Dietary supplementation with calcium peroxide improves methane mitigation potential of finishing beef cattle.

Calcium peroxide (CaO2) offers potential as an anti-methanogenic dietary feed material. The compound has been previously assessed in vitro, with methane (CH4) reductions of > 50% observed. The objective of this study was to assess dietary supplementation of CaO2 at different inclusion levels and physical formats in a finishing beef system on the effects of animal performance, gaseous emissions, rumen fermentation parameters and digestibility. Seventy-two dairy-beef bulls (465 kg; 16 months of age) were randomly allocated to one of four treatments supplemented with CaO2; in a coarse ration (1) CON (0% CaO2), (2) LO (1.35% CaO2), (3) HI (2.25% CaO2), and in a pellet (4) HP (2.25% CaO2) (n = 18). Animals received their respective treatments for a 77 d finishing period, during which DM intake (American Calan Inc., Northwood, NH), average daily gain (ADG), feed efficiency and enteric emissions (GreenFeed emissions monitoring system; C-Lock Inc., Rapid City, SD) were measured. The finishing diet was isonitrogenous and isoenergetic across the four treatment groups, composed of 60:40 grass silage:concentrate. Silage was offered each morning (0900 h), and concentrates were offered twice daily (0800 and 1500 h). Supplementation of CaO2 had no effect on final weight (P = 0.09), ADG (P = 0.22) or feed efficiency (P = 0.13). Regarding DM intake, the HI treatment group consumed in the order of 1 kg less than CON (P < 0.01), while HP did not affect DM intake compared to CON (P = 0.79). Across treatments, DM intake ranged from 8.43 to 9.57 kg/d, equating to 1.6-1.8% of BW. Daily CH4 values for the control were 240 g/d, while CaO2 supplemented diets ranged from 202 to 170 g/d, resulting in daily CH4 reductions of 16, 29 and 27% for LO, HI and HP, respectively, compared to CON (P < 0.0001). Additionally, hydrogen was reduced in CaO2 supplemented animals by 32-36% relative to CON (P < 0.0001), with a simultaneous reduction in volatile fatty acid production (P < 0.01) and an increase in propionate concentration (P < 0.0001). Across all universally accepted CH4 metrics (yield, intensity, production), the dietary inclusion of CaO2 whether at a low or high rate, or indeed, through a coarse ration or pelleted format reduced CH4 in the order of 16-32%. This study also concluded that CaO2 can successfully endure the pelleting process, therefore, improving ease of delivery if implemented at farm level.

Landspreading with co-digested cattle slurry, with or without pasteurisation, as a mitigation strategy against pathogen, nutrient and metal contamination associated with untreated slurry.

North Atlantic European grassland systems have a low nutrient use efficiency and high rainfall. This grassland is typically amended with unprocessed slurry, which counteracts soil organic matter depletion and provides essential plant micronutrients but can be mobilised during rainfall events thereby contributing to pathogen, nutrient and metal incidental losses. Co-digesting slurry with waste from food processing mitigates agriculture-associated environmental impacts but may alter microbial, nutrient and metal profiles and their transmission to watercourses, and/or soil persistence, grass yield and uptake. The impact of EU and alternative pasteurisation regimes on transmission potential of these various pollutants is not clearly understood, particularly in pasture-based agricultural systems. This study utilized simulated rainfall (Amsterdam drip-type) at a high intensity indicative of a worst-case scenario of ~11 mm hr-1 applied to plots 1, 2, 15 and 30 days after grassland application of slurry, unpasteurised digestate, pasteurised digestate (two conditions) and untreated controls. Runoff and soil samples were collected and analysed for a suite of potential pollutants including bacteria, nutrients and metals following rainfall simulation. Grass samples were collected for three months following application to assess yield as well as nutrient and metal uptake. For each environmental parameter tested: microbial, nutrient and metal runoff losses; accumulation in soil and uptake in grass, digestate from anaerobic co-digestion of slurry with food processing waste resulted in lower pollution potential than traditional landspreading of slurry without treatment. Reduced microbial runoff from digestate was the most prominent advantage of digestate application. Pasteurisation of the digestate further augmented those environmental benefits, without impacting grass output. Anaerobic co-digestion of slurry is therefore a multi-beneficial circular approach to reducing impacts of livestock production on the environment.

