Anaerobic digestion of livestock and poultry manures spiked with tetracycline antibiotics.

We investigated the anaerobic degradation of tetracycline antibiotics (tetracycline [TC], oxytetracycline [OTC] and chlortetracycline [CTC]) in swine, cattle, and poultry manures. The manures were anaerobically digested inside polyvinyl chloride batch reactors for 64 days at room temperature. The degradation rate constants and half-lives of the parent tetracyclines were determined following first-order kinetics. For CTC the fastest degradation rate was observed in swine manure (k = 0.016 ± 0.001 d-1; half-life = 42.8 days), while the slowest degradation rate was observed in poultry litter (k = 0.0043 ± 0.001 d-1; half-life = 161 days). The half-lives of OTC ranged between 88.9 (cattle manure) and 99.0 days (poultry litter), while TC persisted the longest of the tetracycline antibiotics studied with half-lives ranging from 92.4 days (cattle manure) to 330 days (swine manure). In general, the tetracyclines were found to degrade faster in cattle manure, which had the lowest concentrations of organic matter and metals as compared to swine and poultry manures. Our results demonstrate that tetracycline antibiotics persist in the animal manure after anaerobic digestion, which can potentially lead to emergence and persistence of antibiotic resistant bacteria in the environment when anaerobic digestion byproducts are land applied for crop production.

Abundances of Tetracycline Resistance Genes and Tetracycline Antibiotics during Anaerobic Digestion of Swine Waste.

The impact of anaerobic digestion of animal waste on the persistence of antibiotic resistance genes (ARGs) and antibiotics is not widely studied. Two identical, 800-L digesters seeded with swine slurry were followed up to 100 d in three separate trials. The trials received varying amounts of antibiotic-free corn ( L.) mixed with water to maintain the digestion process. Biogas production, seven tetracycline resistance () genes, and three tetracyclines and their transformation products were measured. Biogas production proportionally increased as the feeding loads increased between trials. In Trial 1, log gene copies showed small but statistically significant ( < 0.01) increases during digestion. In Trial 2, anaerobic digestion did not have a significant ( > 0.05) effect except for significant reductions in B ( < 0.0001) and G ( = 0.0335) log gene copies. In Trial 3, which received the highest amount of corn mix, log copies of the 16S ribosomal RNA and the genes significantly ( < 0.0001) reduced over time during digestion. Up to 36 µg L tetracycline, 112 µg L chlortetracycline, 11.9 mg L isochlortetracycline, and 30 µg L 4-epitetracycline were detected both in the liquid and solid digestates. Results of this study revealed that although anaerobic digestion of swine waste can produce useful biogas, it does not result in complete removal of bacteria, ARGs, and antibiotics regardless of differences in the feeding loads between trials. Further effluent and sludge treatments are required prior to their downstream use in crop production to minimize emergence and environmental dissemination of antimicrobial-resistant bacteria through animal manure.

Microbiome Diversity of Anaerobic Digesters Is Enhanced by Microaeration and Low Frequency Sound.

Biogas is produced by a consortium of bacteria and archaea. We studied how the microbiome of poultry litter digestate was affected by time and treatments that enhanced biogas production. The microbiome was analyzed at six, 23, and 42 weeks of incubation. Starting at week seven, the digesters underwent four treatments: control, microaeration with 6 mL air L-1 digestate per day, treatment with a 1000 Hz sine wave, or treatment with the sound wave and microaeration. Both microaeration and sound enhanced biogas production relative to the control, while their combination was not as effective as microaeration alone. At week six, over 80% of the microbiome of the four digesters was composed of the three phyla Actinobacteria, Proteobacteria, and Firmicutes, with less than 10% Euryarchaeota and Bacteroidetes. At week 23, the digester microbiomes were more diverse with the phyla Spirochaetes, Synergistetes, and Verrucomicrobia increasing in proportion and the abundance of Actinobacteria decreasing. At week 42, Firmicutes, Bacteroidetes, Euryarchaeota, and Actinobacteria were the most dominant phyla, comprising 27.8%, 21.4%, 17.6%, and 12.3% of the microbiome. Other than the relative proportions of Firmicutes being increased and proportions of Bacteroidetes being decreased by the treatments, no systematic shifts in the microbiomes were observed due to treatment. Rather, microbial diversity was enhanced relative to the control. Given that both air and sound treatment increased biogas production, it is likely that they improved poultry litter breakdown to promote microbial growth.

Effect of continuous in-feed administration of tylosin to feedlot cattle on macrolide and tetracycline resistant enterococci in a randomized field trial.

