Reduction of indicator and pathogenic microorganisms by psychrophilic anaerobic digestion in swine slurries

The objective of this study was to evaluate the efficiency of a low temperature anaerobic treatment to reduce viable populations of indicator microorganisms (total coliforms, Escherichia coli) and the presence of selected pathogens (Salmonella, Yersinia enterocolitica, Cryptosporidium and Giardia) in swine slurries from different sources. Experiments were carried out in 40 l Sequencing Batch Reactors (SBRs). Experimental results indicated that anaerobic digestion of swine manure slurry at 20 degrees C for 20 days in an intermittently fed SBR: (1) reduced indigenous populations of total coliforms by 97.94-100%; (2) reduced indigenous populations of E. coli by 99.67-100%; (3) resulted in undetectable levels of indigenous strains of Salmonella, Cryptosporidium, and Giardia. It can be considered as a promising method for reducing indigenous indicator and pathogenic microorganisms populations in liquid swine manure slurries.     

Comparison of treatment efficacy and stability of microbial populations between raw and anaerobically treated liquid pig manure, using PCR-DGGE and 16S sequencing

The effects of adding an adapted inoculum to liquid pig manure (LPM) prior to anaerobic digestion were evaluated by standard analytical methods. In parallel, the phylogenetic diversity of the microbial community of raw and anaerobically digested pig manure was studied by both denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S rRNA fragments amplified by polymerase chain reaction. Gas production, volative fatty acid production, removal of soluble chemical oxygen demand, and removal of volatile soluble solids were measured on raw and on inoculated liquid pig manure subjected to anaerobic digestion. DGGE profiles of 16S rRNA genes were used to compare the major elements of the bacterial community composition in raw LPM with those present under various incubation conditions. Major bands were excised and sequenced to gain insight into the identities of the bacterial populations from LPM treated under different conditions. The results show that the addition of an adapted inoculum did not have a major impact on the conversion of pig manure into soluble organic matter and did not significantly change the microbial populations present during anaerobic digestion of LPM. Bacterial composition also indicated that Clostridium species are important constituents of the LPM community.     

Anaerobic digestion of pig manure mixed with sewage sludge

This experiment explored the possibility of treating pig manure plus sewage sludge by anaerobic digestion, using batch fermentation at 37°C.The results indicated that the digested materials contained a lower level of putrescible matter when compared to the raw materials. Co-digestion of pig manure and sewage sludge at the ratio of 2:1 seemed to give the best results in terms of reducing the organic load and yielding a higher volume of methane.The experiment should be repeated in digestion tanks of a sewage treatment plant, especially for treating pig manure which has been recognized as the major source of water pollution in Hong Kong.     

Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion.

The extent of reduction in selected microorganisms was tested during both aerobic wastewater treatment and anaerobic digestion of sludge at the wastewater treatment plant in Ottawa to compare the removal of two encysted pathogenic protozoa with that of microbial indicators. Samples collected included the raw wastewater, the primary effluent, the treated wastewater, the mixed sludge, the decanted liquor, and the cake. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested. During aerobic wastewater treatment (excluding the anaerobic sludge digestion), Cryptosporidium and Giardia were reduced by 2.96 log10 and 1.40 log10, respectively. Clostridium perfringens spores, Clostridium perfringens total counts, somatic coliphages, and heterotrophic bacteria were reduced by approximately 0.89 log10, 0.96 log10, 1.58 log10, and 2.02 log10, respectively. All of the other microorganisms were reduced by at least 3.53 log10. Sludge samples from the plant were found to contain variable densities of microorganisms. Variability in microbial concentrations was sometimes great between samples, stressing the importance of collecting a large number of samples over a long period of time. In all cases, the bacterial concentrations in the cake (dewatered biosolids) samples were high even if reductions in numbers were observed with some bacteria. During anaerobic sludge digestion, no statistically significant reduction was observed for Clostridium perfringens, Enterococcus sp., Cryptosporidium oocysts, and Giardia cysts. A 1-2 log10 reduction was observed with fecal coliforms and heterotrophic bacteria. However, the method utilized to detect the protozoan parasites does not differentiate between viable and nonviable organisms. On the other hand, total coliforms and somatic coliphages were reduced by 0.35 log10 and 0.09 log10, respectively. These results demonstrate the relative persistence of the protozoa in sewage sludge during wastewater treatment.     