Cold adaptation and replicable microbial community development during long-term low-temperature anaerobic digestion treatment of synthetic sewage.

The development and activity of a cold-adapting microbial community was monitored during low-temperature anaerobic digestion (LtAD) treatment of wastewater. Two replicate hybrid anaerobic sludge bed-fixed-film reactors treated a synthetic sewage wastewater at 12°C, at organic loading rates of 0.25-1.0 kg chemical oxygen demand (COD) m-3 d-1, over 889 days. The inoculum was obtained from a full-scale anaerobic digestion reactor, which was operated at 37°C. Both LtAD reactors readily degraded the influent with COD removal efficiencies regularly exceeding 78% for both the total and soluble COD fractions. The biomass from both reactors was sampled temporally and tested for activity against hydrolytic and methanogenic substrates at 12°C and 37°C. Data indicated that significantly enhanced low-temperature hydrolytic and methanogenic activity developed in both systems. For example, the hydrolysis rate constant (k) at 12°C had increased 20-30-fold by comparison to the inoculum by day 500. Substrate affinity also increased for hydrolytic substrates at low temperature. Next generation sequencing demonstrated that a shift in a community structure occurred over the trial, involving a 1-log-fold change in 25 SEQS (OTU-free approach) from the inoculum. Microbial community structure changes and process performance were replicable in the LtAD reactors.

Detection, fate and inactivation of pathogenic norovirus employing settlement and UV treatment in wastewater treatment facilities.

It is accepted that discharged wastewaters can be a significant source of pathogenic viruses in receiving water bodies contributing to pollution and may in turn enter the human food chain and pose a risk to human health, thus norovirus (NoV) is often a predominant cause of gastroenteritis globally. Working with NoV poses particular challenges as it cannot be readily identified and detection by molecular methods does not assess infectivity. It has been proposed that the infectivity of NoV may be modelled through the use of an alternative virus; F-specific RNA (FRNA) bacteriophages; GA genotype and other FRNA bacteriophages have been used as a surrogate in studies of NoV inactivation. This study investigated the efficiency of novel pulsed ultraviolet irradiation and low pressure ultraviolet irradiation as a potential pathogen inactivation system for NoV and FRNA bacteriophage (GA) in secondary treated wastewaters. The role of UV dose and the impact of suspended solids concentration on removal efficiency were also examined. The study also investigated the role of settlement processes in wastewater treatment plants in removing NoV. While NoV inactivation could not be determined it was found that at a maximum UV dose of 6.9J/cm(2) (6900mJ/cm(2)) an average 2.4 log removal of FRNA bacteriophage (GA) was observed; indicating the potential need for high UV doses to remove NoV if FRNA bacteriophage prove a suitable indicator for NoV. The study found that increasing concentrations of suspended solids impacted on PUV efficiency however, it appears the extent of the impact may be site specific. Furthermore, the study found that settlement processes can play a significant role in the removal of FRNA bacteriophage, thus potentially NoV.

Carbon amendment and soil depth affect the distribution and abundance of denitrifiers in agricultural soils.

The nitrite reductase (nirS and nirK) and nitrous oxide reductase-encoding (nosZ) genes of denitrifying populations present in an agricultural grassland soil were quantified using real-time polymerase chain reaction (PCR) assays. Samples from three separate pedological depths at the chosen site were investigated: horizon A (0-10 cm), horizon B (45-55 cm), and horizon C (120-130 cm). The effect of carbon addition (treatment 1, control; treatment 2, glucose-C; treatment 3, dissolved organic carbon (DOC)) on denitrifier gene abundance and N2O and N2 fluxes was determined. In general, denitrifier abundance correlated well with flux measurements; nirS was positively correlated with N2O, and nosZ was positively correlated with N2 (P < 0.03). Denitrifier gene copy concentrations per gram of soil (GCC) varied in response to carbon type amendment (P < 0.01). Denitrifier GCCs were high (ca. 10(7)) and the bac:nirK, bac:nirS, bac:nir (T) , and bac:nosZ ratios were low (ca. 10(-1)/10) in horizon A in all three respective treatments. Glucose-C amendment favored partial denitrification, resulting in higher nir abundance and higher N2O fluxes compared to the control. DOC amendment, by contrast, resulted in relatively higher nosZ abundance and N2 emissions, thus favoring complete denitrification. We also noted soil depth directly affected bacterial, archaeal, and denitrifier abundance, possibly due to changes in soil carbon availability with depth.

