Bacterial and archaeal phylogenetic diversity associated with swine sludge from an anaerobic treatment lagoon

Over the last decades, the demand for pork products has increased significantly, along with concern about suitable waste management. Anaerobic-lagoon fermentation for swine-sludge stabilization is a good strategy, although little is known about the microbial communities in the lagoons. Here, we employed a cloning- and sequencing-based analysis of the 16S rRNA gene to characterize and quantify the prokaryotic community composition in a swine-waste-sludge anaerobic lagoon (SAL). DNA sequence analysis revealed that the SAL library harbored 15 bacterial phyla: Bacteroidetes, Cloroflexi, Proteobacteria, Firmicutes, Deinococcus-Thermus, Synergystetes, Gemmatimonadetes, Chlorobi, Fibrobacteres, Verrucomicrobia and candidates division OP5, OP8, WWE1, KSB1, WS6. The SAL library was generally dominated by carbohydrate-oxidizing bacteria. The archaeal sequences were related to the Crenarchaeota and Euryarchaeota phyla. Crenarchaeota predominated in the library, demonstrating that it is not restricted to high-temperature environments, being also responsible for ammonium oxidation in the anaerobic lagoon. Euryarchaeota sequences were associated with the hydrogenotrophic methanogens (Methanomicrobiales and Methanobacteriales). Quantitative PCR analysis revealed that the number of bacterial cells was at least three orders of magnitude higher than the number of archaeal cells in the SAL. The identified prokaryotic diversity was ecologically significant, particularly the archaeal community of hydrogenotrophic methanogens, which was responsible for methane production in the anaerobic lagoon. This study provided insight into the archaeal involvement in the overall oxidation of organic matter and the production of methane. Therefore, the treatment of swine waste in the sludge anaerobic lagoon could represent a potential inoculum for the start-up of municipal solid-waste digesters.     

Estimating the availability of nutrients from processed swine lagoon solids through incubation studies

Potential environmental hazards from the excess accumulation of swine (Sus scrofa domesticus) manure in eastern North Carolina and new state guidelines on treatment alternatives have necessitated the reevaluation of best management practices for disposal of swine waste (manure and effluent) as a fertilizer source on local crop land. Creation of a value-added product is one viable means of utilizing and economically redistributing the nutrients in swine manure. Incubation studies using four agricultural soils from eastern North Carolina were conducted with pelletized processed swine lagoon solids (PSLS) (1.7% N, 2.5% P, 0.12% Cu and 0.18% Zn) composed of dewatered swine lagoon sludge plus rock flour. The PSLS was added at three application rates (0, 200, 400 mg N kg⁻¹ soil) and incubated for 16 weeks at 25°C. The soil was sampled at 0, 1, 2, 4, 8, 12, and 16 weeks and analyzed for NO₃–N, NH₄–N and Mehlich III-extractable P, Zn, and Cu. High concentrations of NH₄–N and low concentrations of NO₃–N were present in the soils shortly after addition of PSLS. After the second week, extractable NH₄–N dropped to <4 mg kg⁻¹, while the soil concentration of NO₃–N increased rapidly. The amount of NO₃–N generated reached 90% of its final value after the fourth week. Across the four soils, 24–35% of the added N, 15–50% of the added P, 20–50% of the added Zn and 15–20% of the added Cu was extractable after 8 weeks. The PSLS is an excellent source of P, but may require additional N if used as a fertilizer source for most row crops.     

Nitrogen source effects on the denitrifying anaerobic methane oxidation culture and anaerobic ammonium oxidation bacteria enrichment process

The co-culture system of denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (Anammox) has a potential application in wastewater treatment plant. This study explored the effects of permutation and combination of nitrate, nitrite, and ammonium on the culture enrichment from freshwater sediments. The co-existence of NO₃ ⁻, NO₂ ⁻, and NH₄ ⁺ shortened the enrichment time from 75 to 30 days and achieved a total nitrogen removal rate of 106.5 mg/L/day on day 132. Even though ammonium addition led to Anammox bacteria increase and a higher nitrogen removal rate, DAMO bacteria still dominated in different reactors with the highest proportion of 64.7% and the maximum abundance was 3.07 ± 0.25 × 10⁸ copies/L (increased by five orders of magnitude) in the nitrite reactor. DAMO bacteria showed greater diversity in the nitrate reactor, and one was similar to M. oxyfera; DAMO bacteria in the nitrite reactor were relatively unified and similar to M. sinica. Interestingly, no DAMO archaea were found in the nitrate reactor. This study will improve the understanding of the impact of nitrogen source on DAMO and Anammox co-culture enrichment.