The survival of Escherichia coli, faecal coliforms and enterobacteriaceae in general in soil treated with sludge from wastewater treatment plants

We monitored the effect of the application of treated sludge on the behaviour of enterobacteriaceae (mainly faecal coliforms and especially Escherichia coli) in the soil, and studied their evolution over time after application. Three different sludges were used: two from a municipal sewage plant, one of them had been subjected to anaerobic digestion and heat drying, and the other to anaerobic digestion and mechanical dehydration, and one from a dairy waste treatment to aerobic digestion and gravity thickening. Two types of tests were carried out: type O, in the open air, with no possibility of controlling humidity or temperature; and type L, under laboratory conditions, with controlled temperature and humidity. Sludge tests were also run on unscreened soil previously treated with chemical fertilizer. After 80 days of experimentation the populations of faecal coliforms and E. coli had decreased considerably or were undetectable in assays carried out on the soil/sludge mixtures, under both open-air and laboratory conditions, but that, over the same period, in the mixtures containing chemical fertilizer (calcium ammonium nitrate) there had been a considerable increase in the micro-organism populations studied.     

Bacterial community analysis of swine manure treated with autothermal thermophilic aerobic digestion

Due to the enviornmental problems associated with disposal of livestock sludge, many stabilization studies emphasizing on the sludge volume reduction were performed. However, little is known about the microbial risk present in sludge and its stabilized products. This study microbiologically explored the effects of anaerobic lagoon fermentation (ALF) and autothermal thermophilic aerobic digestion (ATAD) on pathogen-related risk of raw swine manure by using culture-independent 16S rDNA cloning and sequencing methods. In raw swine manure, clones closely related to pathogens such as Dialister pneumosintes, Erysipelothrix rhusiopathiae, Succinivibrioan dextrinosolvens, and Schineria sp. were detected. Meanwhile, in the mesophilic ALF-treated swine manure, bacterial community clones closely related to pathogens such as Schineria sp. and Succinivibrio dextrinosolvens were still detected. Interestingly, the ATAD treatment resulted in no detection of clones closely related to pathogens in the stabilized thermophilic bacterial community, with the predominance of novel Clostridia class populations. These findings support the superiority of ATAD in selectively reducing potential human and animal pathogens compared to ALF, which is a typical manure stabilization method used in livestock farms.     

Destruction by Anaerobic Mesophilic and Thermophilic Digestion of Viruses and Indicator Bacteria Indigenous to Domestic Sludges

In raw sludges and in mesophilically and thermophilically digested anaerobic sludges, large variations in numbers of viruses occurred over narrow ranges of numbers of fecal coliforms, total coliforms, and fecal streptococci, demonstrating that the bacteria were poor quantitative reflectors of the numbers of the viruses detected. Mesophilic and thermophilic digestion of anaerobic sludges destroyed all three indicator bacteria more rapidly than such digestion destroyed the viruses. The relative rates for the destruction of viruses, fecal coliforms, and fecal streptococci in the digested sludges were consistent over the 17-month study. Fecal coliforms were 7 to 8 times more sensitive than the viruses to mesophilic digestion and 9 to 10 times more sensitive to thermophilic digestion. Total coliforms were even more sensitive. The rates at which fecal streptococci were destroyed by mesophilic and thermophilic digestion of anaerobic sludges approached those at which the viruses were destroyed by those processes; this suggested that the rates at which fecal streptococci in sludges are destroyed by those processes may serve as useful indicators for the rates at which viruses in sludges are destroyed by those processes.     