Liver abscess causes substantial economic loss to the beef cattle industry through liver condemnation, reduced animal performance, and carcass yield. Continuous in-feed use of tylosin is the most effective and a commonly used practice in beef cattle production to prevent liver abscess. However, such mass medication can increase the level of antimicrobial resistant bacteria. We investigated the effect of continuous in-feed use of tylosin in feedlot cattle on (i) concentrations and prevalence of erythromycin-resistant (ERYr) and tetracycline-resistant (TETr) enterococci; (ii) associated antimicrobial resistance genes (ARGs) for resistance; (iii) species distribution; iv) macrolide and tetracycline resistance gene concentrations; and (v) tylosin concentration. A cohort of weaned calves were randomized to receive tylosin-medicated feed (Tylosin; n = 10) or nonmedicated feed (Control; n = 10) for a full feedlot cycle. Feces, feed and pen-surface samples were collected and processed by culture, droplet digital PCR, and liquid chromatography/mass spectroscopy for bacterial enumeration, detection and characterization, ARG quantification, and tylosin concentration, respectively. Data were analyzed by mixed effects linear- or binary-regression models depending on the outcomes. Tylosin administration significantly increased fecal concentration (P < 0.001) and prevalence (P = 0.021) of ERYr enterococci and erm(B) gene concentration (P < 0.001), compared to the control group. Interestingly, tylosin administration significantly reduced (P = 0.037) fecal TETr enterococci concentration compared to the control group, with no significant effect (P = 0.758) on fecal tet(M) concentration. In both treatment groups, enterococci concentrations increased over time, peaking on 174 days in feed before returning to the baseline. ERYr enterococci concentration was significantly (P = 0.012) higher in tylosin medicated feeds, with no significant effect (P = 0.321) on TETr enterococci concentration. Pen-surface concentration of ermB was significantly (P = 0.024) higher in the tylosin group, with no significant effect (P > 0.05) on bacterial concentrations. Increased diversity and a shift in the composition of enterococcal species and ARGs were observed over time, although tylosin use did not significantly affect (P > 0.05) their prevalence. Tylosin concentration was significantly higher in the feces of tylosin administered cattle (P < 0.001) and medicated feed (P = 0.027), with numerically higher pen-surface concentration (P = 0.065) in the tylosin group. In conclusion, continuous in-feed use of tylosin in feedlot cattle increases macrolide resistant enterococci and its fecal excretion, while decreasing tetracycline resistance. Two medically important species, E. faecium and E. faecalis, were predominant regardless of resistance status or sample source. Risk-based approaches including label changes to limit tylosin use such as withdrawal period, and development of effective manure treatments are potential areas of research to reduce environmental and public health impacts.

Lagoon, Anaerobic Digestion, and Composting of Animal Manure Treatments Impact on Tetracycline Resistance Genes.

Increased demand for animal protein is met by increased food animal production resulting in large quantities of manure. Animal producers, therefore, need sustainable agricultural practices to protect environmental health. Large quantities of antimicrobials are used in commercial food animal production. Consequently, antimicrobial-resistant bacteria and the resistance genes emerge and are excreted through feces. Manure management is essential for the safe disposal of animal waste. Lagoons, with or without covers, and anaerobic digesters, with the primary purpose of methane production, and composting, with the primary purpose of producing organic fertilizer, are widely used methods of manure treatment. We reviewed manure management practices and their impact on tetracycline resistance genes. Lagoons are maintained at ambient temperatures; especially uncovered lagoons are the least effective in removing tetracycline resistance genes. However, some modifications can improve the performance of lagoons: sequential use of uncovered lagoons and the use of covered lagoons resulted in a one-log reduction, while post-treatments such as biofiltration following covered lagoon treatment resulted in 3.4 log reduction. Mesophilic digestion of animal manure did not have any significant effect; only a 0.7 log reduction in tet(A) was observed in one study. While thermophilic anaerobic digesters are effective, if properly operated, they are expensive for animal producers. Aerobic thermophilic composting is a promising technology if optimized with its economic benefits. Composting of raw animal manure can result in up to a 2.5 log reduction, and postdigestion composting can reduce tetracycline resistance gene concentration by >80%. In general, manure management was not designed to mitigate antimicrobial resistance; future research is needed to optimize the economic benefits of biogas or organic fertilizer on the one hand and for the mitigation of foodborne pathogens and antimicrobial resistance on the other.

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 1.0x10(7) 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.