     

Spatial and temporal changes in the microbial community in an anaerobic swine waste treatment lagoon

Microorganisms are central to both the beneficial (organic degradation, nutrient removal, biogas production) and detrimental (odor production, pathogen contamination) effects of swine waste storage systems. In this study, both quantitative (real-time polymerase chain reaction) and qualitative (denaturing gradient gel electrophoresis, cloning, sequence analysis) molecular analyses were used to track spatial and temporal changes in the microbial community of swine slurry from a 0.4 ha anaerobic lagoon. The lagoon, located in a region of western Kentucky which has a humid, subtropical environment, was sampled on a monthly basis (n = 10) over a period of one year at four different depths (top, 51 cm from the top, 152 cm from the top, and bottom >198 cm). The concentration and diversity of Bacteroides sp. was seasonal (up to 90% decrease between March and June). Hespellia sp. and other clostridial species, on the other hand, were endemic in the slurry (concentrations up to 1.0 × 10⁷ cells mL^?1 slurry) regardless of time of the year or lagoon depth. Results suggest that there were seasonal effects on the microbial community in the swine lagoon, while the effect of depth was not as pronounced. Seasonal changes in the microbial community in stored wastes may be (directly or indirectly) correlated with changes in malodor emissions from lagoons.     

Denitrification of nitrified and non-nitrified swine lagoon wastewater in the suspended sludge layer of treatment wetlands

One method for managing livestock-wastewater N is the use of treatment wetlands. The objectives of this study were to (1) assess the magnitude of denitrification enzyme activity (DEA) in the suspended sludge layers of bulrush and cattail treatment wetlands, and (2) evaluate the impact of nitrogen pretreatment on DEA in the suspended sludge layer. The study used four wetland cells (3.6 m × 33.5 m) with two cells connected in series. Each wetland series received either untreated or partially nitrified swine wastewater from a single-cell anaerobic lagoon. The DEA of the suspended sludge layers of the constructed wetlands was measured by the acetylene inhibition method. The control DEA treatment for the sludge layer had a mean rate of 18 μg N₂O-N g^?1 sludge h^?1. Moreover, the potential DEA (nitrate-N and glucose-C added) mean was very large, 121 μg N₂O-N g^?1 sludge h^?1. These DEA rates are consistent with the previously reported high levels of nitrogen removal by denitrification from these wetlands, especially when the wastewater was partially nitrified. Stepwise regression using distance within the wetland, wastewater nitrate, and wastewater ammonia explained much of the variation in DEA rates. In both bulrush and cattail wetlands, there were zones of very high potential DEA.     

Struvite precipitation in anaerobic swine lagoon liquid: effect of pH and Mg:P ratio and determination of rate constant

Because of increased concern about surface water eutrophication from nutrient-enriched agricultural runoff, many swine producers are encouraged to decrease application rates of waste-based P. Precipitation and subsequent removal of magnesium ammonium phosphate (MgNH(4)PO(4) x 6H(2)O), commonly known as struvite, is a promising mechanism for N and P removal from anaerobic swine lagoon effluent. The objectives of this research were to (i) quantify the effects of adjusting pH and Mg:P ratio on struvite precipitation and (ii) determine the rate constant pH effect for struvite precipitation in anaerobic swine lagoon liquid. Concentrations of PO(4)-P in liquid from two anaerobic swine lagoons were determined after 24 h of equilibration for a pH range of 7.5-9.5 and Mg:P ratios between 1:1 and 1.6:1. Struvite formation reduced the PO(4)-P concentration in the effluents to as low as 2 mgl(-1). Minimum concentrations of PO(4)-P occurred between pH 8.9 and 9.25 at all Mg:P ratios. Struvite precipitation decreased PO(4)-P concentrations by 85% within 20 min at pH 9.0 for an initial Mg:P ratio of 1.2:1. The rate of PO(4)-P decrease was described by a first-order kinetic model, with rate constants of 3.7, 7.9, and 12.3 h(-1) at pH 8.4, 8.7 and 9.0 respectively. Our results indicate that induced struvite formation is a technically feasible method to remove N and P from swine lagoon liquid and it may allow swine producers to recover nutrients for off-farm sale.     