Inactivation of indicators and pathogens in cattle feedlot manures and compost as determined by molecular and culture assays

Accurate and conservative information about pathogen inactivation rates is needed as the basis for safe manure management on beef cattle feedlots. The survival of indicators and pathogens in faecal pen manure, stockpiled manure and manure compost was measured with autochthonous indicator bacteria (Escherichia coli, Clostridium perfringens, enterococci, total coliforms) and pathogens (Listeria monocytogenes, Campylobacter jejuni) using culture and/or real-time quantitative PCR (qPCR) methods. Additionally, the manures were incubated at 20, 37, 50 and 60 °C in microcosms to quantify the persistence of autochthonous microorganisms and selected process performance surrogates (Clostridium sporogenes, green fluorescent protein-labelled E. coli and L. monocytogenes). Estimated qPCR cell counts indicated that up to four orders of magnitude more target cells were present compared with the culturable counts. Corresponding T(90) estimates were up to sixfold higher. This study demonstrates the benefits of nucleic acid-based quantification of pathogen inactivation in cattle manures and concludes that the concurrent analysis of microorganisms by molecular and culture methods provides complementary value.     

Effect of anaerobic digestion temperature on odour, coliforms and chlortetracycline in swine manure or monensin in cattle manure

Aims: This study evaluated the effect of anaerobic digestion at 22, 38 and 55°C on odour, coliforms and chlortetracycline (CTC) in swine manure or monensin (MON) in cattle manure. Methods and Results: Swine or cattle were fed the respective growth promotant, manure was collected, and 2‐l laboratory methane digesters were established at the various temperatures and sampled over 25 or 28 days. After 21 days, the concentration of CTC in the 22, 38 and 55°C swine digester slurries decreased 7, 80 and 98%, respectively. Coliforms in the 22°C digester slurries were still viable after 25 days; however, they were not detectable in the 38 and 55°C slurries after 3 and 1 days, respectively. After 28 days, the concentration of MON in the 22, 38 and 55°C cattle digester slurries decreased 3, 8 and 27%, respectively. Coliforms in the 22°C cattle digester slurries were still viable after 28 days; however, they were not detectable in the 38 and 55°C slurries after 14 and 1 days, respectively. Conclusions: These studies indicate that anaerobic digestion at 38 or 55°C may be an effective treatment to reduce coliforms and CTC; however, it is not an effective treatment to reduce MON. Significance and Impact of the Study: More studies are needed to determine which pharmaceuticals are susceptible to degradation by a specific manure treatment to prevent negative environmental consequences.     

Occurrence and numbers of bacteriophages and bacterial indicators in faeces of yellow-legged seagull (Larus cachinnans)

Faeces from feral populations of yellow-legged seagulls from the northern coastal area of Catalonia (North-eastern Spain) contained variable amounts of faecal coliforms, faecal streptococci, somatic coliphages, F-specific bacteriophages and Bacteroides fragilis bacteriophages. Occurrence and numbers of bacterial indicators and bacteriophages in the faeces of yellow-legged seagulls are in the ranges described in the faeces of different animals. The ratios between numbers of bacterial indicators and numbers of bacteriophages are much higher in faeces of seagulls than in treated or raw sewage contributed by out-falls of the same area.     

Pathogen removal in farm-scale psychrophilic anaerobic digesters processing swine manure

This study assessed the efficiency of commercial-scale psychrophilic anaerobic digestion in sequencing batch reactors (PADSBRs) for pathogen removal from pig manure. The impact of treatment cycle length and of hydraulic flow regimes on pathogen removal efficiency was investigated. Two conventionally operated SBRs (BR1 and BR2) and two SBRs simultaneously fed during the draw step (BR3 and BR4) were monitored over a two-year period. PADSBRs significantly decreased the concentration of coliforms, Salmonella, Campylobacter spp., and Y. enterocolitica, respectively from about 10⁶, 10³ CFU g⁻¹, 10³, and 10⁴ CFU g⁻¹ to undetectable levels in most samples. Densities of the gram-positive Clostridium perfringens and Enterococcus spp. remained high (10⁵ CFU g⁻¹) in the digesters throughout treatment. The PADSBRs maintained the same level of pathogen removal when the treatment cycle length was reduced from 2 to 1 week. Mass balances on volatile fatty acids (VFAs) revealed short-circuits of inlet flow respectively averaging 6.3% and 6.4% for BR3 and BR4, significantly reducing the overall performance of these reactors regarding pathogens removal. The results obtained in this study show the ability of low temperature anaerobic digestion to remove or significantly reduce indicator and pathogen concentration from raw pig manure.     

