Brandon H. Gilroyed

Bioaugmentation with an anaerobic fungus in a two-stage process for biohydrogen and biogas production using corn silage and cattail

Bioaugmentation with an anaerobic fungus, Piromyces rhizinflata YM600, was evaluated in an anaerobic two-stage system digesting corn silage and cattail. Comparable methane yields of 328.8±16.8mLg(-1)VS and 295.4±14.5mLg(-1)VS and hydrogen yields of 59.4±4.1mLg(-1)VS and 55.6±6.7mLg(-1)VS were obtained for unaugmented and bioaugmented corn silage, respectively. Similar CH4 yields of 101.0±4.8mLg(-1)VS and 104±19.1mLg(-1)VS and a low H2 yield (<1mLg(-1)VS) were obtained for unaugmented and bioaugmented cattail, respectively. However, bioaugmentation resulted in an initial increase in CH4 and H2 production rates and also increased volatile fatty acid degradation rate for both substrates. Our study demonstrates the potential of bioaugmentation with anaerobic fungus for improving the digestibility of lignocellulose substrates for biogas and biohydrogen production.

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.

Prion protein detection via direct immuno-quantitative real-time PCR

We describe a simple and robust assay for the quantitative detection of prions using immuno-quantitative real-time PCR (iQ-RT-PCR) made possible by a direct conjugate of a prion-specific antibody (ICSM35) and a synthetic 99-bp DNA tail. The DNA tail was engineered to include a single ScrFI restriction site, which enabled subsequent quantification of restricted DNA tails using real-time PCR. The assay was tested with scrapie prions bound to polyvinylidene difluoride membranes and to 96-well plates coated with a capturing antibody from a commercially available immuno-based assay (TeSeE). The iQ-RT-PCR assay had a detection limit corresponding to 2.32x10(2) prion epitopes, which represented a 1000-fold increase in detection sensitivity over the commercial assay. Detection of prions from diluted scrapie-positive brain homogenate bound to membranes was linear over a range of 1.06x10(4) to 3.24x10(2) epitopes (R(2)=0.92). Given its sensitivity and versatility, the present assay has potential to enable rapid and reliable detection of agents causing transmissible spongiform encephalopathies.

Biodegradation of Prions in Compost

Composting may serve as a practical and economical means of disposing of specified risk materials (SRM) or animal mortalities potentially infected with prion diseases (transmissible spongiform encephalopathies, TSE). Our study investigated the degradation of prions associated with scrapie (PrP(263K)), chronic waste disease (PrP(CWD)), and bovine spongiform encephalopathy (PrP(BSE)) in lab-scale composters and PrP(263K) in field-scale compost piles. Western blotting (WB) indicated that PrP(263K), PrP(CWD), and PrP(BSE) were reduced by at least 2 log10, 1-2 log10, and 1 log10 after 28 days of lab-scale composting, respectively. Further analysis using protein misfolding cyclic amplification (PMCA) confirmed a reduction of 2 log10 in PrP(263K) and 3 log10 in PrP(CWD). Enrichment for proteolytic microorganisms through the addition of feather keratin to compost enhanced degradation of PrP(263K) and PrP(CWD). For field-scale composting, stainless steel beads coated with PrP(263K) were exposed to compost conditions and removed periodically for bioassays in Syrian hamsters. After 230 days of composting, only one in five hamsters succumbed to TSE disease, suggesting at least a 4.8 log10 reduction in PrP(263K) infectivity. Our findings show that composting reduces PrP(TSE), resulting in one 50% infectious dose (ID50) remaining in every 5600 kg of final compost for land application. With these considerations, composting may be a viable method for SRM disposal.

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.

