has potential negative impact on methanogenesis in anaerobic digestion. In this study, NZVI (average size = 55 ± 11 nm) showed inhibition of methanogenesis due to its disruption of cell integrity. The inhibition was coincident with the fast hydrogen production and accumulation due to NZVI dissolution under anaerobic conditions. At the concentrations of 1 mM and above, NZVI reduced methane production by more than 20%. At the concentration of 30 mM, NZVI led to a significant increase in soluble COD (an indication of cell disruption) and volatile fatty acids in the mixed liquor along with an accumulation of H₂, resulting in a reduction of methane production by 69% (±4% [standard deviation]). By adding a specific methanogenesis inhibitor-sodium 2-bromoethanesulfonate (BES) to the anaerobic sludge containing 30 mM NZVI, the amount of H₂ produced was only 79% (±1%) of that with heat-killed sludge, indicating the occurrence of bacterially controlled hydrogen utilization processes. Quantitative PCR data was in accordance with the result of methanogenesis inhibition, as the level of methanogenic population (dominated by Methanosaeta) in the presence of 30 mM NZVI decreased significantly compared to that of the control. On the contrary, ZVI powder (average size <212 μm) at the same concentration (30 mM) increased methane production presumably due to hydrogenotrophic methanogenesis of hydrogen gas that was slowly released from the NZVI powder. While it is a known fact that NZVI disrupts cell membranes, which inhibited methanogenesis described herein, the results suggest that the rapid hydrogen production due to NZVI dissolution also contribute to methanogenesis inhibition and lead to bacterially controlled hydrogenotrophic processes.