Microbiological contamination of digested products from anaerobic co-digestion of bovine manure and agricultural by-products

Aims: This study was performed to investigate the microbiological contamination of digestate product (DP) obtained from the anaerobic co‐digestion of bovine manure and agricultural by‐products. Methods and results: Microbiological analyses were performed on bovine manure, fresh DP, liquid and solid fractions and stored liquid fraction of DP. A statistically significant reduction of faecal bacterial indicator was found after anaerobic digestion except for enterococci. After liquid/solid DP separation, bacteria tend to be concentrated in the solid fraction. Storage does not seem to influence the indicator parameters, except for enterococci. Escherichia coli O157:H7 and Yersinia were not found in any samples analysed. Salmonella was rarely detected in DP samples and its derivates, while Listeria monocytogenes was encountered in many samples. Conclusions: The results obtained indicate that the hygienic quality of DP is for almost all microbiological parameters better than that of the bovine manure (range of reduction 1.6–3.1 log₁₀) and suggest the need to identify specific pathogen indicators related to the hygienic characteristics of DPs. Significance and Impact of the Study: This study highlights that the anaerobic co‐digestion of bovine manure and agricultural by‐products in a field‐scale biogas plant does not increase human health risk with respect to the use of animal manure for agricultural fertilization.     

Survey of wastewater indicators and human pathogen genomes in biosolids produced by class a and class B stabilization treatments

Accurate modeling of the infectious aerosol risk associated with the land application of biosolids requires an in-depth knowledge of the magnitudes and changes in pathogen concentrations for a variety of class A and class B stabilization methods. The following survey used quantitative PCR (qPCR) and culture assays to detect environmentally resistant bacterial and viral pathogens and biosolid indicator organisms for 36 biosolid grab samples. Biosolids were collected from 14 U.S. states and included 16 class B mesophilic anaerobic digestion (MAD) samples and 20 class A biosolid samples from temperature-phased anaerobic digestion (TPAD), MAD plus composting (COM), and MAD plus heat pelletization processes. The indicator concentrations of fecal coliforms and male-specific coliphages as well as pathogen genome concentrations for human adenovirus species, Legionella pneumophila, Staphylococcus aureus, and Clostridium difficile were significantly lower in the class A samples, and a multivariate analysis of variance ranked the stabilization processes from the lowest pathogen/indicator load to the highest as (i) class A COM, (ii) class A TPAD, and (iii) class B MAD. Human adenovirus genomes were found in 88% of the class B samples and 70 to 100% of the class A samples. L. pneumophila, S. aureus, and C. difficile genomes were detected at the qPCR assay detection limits in 19 to 50% of the class B and class A anaerobic digestion samples, while L. pneumophila was detected in 50% of the class A compost samples. When considering all the stabilization methods, both the fecal coliform and the male-specific coliphage concentrations show a significant linear correlation with the pathogen genome concentrations. This survey provides the necessary pathogen concentrations to add to biosolid aerosol risk and pathogen exposure analyses and clarifies the effectiveness of class A stabilization methods with the pathogen and indicator loads in biosolids.     

Fate of Escherichia coli O157:H7 in manure-amended soil

Escherichia coli O157:H7 cells survived for up to 77, >226, and 231 days in manure-amended autoclaved soil held at 5, 15, and 21 degrees C, respectively. Pathogen populations declined more rapidly in manure-amended unautoclaved soil under the same conditions, likely due to antagonistic interactions with indigenous soil microorganisms. E. coli O157:H7 cells were inactivated more rapidly in both autoclaved and unautoclaved soils amended with manure at a ratio of 1 part manure to 10 parts soil at 15 and 21 degrees C than in soil samples containing dilute amounts of manure. The manure-to-soil ratio, soil temperature, and indigenous microorganisms of the soil appear to be contributory factors to the pathogen's survival in manure-amended soil.     