Anaerobic Digestion of Tetracycline Spiked Livestock Manure and Poultry Litter Increased the Abundances of Antibiotic and Heavy Metal Resistance Genes.

Anaerobic digestion is used for the treatment of animal manure by generating biogas. Heavy metals cause environmental pollutions and co-select for antimicrobial resistance. We evaluated the impact of mesophilic anaerobic digestion of cattle manure (CM), swine manure (SM) and poultry litter (PL) on the concentrations of seven tetracycline [tet(A), tet(B), tet(G), tet(M), tet(O), tet(Q), and tet(W)], macrolide [erm(B)], methicillin (mecA and mecC), copper (copB, pcoA, pcoD, and tcrB) and zinc (czrC) resistance genes, and three bacterial species (E. coli, Enterococcus spp. and Staphylococcus aureus). The total bacterial population and total abundance of the seven tet genes significantly increased in the three manure types after digestion. Concentration of tet(M) was strongly correlated with that of erm(B) and enterococci. As concentration of tetracyclines declined during anaerobic digestion, that of four tet genes (A, B, Q, and W) and 16S rRNA increased, that of tet(M) decreased, and that of tet(G) and tet(O) did not change. Concentrations of copB and pcoA did not change; while that of pcoD did not change in the PL, it increased in the SM and CM. While the concentration of enterococci remained unchanged in CM, it significantly increased in the PL and SM. Concentrations of tcrB significantly increased in the three manure types. While concentrations of S. aureus significantly increased in the CM and PL, that of SM was not affected. Concentrations of mecC significantly increased in all manure types after digestion; while mecA concentrations did not change in the SM, they significantly increased in CM and PL. While concentration of czrC remained low in the CM, it increased in the PL but declined in the SM. In conclusion, while mesophilic anaerobic digestion of animal manure decreased concentration of tetracyclines, it increased the concentrations of total bacteria, tet genes, E. coli, enterococci and S. aureus and methicillin resistance genes. It did not have any effect on concentrations of heavy metals; concentrations of heavy metal resistance genes either increased or remained unaffected depending on the animal species. This study showed the need for post-digestion treatments of animal manure to remove bacteria, antibiotic resistance genes, heavy metals and their resistance genes.

Evaluation of second-generation multistage wastewater treatment system for the removal of malodors from liquid swine waste.

Wastewater quality and malodors in a second generation implementation of environmentally superior technology (EST) were monitored over three cycles of pig (Sus scrofa) production and 15 mo. The wastewater treatment system consisted of three modules: solids separation, biological N removal, and P recovery/wastewater disinfection. While approximately more than 90% of the wastewater suspended solids were removed in the first stage of treatment, little reduction in malodorous compounds occurred, indicating that malodors largely remained with the liquid waste stream. The greatest improvements in wastewater quality occurred in the N treatment module where ammonium was removed through nitrification/denitrification processes: there was more than 99% reduction in aromatic malodorous compounds (e.g., p-cresol, skatole) and almost 90% reduction in volatile fatty acids (e.g., propanoate and butanoate) in N module effluent as compared to raw flushed manure. The system performed consistently well in wastewater odor removal, even during the first cycle of livestock production when system performance was being optimized. These findings showed that the combination of the processes of solids removal and biological N treatment into a practical treatment system can be very effective in reducing malodors from livestock wastewater.

Equilibrium sampling used to monitor malodors in a Swine waste lagoon.

The concentrations of malodorous compounds in a 0.4-ha anaerobic lagoon that received waste from approximately 2000 sows were monitored during the late summer to late fall of 2006 to gain insight into the factors influencing their concentrations. Selected malodorous compounds were measured by the use of equilibrium samplers consisting of submersible stir plates and stir bar sorbtive sampling with polydimethylsiloxane-coated magnetic stir bars. During the same period, air and water temperatures, suspended solids, total organic carbon and nitrogen content, and wastewater pH were recorded. Concentrations of malodorous compounds were higher at the surface of the lagoon than at the middle or bottom of the lagoon. Skatole concentration, for instance, averaged 54, 24, and 38 microg L(-1) near the surface, in the middle, and at the lowest sampling depths, respectively. While the lagoon was being pumped down during field application of wastewater, concentrations of malodorous compounds fluctuated widely, increased 16-fold as compared with the sampling period before pumping, and continued to increase as fall progressed and temperatures cooled. Suspended solids, volatile suspended solids, and total organic carbon increased near the bottom of the lagoon during this same period. The increases in the concentrations of malodorous compounds in the wastewater during the fall could have been due to a combination of several factors. These factors include reduced degradation by lagoon bacteria, less wind stripping of volatile compounds from the lagoon surface due to lowering of the lagoon surface after crop application, and/or reduced evaporation of malodorous compounds due to falling temperatures.

