Weiping Xu

A biosecure composting system for disposal of cattle carcasses and manure following infectious disease outbreak.

During outbreaks of infectious animal diseases, composting may be an effective method of disposing of mortalities and potentially contaminated manure. Duplicate biosecure structures containing 16 cattle (Bos taurus) mortalities (343 kg average weight) were constructed with carcasses placed on a 40-cm straw layer and overlaid with 160 cm of feedlot manure. At a depth of 80 cm (P80), compost heated rapidly, exceeding 55 degrees C after 8 d and maintained temperatures of 55 to 65 degrees C for > 35 d. Temperatures at 160 cm (P160) failed to exceed 55 degrees C, but remained above 40 degrees C for >4 mo. To investigate rates of microbial inactivation, Escherichia coli O157:H7, Campylobacter jejuni, and Newcastle disease virus (NDV) were inoculated in manure (E. coli O157:H7 and C. jejuni approximately 10(8) CFU g(-1); NDV, approximately 10(6) EID(50) g(-1)), embedded at P80 and P160 and retrieved at intervals during composting. Escherichia coli O157:H7 and NDV were undetectable after 7 d at both depths. The C. jejuni DNA was detected up to 84 d at P80 and >147 d at P160. To estimate degradation of recalcitrant substrates, bovine brain, hoof, and rib bones were also embedded at P80 and P160 and retrieved at intervals. Residues of soft tissues remained in carcasses after opening at 147 d and bovine tissue decomposition ranked as brain > hoof > bone. More than 90% dry matter (DM) of brain disappeared after 7 d and 80% DM of hoof decomposed after 56 d. High degradation of cattle carcasses, rapid suppression of E. coli O157:H7 and NDV and reduction in viable cell densities of >6 logs for C. jejuni demonstrates that the biosecure composting system can dispose of cattle carcasses and manure in an infectious disease outbreak.

Anaerobic digestion of specified risk materials with cattle manure for biogas production.

Biogas production from anaerobic digestion (AD) of specified risk materials (SRM) co-digested with cattle manure was assessed in a 3 x 2 factorial design. SRM replaced manure at 0 (control), 10% or 25% (w/w) as the substrate fed to six 2-L biodigesters maintained at 37 degrees C or 55 degrees C. Digesters were fed substrate (30 g L(-1) total volatile solids) at 6-d intervals for 90 d, with a retention time of 30 d. Keratin (<20mg) was added to each digester to model the degradation of beta-sheet rich proteins. Methane production was measured daily, and effluent was collected at feeding to monitor SRM degradation using real-time PCR analysis of bovine-specific DNA fragments. Compared with control, methane production increased by 83% or 161% (P<0.05) with 10% or 25% SRM at 37 degrees C, and by 45% and 87%, respectively, at 55 degrees C (P<0.05). Bovine DNA degradation over 6d was higher (P<0.05) at 37 degrees C as compared to 55 degrees C. Dry matter degradation of keratin at 37 degrees C decreased with increasing SRM concentration (P<0.05), whereas at 55 degrees C no difference between treatments was observed (P>0.05). Inclusion of SRM increases the production of methane during the anaerobic digestion of manure and may offer a means of deriving economic value from the disposal of SRM.

Purification of polymerase chain reaction (PCR)-amplifiable DNA from compost piles containing bovine mortalities

Livestock production systems utilize composting as a method of disposal of livestock mortalities, but there is limited information on the rate and extent of carcass decomposition. Detection of specific DNA fragments by PCR offers a method for investigating the degradation of carcasses and other biological materials during composting. However, the purity of extracted DNA is critical for successful PCR analysis. We applied a method to purify DNA from compost samples and have tested the method by analyzing bovine and plant DNA targets after 0, 4, and 12 month of composting. The concentration of organic matter from composted material posed a particular challenge in obtaining pure DNA for molecular analysis. Initially extracted DNA from composted piles at day 147 was discoloured, and PCR inhibitors prevented amplification of target plant or bovine gene fragments. Bovine serum albumin improved detection by PCR (25-50 microl final volume) through the removal of inhibitors, but only when concentrations of humic acids in extracted DNA were 1.0 ng microl(-1) or less. Optimal purification of DNA from compost was achieved by chromatography using Sepharose 4B columns. The described DNA purification protocol enabled molecular monitoring of otherwise cryptic bovine and plant target genes throughout the composting process. The assay could likely be used to obtain PCR-amplifiable DNA that could be used for the detection of microbial pathogens in compost.