Quantifying faecal indicator organism hydrological transfer pathways and phases in agricultural catchments.

Faecal indicator organisms (FIOs) can impact on water quality and pose a health and environmental risk. The transfer of FIOs, such as Escherichia coli (E. coli), from land to water is driven by hydrological connectivity and may follow the same flowpaths as nutrients, from agricultural and human sources. This study investigated E. coli transfer in two catchment areas with high source and transport pressures. These pressures were: organic phosphorus (P) loading; human settlement; conduits and fissures in a grassland karst area; and clay rich and impermeable soils in a mixed arable area. The occurrence of E. coli and its transport pathways, along with the pathways of nutrients, were studied using a combination of targeted FIO sampling, during different hydrological phases and events, and high resolution nutrient analysis. The quick flow component in both catchments was found to be a more potent vector for E. coli, and was coincident with the total P flowpaths using a P Loadograph Recession Analysis (LRA). The karst grassland catchment was found to be a transport limited system and the mixed arable catchment a source limited system. Hence, despite the grassland catchment being a potentially higher FIO source, the E. coli loads leaving the catchment were low compared to the mixed arable catchment. E. coli load whole-event comparisons also indicated that the grassland karst transfers tended to be much lower on falling phases of runoff, while the arable catchment, over greywacke and mudstone geology, showed little change between the phases. Furthermore, the arable catchment showed asymptotic decline of sustained E. coli loads towards low flows, which may be indicative of chronic point sources. These results indicate the dominance of transport mechanisms over source mechanisms for mass E. coli loads and also chronic loads during low flow. These will be important considerations for risk assessment and mitigation.

Simulated sunlight inactivation of norovirus and FRNA bacteriophage in seawater.

AIMS: To investigate norovirus (NoV) and F-specific RNA (FRNA) bacteriophage inactivation in seawater under simulated sunlight and temperature conditions representative of summer (235 W m(-2) ; 17°C) and winter (56 W m(-2) ; 10°C) conditions in Ireland. METHODS AND RESULTS: Inactivation experiments were carried out using a collimated beam of simulated sunlight and 100 ml of filtered seawater seeded with virus under controlled temperature conditions. NoV concentrations were determined using RT-qPCR, and FRNA bacteriophage concentrations were determined using RT-qPCR and by plaque assay. For all virus types, the fluence required to achieve a 90% reduction in detectable viruses (S90 value) using RT-qPCR was not significantly different between summer and winter conditions. S90 values for FRNA bacteriophage determined by plaque assay were significantly less than those determined by RT-qPCR. Unlike S90 values determined by RT-qPCR, a significant difference existed between summer and winter S90 values for infectious FRNA bacteriophage. CONCLUSIONS: This study demonstrated that RT-qPCR significantly overestimates the survival of infectious virus and is therefore unsuitable for determining the inactivation rates of viruses in seawater. SIGNIFICANCE AND IMPACT OF THE STUDY: Results from this study provide initial data on the inactivation of NoV and FRNA bacteriophage in seawater under representative summer and winter conditions and will be of interest to shellfish and water management agencies alike.

The hydrolysis and biogas production of complex cellulosic substrates using three anaerobic biomass sources.

In this study, the ability of various sludges to digest a diverse range of cellulose and cellulose-derived substrates was assessed at different temperatures to elucidate the factors affecting hydrolysis. For this purpose, the biogas production was monitored and the specific biogas activity (SBA) of the sludges was employed to compare the performance of three anaerobic sludges on the degradation of a variety of complex cellulose sources, across a range of temperatures. The sludge with the highest performance on complex substrates was derived from a full-scale bioreactor treating sewage at 37 °C. Hydrolysis was the rate-limiting step during the degradation of complex substrates. No activity was recorded for the synthetic cellulose compound carboxymethylcellulose (CMC) using any of the sludges tested. Increased temperature led to an increase in hydrolysis rates and thus SBA values. The non-granular nature of the mesophilic sludge played a positive role in the hydrolysis of solid substrates, while the granular sludges proved more effective on the degradation of soluble compounds.