Evolution of the pathogen content during co-composting of winery and distillery wastes

The aim of this study was to monitor some microbial indicators and pathogen contents (sulphite reducers clostridia, total enterobacteriaceae, total coliforms, faecal coliforms (Escherichia coli), enterococci, Staphylococcus aureus and Salmonella spp.) throughout the co-composting of wastes from the winery and distillery industry with other organic residues, as well as the effect of the composting system used. Seven different piles using mixtures of winery-distillery wastes with other organic materials were prepared. P1 and P2 were made using grape stalk (GS), grape marc (GM), exhausted grape marc (EGM) and sewage sludge (SS), whereas in P3 and P4 were also used exhausted grape marc with cow manure (CW) and poultry manure (PM), respectively, using the Rutgers system. Additionally, P2 was watered with vinasse (V). The rest of piles (P5, P6 and P7) were prepared with grape marc, exhausted grape marc, cow manure and poultry manure, using the turning system. The effectiveness of the composting process to reduce the pathogen content was higher in the static aerated piles than in those elaborated with the turning. The relatively high temperatures (50-60 degrees C) reached in some of the piles produced a notable decrease in some microbial groups, such as total and faecal coliforms (E. coli), but the characteristics of the raw materials used notably influenced the pathogen contents of the end-product.     

Elevated methane emissions from a paddy field in southeast China occur after applying anaerobic digestion slurry

One hundred million tons of farm stalk waste and livestock and poultry excrement are used every year in China for the production of clean energy (biogas) by anaerobic digestion. Consequently, a large amount of fermented liquid is produced, and if disposed of improperly, it will result in secondary pollution. Agricultural application of this anaerobic slurry as a liquid fertilizer would reduce possible eutrophication of water sources from random slurry discharge and supply a superior organic fertilizer for farming. This study investigated the effect of applying anaerobic digestion slurry as a liquid fertilizer on the methane emitted from a paddy field. A two‐year (2008–2009) field experiment replacing chemical fertilizer with liquid fertilizer from anaerobically digested pig manure slurry was conducted in a paddy field in Yixing, Jiangsu, China. A static closed chamber method was used to measure methane fluxes over the period from June 2008 to October 2009. All fertilizer treatments increased methane emissions relative to untreated controls, with increases in methane ranging from 40–70% in 2008 to 48–84% in 2009. Paddy fields treated with anaerobically digested pig manure slurry had greater methane emissions (8–84% in 2008 and 3–26% in 2009) than those treated with chemical fertilizer. This suggests that the anaerobic digestion slurry would increase methane emission and so is unsuitable as a liquid fertilizer in paddy fields without development of cultivation practices to limit these emissions.     

The composition and persistence of faecal coliforms and enterococcal populations in sewage treatment plants

AIMS: The changes in structure and composition of faecal coliforms and enterococcal populations in sewage from different treatment plants, and the elimination of vancomycin- and erythromycin-resistant enterococci (VRE and ERE, respectively) in these treatment plants was analysed to determine any selective reduction. METHODS AND RESULTS: Faecal coliforms, enterococci, VRE, ERE and spores of sulphite-reducing bacteria were enumerated using standard methods. Samples were enriched where necessary in order to isolate antibiotic resistant strains. The structure and composition of these bacterial populations were determined by biochemical fingerprinting and clustering analysis. High diversity and similarity indexes were detected among all the bacterial populations in raw and treated sewage, independently of their origin and the treatment processes employed. Antibiotic resistant strains were detected in all sewage tested and no selective reduction was observed. CONCLUSIONS: The faecal coliforms and enterococci populations did not differ in the sewage samples studied. The vancomycin and erythromycin resistances of the enterococcal populations were similar in the sewage samples. Resistance to both antibiotics persisted after the treatment process independently of raw sewage flow, faecal origin or size of the human population contributing to sewage. However, sewage of mixed origin (human and animal) presented a lower similarity index for the two bacterial populations compared with that of the other human sewage analysed. SIGNIFICANCE AND IMPACT OF THE STUDY: Although a significant reduction in bacterial populations was observed, the persistence of VRE and ERE strains in the same proportions in sewage suggests that there is no selective elimination of bacterial populations during the treatment processes. The ability of antibiotic resistance strains to survive sewage treatment systems should be considered in certain water reuse programmes.     

Sunlight inactivation of fecal bacteriophages and bacteria in sewage-polluted seawater.