An Improved Design for Biocontained Composting of Cattle Mortalities

Lack of uniform heating and peak temperatures lower than 55°C reduce the utility of beef cattle mortality composting. In a previous study, beef cattle mortalities were placed on a 40-cm straw base and covered with 160-cm manure, and compost reached 50°C with 20% reduction in bovine mitochondrial DNA (a 171-bp fragment; Mt171) at 160-cm depth after 147 d of static composting. Two modifications from the previous studys methodology were made: (1) carcasses were raised to 100-cm depth by placing them on an additional 60-cm layer of manure and (2) feedlot manure with 60% moisture content at construction was used. Temperature profiles at depths of 40, 100, and 160 cm all reached 55°C after 7 d, and remained above 55°C for at least 70 d. The bovine Mt171 fragment was degraded 75% by d 112 and 86% by d 230 at 100-cm depth. Carcass soft tissues were almost completely decomposed, with a 99% reduction in genomic DNA and a 92% decrease in Mt171 fragment after 230 d. Levels of coliform bacteria were below 10 CFU g−1 dry wt at all depths on d 230. The modified cattle mortality composting system was characterized by higher peak temperature, longer uniform heating, and faster bovine tissue degradation compared to the previous study. This biocontained composting system could offer effective containment and control of a disease outbreak in which the infectious agent is sensitive to temperatures between 55-62°C.

Biodegradation of genetically modified seeds and plant tissues during composting

BACKGROUND The increasing global market of genetically modified (GM) crops amplifies the potential for unintentional contamination of food and feed with GM plants. Methods proposed for disposal of crop residues should be assessed to prevent unintended distribution of GM materials. Composting of organic material is inexpensive and location-independent. The objective of this study was to determine the effectiveness of composting for disposal of GM plants in terms of reducing seed viability and promoting the degradation of endogenous as well as transgenic DNA. RESULTS Duplicate samples of corn kernels, alfalfa leaves, and GM canola seeds, meal and pellets were sealed in porous nylon bags and implanted in duplicate 85,000 kg (initial weight) feedlot manure compost piles. Samples were collected at intervals over 230 days of composing. Canola seeds and corn kernels were not viable after 14 days of composting with temperatures in the piles exceeding 50 degrees C. In all samples, PCR analyses revealed that plant endogenous and transgenic fragments were substantially degraded after 230 days of composting. Southern blotting of genomic DNA isolated from canola seeds identified differences in the persistence of endogenous, transgenic, and bacterial DNA. CONCLUSION Composting GM and non-GM plant materials with manure rendered seeds non-viable, and resulted in substantial, although not complete, degradation of endogenous and transgenic plant DNA. This study demonstrates that composting could be effective for disposing of GM crops in the event of their inadvertent entry into the food or feed chain.

Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock

Intensive livestock production systems are particularly vulnerable to natural or intentional (bioterrorist) infectious disease outbreaks. Large numbers of animals housed within a confined area enables rapid dissemination of most infectious agents throughout a herd. Rapid containment is key to controlling any infectious disease outbreak, thus depopulation is often undertaken to prevent spread of a pathogen to the larger livestock population. In that circumstance, a large number of livestock carcasses and contaminated manure are generated that require rapid disposal. Composting lends itself as a rapid-response disposal method for infected carcasses as well as manure and soil that may harbor infectious agents. We designed a bio-contained mortality composting procedure and tested its efficacy for bovine tissue degradation and microbial deactivation. We used materials available on-farm or purchasable from local farm supply stores in order that the system can be implemented at the site of a disease outbreak. In this study, temperatures exceeded 55°C for more than one month and infectious agents implanted in beef cattle carcasses and manure were inactivated within 14 days of composting. After 147 days, carcasses were almost completely degraded. The few long bones remaining were further degraded with an additional composting cycle in open windrows and the final mature compost was suitable for land application. Duplicate compost structures (final dimensions 25 m x 5 m x 2.4 m; L x W x H) were constructed using barley straw bales and lined with heavy black silage plastic sheeting. Each was loaded with loose straw, carcasses and manure totaling ~95,000 kg. A 40-cm base layer of loose barley straw was placed in each bunker, onto which were placed 16 feedlot cattle mortalities (average weight 343 kg) aligned transversely at a spacing of approximately 0.5 m. For passive aeration, lengths of flexible, perforated plastic drainage tubing (15 cm diameter) were placed between adjacent carcasses, extending vertically along both inside walls, and with the ends passed though the plastic to the exterior. The carcasses were overlaid with moist aerated feedlot manure (~1.6 m deep) to the top of the bunker. Plastic was folded over the top and sealed with tape to establish a containment barrier and eight aeration vents (50 x 50 x 15 cm) were placed on the top of each structure to promote passive aeration. After 147 days, losses of volume and mass of composted materials averaged 39.8% and 23.7%, respectively, in each structure.