Temperature effects on the trophic stages of perennial rye grass anaerobic digestion.

Continuous Stirred Tank Reactors (CSTRs), operated in batch mode, were used to evaluate the feasibility of psychrophilic (low temperature) digestion of perennial rye grass in a long term experiment (150 days) for the first time. The reactors were operated in parallel at 3 different temperatures, 10, 15 and 37 degrees C. Hydrolysis, acidification and methanogenesis were assessed by VS degradation, by soluble chemical oxygen demand (SCOD) and volatile fatty acids (VFA) production, and by methane production, respectively. Hydrolysis was the rate-limiting step at all temperatures and the rates and extent of hydrolysis were considerably lower at 15 and 10 degrees C, than at 37 degrees C. The total VS degradation was 53%, 34% and 19% at 37, 15 and 10 degrees C, respectively. Acidification was not affected by temperature and VFA production and consumption was balanced in all cases, except at 10 degrees C. Methane yields were 0.215 m3 CH4 kg(-1) VS(-1) added, 0.160 m3 CH4 kg(-1) VS(-1) added and 0.125 m3 CH4 kg(-1) VS(-1) added at 37, 15 and 10 degrees C, respectively. Methanogenesis was not strongly affected at 15 C but it became rate-limiting at 10 degrees C. Overall, the solid degradation and methane production performance under psychrophilic conditions was encouraging and greater than previously reported. Considering the non-acclimated, mesophilic nature of the inoculum, there are grounds to believe that low-temperature anaerobic digestion of grass could be feasible if coupled to efficient hydrolysis of the biomass.

A metaproteomic approach gives functional insights into anaerobic digestion.

AIMS: The objective of this work was to provide functional evidence of key metabolic pathways important for anaerobic digestion processes through the identification of highly expressed proteins in a mixed anaerobic microbial consortium. METHODS AND RESULTS: The microbial communities from an anaerobic industrial-like wastewater treatment bioreactor were characterized using phylogenetic analyses and metaproteomics. Clone libraries indicated that the bacterial community in the bioreactor was diverse while the archaeal population was mainly composed of Methanocorpusculum-like (76%) micro-organisms. Three hundred and eighty-eight reproducible protein spots were obtained on 2-D gels, of which 70 were excised and 33 were identified. The putative functions of the proteins detected in the anaerobic bioreactor were related to cellular processes, including methanogenesis from CO(2) and acetate, glycolysis and the pentose phosphate pathway. Metaproteomics also indicated, by protein assignment, the presence of specific micro-organisms in the bioreactor. However, only a limited overlap was observed between the phylogenetic and metaproteomic analyses. CONCLUSIONS: This study provides some direct evidence of the microbial activities taking place during anaerobic digestion. SIGNIFICANCE AND IMPACT OF STUDY: This study demonstrates metaproteomics as a useful tool to uncover key biochemical pathways underpinning specific anaerobic bioprocesses.

Cultivation of low-temperature (15 degrees C), anaerobic, wastewater treatment granules.