Sunlight inactivation rates of somatic coliphages, F-specific RNA bacteriophages (F-RNA phages), and fecal coliforms were compared in seven summer and three winter survival experiments. Experiments were conducted outdoors, using 300-liter 2% (vol/vol) sewage-seawater mixtures held in open-top chambers. Dark inactivation rates (k(D)s), measured from exponential survival curves in enclosed (control) chambers, were higher in summer (temperature range: 14 to 20 degrees C) than in winter (temperature range: 8 to 10 degrees C). Winter k(D)s were highest for fecal coliforms and lowest for F-RNA phages but were the same or similar for all three indicators in summer. Sunlight inactivation rates (k(S)), as a function of cumulative global solar radiation (insolation), were all higher than the k(D)s with a consistent k(S) ranking (from greatest to least) as follows: fecal coliforms, F-RNA phages, and somatic coliphages. Phage inactivation was exponential, but bacterial curves typically exhibited a shoulder. Phages from raw sewage exhibited k(S)s similar to those from waste stabilization pond effluent, but raw sewage fecal coliforms were inactivated faster than pond effluent fecal coliforms. In an experiment which included F-DNA phages and Bacteroides fragilis phages, the k(S) ranking (from greatest to least) was as follows: fecal coliforms, F-RNA phages, B. fragilis phages, F-DNA phages, and somatic coliphages. In a 2-day experiment which included enterococci, the initial concentration ranking (from greatest to least: fecal coliforms, enterococci, F-RNA phages, and somatic coliphages) was reversed during sunlight exposure, with only the phages remaining detectable by the end of day 2. Inactivation rates under different optical filters decreased with the increase in spectral cutoff wavelength (50% light transmission) and indicated that F-RNA phages and fecal coliforms are more susceptible than somatic coliphages to longer solar wavelengths, which predominate in seawater. The consistently superior survival of somatic coliphages in our experiments suggests that they warrant further consideration as fecal, and possibly viral, indicators in marine waters.     

Effect of oxygen on survival of faecal pollution indicators in drinking water

AIMS: The aim of this study was to determine the effect of oxygen on the survival of faecal pollution indicators including Escherichia coli in nondisinfected drinking water. METHODS AND RESULTS: Aerobic and anaerobic drinking water microcosms were inoculated with E. coli ATCC 25922 or raw sewage. Survival of E. coli was monitored by membrane filtration combined with cultivation on standard media, and by in situ hybridization with 16S rRNA-targeted fluorescent oligonucleotide probes. Anaerobic conditions significantly increased the survival of E. coli in drinking water compared with aerobic conditions. Escherichia coli ATCC 25922 showed a biphasic decrease in survival under aerobic conditions with an initial first-order decay rate of -0.11 day(-1) followed by a more rapid rate of -0.35 day(-1). In contrast, the first-order decay rate under anaerobic conditions was only -0.02 day(-1). After 35 days, <0.01% of the initial E. coli ATCC 25922 population remained detectable in aerobic microcosms compared with 48% in anaerobic microcosms. A poor survival was observed under aerobic conditions regardless of whether E. coli ATCC 25922 or sewage-derived E. coli was examined, and regardless of the detection method used (CFU or fluorescent in situ hybridization). Aerobic conditions in drinking water also appeared to decrease the survival of faecal enterococci, somatic coliphages and coliforms other than E. coli. CONCLUSIONS: The results indicate that oxygen is a major regulator of the survival of E. coli in nondisinfected drinking water. The results also suggest that faecal pollution indicators other than E. coli may persist longer in drinking water under anaerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of oxygen should be considered when evaluating the survival potential of enteric pathogens in oligotrophic environments.     

Bipolar effects of settling time on active biomass retention in anaerobic sequencing batch reactors digesting flushed dairy manure

Active biomass retention is a technical challenge in anaerobic digester treating dilute animal manure that contains solids particles. A strategy was tested using fibers in the dairy manure as biomass carriers by controlling settling time. Settling time ranging from 0.5 to 60 min were applied to eight anaerobic sequencing batch reactors to investigate their effects on active biomass retention in anaerobic digestion of flushed dairy manure. Results revealed that there existed a critical settling time at 2 min at which only minimum amount of active biomass was retained, and as settling time increased or decreased from this threshold, more active biomass could be retained. Gravity settling and selection pressure theories were suggested to account for the results. A model integrating these two effects was developed and verified with the experimental data. Knowledge derived from this study may lead to innovative bacterial retention technology for cost-effective anaerobic digestion of dairy wastes.