Biocontained Mortality Compost Using Liquid Manure

During disease outbreaks, composting has been used to safely dispose of carcasses and infectious solid manure. However, optimized methods have not been established to use liquid manure (> 80% water content, WC) from dairies as the substrate for mortality composting. In April of 2007, a 3×2 factorial study was conducted in wooden compost bins (240 × 240 × 140 cm) lined with 0.5 mm plastic to a height of 50 cm. Three levels (LO, 95 kg; MED, 236 kg; HI, 606 kg wet wt.) of liquid manure (91% WC) were applied to barley straw containing a single calf mortality (avg. wt 130 kg). Compost temperature and carcass degradation were evaluated in each bin over 52 d. As an indication of compost efficiency, viability of 4 types of weed seeds (wild buckwheat, Polygonium convolvulus L, BW; dandelion, Taraxacum officinale, DL; stinkweed, Thlaspi arvense L., SW; and wild oat, Avena fatua, WO) was determined from seeds retained at the laboratory (Control) or from those placed in sealed nylon bags (50 μm pore size) embedded at 3 locations in each bin. Initial C: N ratios ranged from 58:1 (HI) to 68:1 (LO), while initial WC was 64, 52 and 42% for the HI, MED and LO treatments, respectively. Rate of compost heating and peak temperature (58.6 vs. 46.5°C) was increased (P < 0.05) for HI as compared to LO. Rate of temperature decline was lower (P < 0.05) and calf decomposition was visibly superior for HI as compared to LO. Viability of WO was eliminated by all compost treatments, but that of BW increased (P < 0.05) in MED and LO as compared to Control. Although HI did not eliminate BW viability, this treatment showed the the most promise for incorporating liquid manure as a substrate in contained mortality compost.

Field scale evaluation of bovine-specific DNA as an indicator of tissue degradation during cattle mortality composting

Currently, mortality compost is managed by temperature as extent of tissue degradation is difficult to assess. In the present study, field-scale mortality compost was constructed with composted brain tissue (Brain) and compost adjacent to brain tissue (CAB) sampled over 230 d. Following genomic DNA extraction, bovine-specific mitochondrial DNA (Mt-DNA) and bacterial 16S rDNA fragments were quantified using real-time PCR. Genomic DNA yield of Brain and CAB decreased rapidly (89-98%) and stabilized after 7 d. Compared to d 0, Brain Mt-DNA rapidly decreased (84-91% reduction on d 7). In CAB, Mt-DNA dramatically increased until d 28 (up to 34,500 times) thereafter decreasing by 77-93% on d 112. Quantification of bovine Mt-DNA indicates tissue degradation was initially characterized by rapid decomposition and release of cell contents into surrounding compost matrix followed by further degradation of Mt-DNA by flourishing microorganisms. Consequently, bovine Mt-DNA copies in compost matrix were reliable indicators of tissue degradation.

Evaluation of compost, vegetable and food waste as amendments to improve the composting of NaOH/NaClO-contaminated poultry manure

Regular usage of NaOH/NaClO disinfectants results in high sodium salt and alkalinity of poultry manure. This study compared three amendments: vegetable waste (V), food waste (F) and mature compost (C) for their ability to improve the composting of NaOH/NaClO-contaminated poultry manure. C compost resulted in the highest compost temperatures (p<0.001) and greatest reduction in OM, TC, TN and NH4-N (p<0.05). C and V composts were more efficient at lowering extractable-Na (ext-Na) and electrical conductivity (EC) than F (p<0.05). Maturity was primarily indicated by NH4-N, EC and ext-Na. Bacterial dynamics was profoundly influenced by NH4-N, EC and TC, with the decrease leading to discriminate genera shift from Sinibacillus and Thiopseudomonas to Brevbacterium, Brachybacterium, and Microbacterium. These findings suggest that mature compost was more desirable amendment than vegetable and food waste in the composting of NaOH/NaClO-contaminated poultry manure, and the decrease of ext-Na indicated compost maturity but did not influence bacterial dynamics.

Compost biodegradation of recalcitrant hoof keratin by bacteria and fungi.

Compost activities efficiently break down a wide range of organic substances over time. In this study, bovine hoof was used as recalcitrant protein model to gain so far cryptic information on biodegradation during livestock mortalities composting.