Microbial communities and greenhouse gas emissions associated with the biodegradation of specified risk material in compost

Provided that infectious prions (PrP(Sc)) are inactivated, composting of specified risk material (SRM) may be a viable alternative to rendering and landfilling. In this study, bacterial and fungal communities as well as greenhouse gas emissions associated with the degradation of SRM were examined in laboratory composters over two 14 day composting cycles. Chicken feathers were mixed into compost to enrich for microbial communities involved in the degradation of keratin and other recalcitrant proteins such as prions. Feathers altered the composition of bacterial and fungal communities primarily during the first cycle. The bacterial genera Saccharomonospora, Thermobifida, Thermoactinomycetaceae, Thiohalospira, Pseudomonas, Actinomadura, and Enterobacter, and the fungal genera Dothideomycetes, Cladosporium, Chaetomium, and Trichaptum were identified as candidates involved in SRM degradation. Feathers increased (P<0.05) headspace concentrations of CH4 primarily during the early stages of the first cycle and N2O during the second. Although inclusion of feathers in compost increases greenhouse gas emissions, it may promote the establishment of microbial communities that are more adept at degrading SRM and recalcitrant proteins such as keratin and PrP(Sc).

Biodegradation of specified risk material and fate of scrapie prions in compost

Composting may be a viable alternative to rendering and land filling for the disposal of specified risk material (SRM) provided that infectious prion proteins (PrPTSE) are inactivated. This study investigated the degradation of SRM and the fate of scrapie prions (PrPSc) over 28 days in laboratory-scale composters, with and without feathers in the compost matrices. Compost was mixed at day 14 to generate a second heating cycle, with temperatures exceeding 65°C in the first cycle and 50°C in the second cycle. Approximately 63% and 77% of SRM was degraded after the first and second cycles, respectively. Inclusion of feathers in the compost matrices did not alter compost properties during composting other than increasing (P < 0.05) total nitrogen and reducing (P < 0.05) the C/N ratio. However, addition of feathers enhanced (P < 0.05) SRM degradation by 10% upon completion of experiment. Scrapie brain homogenates were spiked into manure at the start of composting and extracted using sodium dodecyl sulphate followed by detection using Western blotting (WB). Prior to composting, PrPSc was detectable in manure with 1–2 log10 sensitivity, but was not observable after 14 or 28 days of composting. This may have been due to either biological degradation of PrPSc or the formation of complexes with compost components that precluded its detection.

Can plants serve as a vector for prions causing chronic wasting disease

Prions, the causative agent of chronic wasting disease (CWD) enter the environment through shedding of bodily fluids and carcass decay, posing a disease risk as a result of their environmental persistence. Plants have the ability to take up large organic particles, including whole proteins, and microbes. This study used wheat (Triticum aestivum L.) to investigate the uptake of infectious CWD prions into roots and their transport into aerial tissues. The roots of intact wheat plants were exposed to infectious prions (PrPTSE) for 24 h in three replicate studies with PrPTSE in protein extracts being detected by western blot, IDEXX and Bio-Rad diagnostic tests. Recombinant prion protein (PrPC) bound to roots, but was not detected in the stem or leaves. Protease-digested CWD prions (PrPTSE) in elk brain homogenate interacted with root tissue, but were not detected in the stem. This suggests wheat was unable to transport sufficient PrPTSE from the roots to the stem to be detectable by the methods employed. Undigested PrPTSE did not associate with roots. The present study suggests that if prions are transported from the roots to the stems it is at levels that are below those that are detectable by western blot, IDEXX or Bio-Rad diagnostic kits.

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.

Synthesis of O-serogroup specific positive controls and real-time PCR standards for nine clinically relevant non-O157 STECs.

Non-O157 Shiga toxin producing Escherichia coli (STEC) are gaining recognition as human pathogens, but no standardized method exists to identify them. Sequence analysis revealed that STEC can be classified on the base of variable O antigen regions into different O serotypes. Polymerase chain reaction is a powerful technique for thorough screening and complex diagnosis for these pathogens, but requires a positive control to verify qualitative and/or quantitative DNA-fragment amplification. Due to the pathogenic nature of STEC, controls are not readily available and cell culturing of STEC reference strains requires biosafety conditions of level 2 or higher. In order to bypass this limitation, controls of stacked O-type specific DNA-fragments coding for primer recognition sites were designed to screen for nine STEC serotypes frequently associated with human infection. The synthetic controls were amplified by PCR, cloned into a plasmid vector and transferred into bacteria host cells. Plasmids amplified by bacterial expression were purified, serially diluted and tested as standards for real-time PCR using SYBR Green and TaqMan assays. Utility of synthetic DNA controls was demonstrated in conventional and real-time PCR assays and validated with DNA from natural STEC strains.