Improved water quality and reduction of odorous compounds in anaerobic lagoon columns receiving pre-treated pig wastewater.

Large volumes of wastewater from confined pig production are stored in anaerobic lagoons. Control methods are needed to reduce air pollution by foul odors released from these lagoons. In a pilot-scale experiment, we evaluated the effect of pig wastewater pre-treatment on reducing the concentration of selected malodor compounds in lagoons receiving liquid from: (1) flocculant enhanced solid-liquid separation (SS), and (2) solid-liquid separation plus biological N treatment using nitrification-denitrification (SS+NDN). A conventional anaerobic lagoon was included as a control. Concentrations of five selected malodorous compounds (phenol, p-cresol, 4-ethylphenol, indole, and skatole) and water quality parameters (ammonia-nitrogen and chemical oxygen demand) were determined in lagoon effluents. The SS+NDN pretreatment was more efficient than the SS in reducing odorous compounds in the lagoon liquid. The SS+NDN reduced by about 99% the liquid concentrations of all selected compounds. An odor panel test revealed that SS was ineffective to reduce the human sense of malodor with respect to the control. Whereas the SS+NDN had the significant lowest odor intensity and unpleasantness. These results are supported by the strong correlations found between the sum of odorous compound concentration with odor panel results and concentrations of both ammonium-nitrogen and chemical oxygen demand in lagoon liquid samples.

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.

Comparison of solid-phase microextraction and stir bar sorptive extraction for the quantification of malodors in wastewater.

Malodors in wastewater from animal-rearing facilities are due to the presence of characteristic polar compounds. The efficiency and reproducibility of three solid-phase microextraction (SPME) fibers (Carboxen-PDMS, polyacrylate, and PDMS) as well as PDMS-coated stir bars for the measurement of some of these compounds in the liquid phase were compared. In initial experiments, the SPME fibers and stir bars were exposed to a standard water solution containing a mixture of 18 compounds with a range of octanol-water partition coefficients. The polyacrylate SPME fibers and PDMS-coated stir bars, having been found to possess the best combination of extraction efficiency and reproducibility of measurement, were compared for the extraction of a high-strength swine wastewater. Ten compounds, which are known contributors to malodors in wastewater, were quantified by both methods of extraction. For most compounds, greater levels were estimated by the PDMS-coated stir bars than by SPME, and measurement reproducibility was also greater. For both methods of extraction, there was greater variation in the measurement of volatile fatty acids than there was for aromatics.

Reduction of malodorous compounds from a treated swine anaerobic lagoon.

There is a need for treatment technologies that can eliminate environmental problems associated with anaerobic lagoons. These technologies must be able to capture nutrients, kill pathogens, and reduce emissions of ammonia and nuisance odors. To meet these needs, a full-scale wastewater treatment plant was installed as a demonstration project on one of three 4360-pig (Sus scrofa) production units in a finishing farm in Duplin County, North Carolina. Once the treatment plant was operational, flow of raw manure into the unit's corresponding lagoon was discontinued and the lagoon was used to store treated wastewater. Water quality was monitored in the converted lagoon and in the two conventional lagoons. A gas chromatographic method was developed to measure concentration of five selected malodorous compounds (phenol, p-cresol, 4-ethylphenol, indole, and skatole) in liquid lagoon samples. Dramatic improvements in the water quality parameters TKN, NH3-N, solids, COD, and BOD in the converted waste lagoon paralleled reductions in malodorous compounds. Nine months after conversion, identified malodorous compounds in liquid extractions averaged 6.6 and 38.8 ng mL(-1) in water from the converted lagoon and the conventional lagoons, respectively. The reduction was particularly marked for p-cresol, 4-ethylphenol, and skatole, all of which make important contributions to swine waste odors due to their characteristic odors and low detection thresholds.

Aroma content of fresh basil (Ocimum basilicum L.) leaves is affected by light reflected from colored mulches.