AIMS: Anaerobic sludge granules underpin high-rate waste-to-energy bioreactors. Granulation is a microbiological phenomenon involving the self-immobilization of several trophic groups. Low-temperature anaerobic digestion of wastes is of intense interest because of the economic advantages of unheated bioenergy production technologies. However, low-temperature granulation of anaerobic sludge has not yet been demonstrated. The aims of this study were to (i) investigate the feasibility of anaerobic sludge granulation in cold (15 degrees C) bioreactors and (ii) observe the development of methanogenic activity and microbial community structure in developing cold granules. METHODS AND RESULTS: One mesophilic (R1; 37 degrees C) and two low-temperature (R2 and R3, 15 degrees C) laboratory-scale, expanded granular sludge bed bioreactors were seeded with crushed (diameter <0.4 mm) granules and were fed a glucose-based wastewater for 194 days. Bioreactor performance was assessed by chemical oxygen demand removal, biogas production, granule growth and temporal methanogenic activity. Granulation was observed in R2 and R3 (up to 33% of the sludge). Elevated hydrogenotrophic methanogenesis was observed in psychrophilically cultivated biomass, but acetoclastic methanogenic activity was also retained. Denaturing gradient gel electrophoresis of archaeal 16S rRNA gene fragments indicated that a distinct community was associated with developing and mature granules in the low-temperature (LT) bioreactors. CONCLUSIONS: Granulation was observed at 15 degrees C in anaerobic bioreactors and was associated with H(2)/CO(2)-mediated methanogenesis and distinct community structure development. SIGNIFICANCE AND IMPACT OF THE STUDY: Granulation underpins high-rate anaerobic waste treatment bioreactors. Most LT bioreactor trials have employed mesophilic seed sludge, and granulation <20 degrees C was not previously documented.

Low temperature anaerobic biotreatment of priority pollutants.

The effect of low operating temperature and pollutant concentration on the performance of five anaerobic hybrid reactors was investigated. Stable and efficient long-term (>400 days) treatment of a cold (6-13 degrees C), volatile fatty acid (VFA)-based, wastewater was achieved at applied organic loading rates (OLRs) of 5 kg chemical oxygen demand (COD) m(-3) d(-1) with COD removal efficiencies c. 84% at 6 degrees C (sludge loading rate (SLR) 1.04-1.46 kg COD kg [VSS](-1) d(-1)). VFA-based wastewaters, containing up to 14 g pentachlorophenol (PCP) m(-3) d(-1) or 155 g toluene m(-3) d(-1) were successfully treated at applied OLRs of 5-7 kg COD m(-3) d(-1). Despite transient declines in reactor performance in response to increasing toxicant loading rates, stable operation (COD removal efficiencies > 90%) and satisfactory toxicant removal efficiencies (>88%) were demonstrated by the systems.

Determination and localisation of in situ substrate uptake by anaerobic wastewater treatment granular biofilms.

Radiotracer incubation experiments and beta microimaging, along with fluorescent in situ hybridisation (FISH), are proposed as a complementary approach to specific methanogenic activity testing and measurement of in vitro substrate utilisation rates to understand better the ecophysiology of anaerobic granular biofilms from wastewater treatment reactors.

Granular biofilm-based anaerobic digestion: molecular biomonitoring and high-rate psychrophilic treatment of phenolic wastewater.

Two pairs of expanded granular sludge bed (EGSB) bioreactors, R1/R2 and R3/R4, were designed. R1/R2 were used for mesophilic (37 degrees C) treatment of synthetic wastewater over a 100-day trial. A successful start-up was achieved by R1 and R2, with COD removal over 90%. Both reactors were operated under identical parameters; however, increased organic loading induced a reduction in COD removal by R1, while R2 maintained satisfactory performance throughout the experiment. R3/R4 were operated at 15 degrees C throughout a 422-day trial and were used for the stabilisation of volatile fatty acid-based wastewater. Phenol was introduced to R4 at an applied loading rate of 1 kg phenol m(-3)d(-1), which was increased to 2 kg phenol m(-3)d(-1). No phenol was supplied to R3. Efficient COD conversion was recorded in both R3 and R4, thus demonstrating the feasibility of high-rate phenol degradation under psychrophilic conditions. Terminal restriction fragment length polymorphism analysis was applied to the characterisation of microbial community dynamics within each of the reactors. The results indicated a microbiological basis for the deviation, in terms of operational performance, of R1 and R2. TRFLP analyses indicated stable microbial communities in R3 and R4, but detected changes in the abundance of specific ribotypes in response to phenol mineralisation.

Application and biomolecular monitoring of psychrophilic anaerobic digestion.