The effect of stratification and seasonal variability on the profile of an anaerobic swine waste treatment lagoon

In this study, the characterization of an anaerobic swine waste treatment lagoon from a farrowing operation (approximately 2000 sows) was carried out to examine the dynamics of the system due to stratification and seasonal variability. Swine waste samples were taken at different depths with a pulley system equipped with a special sampler that allows for sampling exclusively at certain depth. Chemicals and microbial dynamics were monitored throughout a one-year-period. Results showed that nutrient (C, N, P, S) concentrations varied according to stratified lagoon layers and season. Trace minerals (Al, Ca, Fe, and Mg), on the other hand, appeared to be affected more by stratification than seasonal variability. Molecular analysis also showed that microbial community structure appeared to be affected by the stratification and seasonal variability. Based on these data, it is important to consider the effect of stratification and seasonal variability in managing these open lagoons.     

Influence of a supplemental carbon source on anaerobic dechlorination of pentachlorophenol in granular sludge

[This corrects the article on p. 367 in vol. 58.].     

Molecular ecology of a facultative swine waste lagoon

Aims:  To investigate the microbial ecology of three facultative swine waste lagoons.
Methods and Results:  Phylogenetic analysis of sequences in a 16S rRNA gene clone library and fluorescence in situ hybridization (FISH) analyses were used to assess bacterial diversity in a swine waste lagoon. FISH analysis and Gram-staining were used to compare the microbial communities of all three swine waste lagoons. Six operational taxonomic units were in high relative abundance and corresponded to the following phylotypes; Thiolamprovum , Verrucomicrobia , Acholeplasma , Turicibacter , Clostridium and Bacteroides . PCR was employed to detect the genes apsA and dsrAB which encode for enzymes specifically associated with dissimilatory sulfate-reduction within sulfate-reducing bacteria (SRB). Amplification of these genes confirmed their presence within the lagoons.
Conclusions:  All lagoons were dominated by purple sulfur bacteria, affiliated to Thiolamprovum pedioforme . The molecular identification of fermentative bacteria and SRB indicate the following metabolic processes within such facultative ponds: sulfur-cycling, fermentation, inter-species hydrogen transfer and carbon cycling.
Significance and Impact of the Study:  This study provides the first molecular evidence for the existence of a sulfur cycle which is linked to phototrophic sulfide oxidation by purple bacteria and organotrophic sulfate-reduction by SRB.     

Ultrasonic sludge disintegration for enhanced methane production in anaerobic digestion: effects of sludge hydrolysis efficiency and hydraulic retention time

Hydrolysis of waste activated sludge (WAS) has been regarded as the rate limiting step of anaerobic sludge digestion. Therefore, in this study, the effect of ultrasound and hydraulic residence time during sludge hydrolysis was investigated with the goal of enhancing methane production from anaerobic digestion (AD). WAS was ultrasonically disintegrated for hydrolysis, and it was semi-continuously fed to an anaerobic digesters at various hydraulic retention times (HRTs). The results of these experiments showed that the solids and chemical oxygen demand (COD) removal efficiencies when using ultrasonically disintegrated sludge were higher during AD than the control sludge. The longer the HRT, the higher the removal efficiencies of solids and COD, while methane production increased with lower HRT. Sludge with 30% hydrolysis produced 7 × more methane production than the control sludge. The highest methane yields were 0.350 m(3)/kg volatile solids (VS)(add) and 0.301 m(3)/kg COD(con) for 16 and 30% hydrolyzed sludge, respectively. In addition, we found that excess ultrasound irradiation may inhibit AD since the 50% hydrolyzed sludge produced lower methane yields than 16 and 30% hydrolyzed sludge.     

Influence of a supplemental carbon source on anaerobic dechlorination of pentachlorophenol in granular sludge.

Anaerobic dechlorination of pentachlorophenol (PCP) was studied in two upflow anaerobic sludge blanket reactors. One reactor received glucose (0.9 g liter-1) as an additional carbon source; the other one served as a control. The concentration of PCP in the medium was 4.5 and 3.0 mg liter-1 in the experimental and control reactors, respectively. The reactors were inoculated with granular sludge previously grown on sugar-containing wastewater. After 10 months of continuous operation, the removal of PCP was 99% in the glucose-amended reactor, whereas the removal in the control reactor varied between 32 and 77%. Furthermore, 94% of the PCP was completely dechlorinated in the glucose reactor compared with a maximum of 20% in the control reactor. In the same period, the amount of biomass in the glucose reactor had increased by approximately 150% compared with that in the control reactor, where no growth of the sludge bed occurred. Batch culture activity tests showed that the addition of glucose had a stimulatory effect on the dechlorination rate of PCP per gram of volatile solids. This indicated that the better performance of the glucose-amended reactor was due to a higher concentration of biomass and a direct stimulatory effect of glucose on the dechlorination rate. The pattern of dechlorination of PCP showed that an initial para cleavage was followed by two ortho cleavages.     