Sweet basil (Ocimum basilicum L.) is an herb that is used to add a distinct aroma and flavor to food. Volatile compounds emitted from fully expanded fresh leaves grown in drip-irrigated plots that were covered with six colors of mulch were compared. The colors reflected a range of photosynthetic photon flux, far-red, red, and blue light from the soil surface to developing leaves. Our objective was to determine whether reflection from the different colors could influence concentrations of volatile compounds emitted from the fresh leaves. Volatile compounds were isolated by headspace sampling and quantified by gas chromatography. Twenty-six compounds were identified, of which the terpenoids linalool and 1,8-cineole comprised more than 50% of the total yield. Concentrations of volatile compounds from leaves that developed over green, blue, yellow, white, and red mulches followed the same patterns as they did for air-dried leaves of the same cultivar. However, the concentration of volatile compounds from fresh leaves was about 50-fold higher than those found in the previous study of air-dried leaves.

Aroma of fresh strawberries is enhanced by ripening over red versus black mulch.

Strawberry (Fragaria ananassa Duch) is a high-value food crop, the aroma of which is important in consumer satisfaction. We hypothesized that the concentration of fresh strawberry aroma compounds could be enhanced by growing the berries over a red plastic mulch that was formulated to reflect more far-red (FR) and red light (R) and a higher FR/R photon ratio than is reflected by standard black plastic mulch. Strawberries of the cultivars "Chandler" and "Sweet Charlie" were grown in trickle-irrigated field plots over the two colors of mulch. The berries were harvested when ripe, and aroma compounds were isolated by dynamic headspace sampling. Entrained compounds were quantified by gas chromatography and identified by gas chromatography/mass spectroscopy. Twenty-three compounds were identified, and most of them were aliphatic esters. Strawberries that ripened over the red mulch during periods of sunny weather had significantly higher concentrations of aroma compounds than berries grown over the black plastic mulch. Total levels of aroma compounds from berries grown over red plastic mulch were higher for both Chandler and Sweet Charlie. We postulate that FR and R in light reflected from the red mulch acted through the natural phytochrome system to modify gene expression enough to result in greater concentration of aroma compounds in fresh strawberries.

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.

Development of a second-generation environmentally superior technology for treatment of swine manure in the USA.

New swine waste management systems in North Carolina need to meet high performance standards of an environmentally superior technology (EST) regarding nitrogen, phosphorus, heavy metals, pathogens, ammonia and odor emissions, and remain affordable and simple to operate. The objective of this study was to develop a second-generation treatment system that can achieve high EST standards at reduced costs. The system used solids separation, nitrification/denitrification and phosphorus removal/disinfection, and was demonstrated at full-scale on a 5145-head swine farm during three production cycles (15-months). Removal efficiencies were: 98% suspended solids, 97% ammonia, 95% phosphorus, 99% copper and zinc, 99.9% odors, and 99.99% pathogens. The system met EST standards at 1/3 the cost of the previous version. Animal health and productivity were enhanced; hog sales increased 32,900 kg/cycle (5.6%). These results demonstrated that: (1) significant cost reductions were achieved by on-farm implementation and continued engineering improvements, and (2) the new waste management system substantially benefited livestock productivity.

Free Fatty acids and sterols in swine manure.

Free fatty acids and sterols were assessed in fresh manure and anaerobic lagoon sludge from swine production facilities in North Carolina. Eight free fatty acids and five sterols were identified and quantified in both manure and sludge samples. Compound identification was performed by gas chromatography/mass spectroscopy (GC-MS), and compound quantities were determined by gas chromatography after solid phase extraction with a 50:50 mixture of diethyl ether and hexane. The free fatty acids occurring in greatest abundance in both fresh manure and lagoon sludge were palmitic, oleic, and stearic. Free fatty acid content in fresh manure ranged from approximately 3 microg g(-1) dry weight (dw) to over 45 microg g(-1) dw. In lagoon sludge, free fatty acid content ranged from about 0.8 microg g(-1) dw to nearly 4 microg g(-1) dw. Coprostanol and epicoprostanol were the sterols in largest concentrations in fresh manure and lagoon sludge samples. Total sterol content ranged from approximately 0.5 microg g(-1) dw to around 11 microg g(-1) dw in fresh manure and from 3.5 microg g(-1) dw to almost 9 microg g(-1) dw in lagoon sludge. Fresh manure and lagoon sludge both had high levels of inorganic cations (e.g., Ca, Mg, Fe) capable of binding free fatty acids and forming insoluble complexes, thereby potentially reducing fatty acid biodegradation. In anaerobic lagoons, sterols are an organic fraction of sludge that are resistant to bacterial degradation. In the case of fresh manure, fatty acids could represent a potential source of energy via the manufacture of biodiesel fuel, if efficient means for their extraction and transesterification can be devised.