Thirteen anaerobic hybrid expanded granular sludge bed-anaerobic filter bioreactors were used for psychrophilic (15-18 degrees C) anaerobic digestion of a variety of synthetic and non-synthetic wastewaters, including: food-processing, dairy, aromatic- and aliphatic-containing and brewery discharges. Specific methanogenic activity assays were employed to assess temporal physiological activity dynamics. Terminal restriction fragment length polymorphism genetic fingerprinting and fluorescent in situ hybridization were used to monitor shifts in the structure of the microbial communities in the bioreactors in response to operating conditions. Treatment efficiencies obtained were comparable to previous mesophilic (37 degrees C) trials. Methanogenic activity developed under psychrophilic conditions and putative psychrophilic populations were detected within otherwise psychrotolerant mesophilic communities. Shifts in the population structure of archaeal (methanogenic) communities were more indicative of process disruption than bacterial communities. Biomolecular techniques were demonstrated as valuable tools for anaerobic wastewater treatment plant monitoring.

Assessment of anaerobic wastewater treatment failure using terminal restriction fragment length polymorphism analysis.

AIMS: The suitability of genetic fingerprinting to study the microbiological basis of anaerobic bioreactor failure is investigated. METHODS AND RESULTS: Two laboratory-scale anaerobic expanded granular sludge bed bioreactors, R1 and R2, were used for the mesophilic (37 degrees C) treatment of high-strength [10 g chemical oxygen demand (COD) l(-1)] synthetic industrial-like wastewater over a 100-day trial period. A successful start up was achieved by both bioreactors with COD removal over 90%. Both reactors were operated under identical parameters; however, increased organic loading during the trial induced a reduction in the COD removal of R1, while R2 maintained satisfactory performance (COD removal >90%) throughout the experiment. Specific methanogenic activity measurements of biomass from both reactors indicated that the main route of methane production was hydrogenotrophic methanogenesis. Terminal restriction fragment length polymorphism (TRFLP) analysis was applied to the characterization of microbial community dynamics within the system during the trial. The principal differences between the two consortia analysed included an increased abundance of Thiovulum- and Methanococcus-like organisms and uncultured Crenarchaeota in R1. CONCLUSIONS: The results indicated that there was a microbiological basis for the deviation, in terms of operational performance, of R1 and R2. SIGNIFICANCE AND IMPACT OF THE STUDY: High-throughput fingerprinting techniques, such as TRFLP, have been demonstrated as practically relevant for biomonitoring of anaerobic reactor communities.

Full-scale and laboratory-scale anaerobic treatment of citric acid production wastewater.

This paper reviews the operation of a full-scale, fixed-bed digester treating a citric acid production wastewater with a COD:sulphate ratio of 3-4:1. Support matrix pieces were removed from the digester at intervals during the first 5 years of operation in order to quantify the vertical distribution of biomass within the digester. Detailed analysis of the digester biomass after 5 years of operation indicated that H2 and propionate-utilising SRB had outcompeted hydrogenophilic methanogens and propionate syntrophs. Acetoclastic methanogens were shown to play the dominant role in acetate conversion. Butyrate and ethanol-degrading syntrophs also remained active in the digester after 5 years of operation. Laboratory-scale hybrid reactor treatment at 55 degrees C of a diluted molasses influent, with and without sulphate supplementation, showed that the reactors could be operated with high stability at volumetric loading rates of 24 kgCOD.m-3.d-1 (12 h HRT). In the presence of sulphate (2 g/l-1; COD/sulphate ratio of 6:1), acetate conversion was severely inhibited, resulting in effluent acetate concentrations of up to 4000 mg.l-1.

Measurement of intracellular calcium levels of human spermatozoa acrosome reacted by electropermeabilization.

Human spermatozoa readily undergo in vitro capacitation and the acrosome reaction when exposed to a single, brief, high-voltage electric pulse in a medium containing calcium. Using the fluorescent calcium indicator, Fura-2, the changes in the intracellular calcium concentration of human sperm following exposure to voltages in the range of 200-2000 V cm-1 were measured. The induction of the acrosome reaction by the electropermeabilization pulse was also monitored. In comparison, changes in calcium uptake during a standard capacitation procedure in which sperm samples were incubated for 7 h were analyzed. At hourly intervals, acrosome reaction induction and intracellular calcium uptake were measured. The results indicate that electropermeabilization rapidly and efficiently produces high populations of acrosome-reacted sperm and a corresponding and associated increase in the intracellular calcium concentration.