Influence of a supplemental carbon source on anaerobic dechlorination of pentachlorophenol in granular sludge.

Anaerobic dechlorination of pentachlorophenol (PCP) was studied in two upflow anaerobic sludge blanket reactors. One reactor received glucose (0.9 g liter-1) as an additional carbon source; the other one served as a control. The concentration of PCP in the medium was 4.5 and 3.0 mg liter-1 in the experimental and control reactors, respectively. The reactors were inoculated with granular sludge previously grown on sugar-containing wastewater. After 10 months of continuous operation, the removal of PCP was 99% in the glucose-amended reactor, whereas the removal in the control reactor varied between 32 and 77%. Furthermore, 94% of the PCP was completely dechlorinated in the glucose reactor compared with a maximum of 20% in the control reactor. In the same period, the amount of biomass in the glucose reactor had increased by approximately 150% compared with that in the control reactor, where no growth of the sludge bed occurred. Batch culture activity tests showed that the addition of glucose had a stimulatory effect on the dechlorination rate of PCP per gram of volatile solids. This indicated that the better performance of the glucose-amended reactor was due to a higher concentration of biomass and a direct stimulatory effect of glucose on the dechlorination rate. The pattern of dechlorination of PCP showed that an initial para cleavage was followed by two ortho cleavages.     

Oxygen stress in Salvinia natans: Interactive effects of oxygen availability and nitrogen source

The ability of Salvinia natans (L.) All. to tolerate growth in oxic, hypoxic and anoxic nutrient solutions when supplied with either NH₄⁺ or NO₃^? were studied in the laboratory to test the hypothesis that inorganic N-source affects the response of the plants to O₂ deprivation. The relative growth rate (RGR) was significantly reduced in the anoxic treatment, but in the hypoxic treatment RGR was only slightly affected. The NH₄⁺ fed plants generally had a higher shoot to root ratio than the NO₃^? fed plants, and highest in the anoxic treatment. Plants had more roots and larger leaves when supplied with NH₄⁺ as compared with NO₃^?, particularly in the oxic treatment, and root length was most affected by O₂ deprivation for NO₃^? fed plants. Cell walls in the endodermis, the bundle sheath and the cortex adjacent to endodermis developed thickened sclerenchymatous walls when deprived of O₂, and more so in plants supplied with NO₃^?. Plants lost chlorophylls, had lower rates of photosynthetic electron transport (ETR_max) and lower quantum yields (Fv/Fm ratios) when grown in anoxic solutions, and the negative effects were mildest for NO₃^? fed plants suggesting that NO₃^? may be used as an alternative e^?-acceptor in non-cyclic electron transport in the chloroplasts. Overall S. natans grew best on NH₄⁺, but it also grew well on NO₃^?, and the O₂ stress symptoms differed somewhat between NH₄⁺ fed and NO₃^? fed plants. However, because N-form itself significantly influenced morphology and cell metabolism, it was impossible to conclusively identify the role of N-form for the O₂ stress reactions. S. natans is not well-adapted to grow in O₂ deficient waters and will not tolerate completely anoxic conditions as will prevail in waters receiving high loadings of organic pollutants such as livestock wastewater.     

Preservation characteristics of anaerobic granular sludge

Summary The preservation characteristics of granular sludges were investigated in terms of storing age and environmental condition. As a preservative index, physicochemical, microbiological, and morphological parameters were measured in order to quantify the change in granular sludge. Results from this research indicated that, as the anaerobic granular sludge had an outstanding preservation characteristic, UASB system could be effectively applied to the industries operating seasonally or intermittently.     

Characterization of skatole-producing microbial populations in enriched swine lagoon slurry

Skatole is one of the most malodorous compounds produced from the anaerobic degradation of animal waste. Little is known about the biochemistry of skatole production, the phylogeny of skatole-producing microorganisms or the conditions that favor their growth. These deficiencies hamper attempts to reduce skatole production. Our goals were to enrich for skatole producers in swine lagoon slurry (SLS) and evaluate the resulting microbial community structure using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene sequence analysis. Skatole producers were enriched by incubating dilutions of SLS with 100 muM indole-3-acetic acid (IAA). GC-MS was used to measure skatole production in the slurries after 0, 7 and 17 days' incubation. Based on most probable number analysis, skatole producers increased 100-fold in SLS samples supplemented with IAA. Based on DGGE fingerprint patterns from day 0, 7 and 17 treatments with high, mid or low levels of skatole production, changes in the SLS population occurred as skatole production increased. Changes in the bacterial community fingerprints were associated with an increase in the low-GC gram-positive and Bacteroides groups. Results from this study provides valuable new information concerning the organisms responsible for production of this odorant, a necessary first step towards controlling skatole production.     

The inhibitory effects and removal of dieldrin in continuous upflow anaerobic sludge blanket reactors

The inhibitory effects and removal efficiency of dieldrin (DLD) in anaerobic reactors were investigated. Anaerobic toxicity assay (ATA) experiments conducted in batch reactors revealed that 30 mg/l DLD had inhibitory effects on the unacclimated mixed anaerobic cultures. Continuous reactor experiments performed in a lab-scale two-stage upflow anaerobic sludge blanket (UASB) reactor system which was fed with ethanol as the sole carbon source, indicated that anaerobic granular cultures could be successfully acclimated to DLD. Chemical oxygen demand (COD) removal efficiencies were 88-92% for the two-stage system. The influent DLD concentration of 10 mg/l was removed by 44-86% and 86-94% in the second stage and overall UASB system, respectively. Biosorption of DLD on granular anaerobic biomass was found to be a significant mechanism for DLD removal in the UASB system. The maximum DLD loading rate and minimum HRT achievable for the first stage UASB reactor were 0.5 mg/lday (76 microg DLD/g VSS.day) and 10 h, respectively, which resulted in the overall COD removal efficiency of 85%.     

Physical characterization of a phytodewatered anaerobic sludge

Some physical parameters were determined on an organic product derived from a new phytodewatering process of anaerobic sludge, in order to verify its possible use as a substrate. Wetting-drying cycles were carried out with a sandy soil, and with mixtures of this soil with the organic product taken before and after the phytodewatering process. The phytodewatering process decreased bulk density while raising aggregate stability and friability.     

Chemical properties of biocrude oil from the hydrothermal liquefaction of Spirulina algae, swine manure, and digested anaerobic sludge

This study explores the influence of wastewater feedstock composition on hydrothermal liquefaction (HTL) biocrude oil properties and physico-chemical characteristics. Spirulina algae, swine manure, and digested sludge were converted under HTL conditions (300 °C, 10-12 MPa, and 30 min reaction time). Biocrude yields ranged from 9.4% (digested sludge) to 32.6% (Spirulina). Although similar higher heating values (32.0-34.7 MJ/kg) were estimated for all product oils, more detailed characterization revealed significant differences in biocrude chemistry. Feedstock composition influenced the individual compounds identified as well as the biocrude functional group chemistry. Molecular weights tracked with obdurate carbohydrate content and followed the order of Spirulina < swine manure < digested sludge. A similar trend was observed in boiling point distributions and the long branched aliphatic contents. These findings show the importance of HTL feedstock composition and highlight the need for better understanding of biocrude chemistries when considering bio-oil uses and upgrading requirements.     

Survival of enteroviruses and coliform bacteria in a sludge lagoon.

Enteroviruses associate with aerobically and anaerobically digested sludge were determined before the addition of the sludge to a sludge lagoon. The fate of sludge-associated viruses was followed during detention of sludge in the lagoon and after application of sludge to land for disposal. While digested sludge was being added to the lagoon, enteroviruses were readily detected in grab samples of sludge from the lagoon. Sludge-associated viruses dropped to low or undetectable levels after disposal of sludge on land and during periods when addition of digested sludge to the lagoon was suspended. Changes in the levels of fecal coliforms in the lagooned sludge paralleled changes in the numbers of enteroviruses. Enteroviruses were not detected in water from deep wells located on the sludge disposal site or near the lagoon. During the initial part of the study, poliovirus serotypes accounted for greater than 90% of the viruses identified. Later, poliovirus serotypes comprised less than 40% of the virus isolates, and echoviruses and Coxsackieviruses were the most common enteroviruses identified.