Highly effective water hyacinth (Eichhornia crassipes) waste-based functionalized sustainable green adsorbents for antibiotic remediation from wastewater.

Azithromycin (AZIM) is considered as one of the most frequently prescribed antibiotics (ABs) in the world by medical professionals. This study explored, two novel, cheap and environmentally beneficial adsorbents i.e., alkali treated water hyacinth powder (AT-WHP) and graphene oxide-water hyacinth-polyvinyl alcohol (GO-WH-PVA) composite, fabricated from water hyacinth (Eichhornia crassipes) waste to remediate AZIM from wastewater. Biosorption experiments were performed by batch and packed-bed column studies and the adsorbents were characterized using various instrumental methods. The morpho-chemical profile of the adsorbents suggested noteworthy AZIM adsorption. AZIM adsorption data can be reasonably explained by pseudo second order (PSO) kinetic model with maximum regression coefficient (R2 > 0.99) and lowest Marquardt's present standard deviation (MPSD) and root mean squared error (RMSE) values. The isotherm models recommended Langmuir and Temkin to be the best-fitted, providing highest regression coefficient and lowest error values. Conferring to Langmuir model, the theoretical highest adsorption potentials (qmax) were accounted to be 244.498 and 338.115 mg/g for AT-WHP and GO-WH-PVA, correspondingly, very close to experimental values (qe, exp). AZIM adsorption processes were governed by the chemisorption mechanisms. The adsorbents had excellent regeneration potential and could be reused several times. In order to scale-up application of the adsorbents, performance of a 100 L packed-bed reactor was assessed and a breakthrough time of adsorption for GO-WH-PVA was 15 min in 5000 mg/L AZIM concentration. Thus, the absorbents synthesized in this study can be considered highly effective at removal of AZIM from wastewater.

Evaluation and modelling of methane production from corn stover pretreated with various physicochemical techniques.

Lignocellulosic by-products from agricultural crops represent an important raw material for anaerobic digestion and clean renewable, which is a key component of the circular economy. Lignocellulose is recalcitrant to biodegradation and pretreatments are required to increase methane yield during anaerobic digestion. In this work, the efficacy of different physicochemical pretreatments was compared using corn stover biomass as substrate. Anaerobic digestion of untreated and pretreated corn stover was performed in batch mode at mesophilic temperature (38°C) and organic matter solubilization of pretreated substrates was also investigated. The highest organic matter solubilization occurred in autoclave pretreatment (soluble chemical oxygen demand = 5630 ± 42 mg O2 L-1). However, the highest methane yield was obtained using alkaline pretreatment (367 ± 35 mL CH4 g-1 VSadded). Alkaline pretreatment increased methane yield by 43.3% compared to untreated control (256 ± 15 mL CH4 g-1 VSadded). Two mathematical models (i.e. first-order kinetics and transfer function) were utilized to fit the experimental data with the aim of assessing anaerobic biodegradation and to obtain the kinetic constants in all cases studied. Both models adequately fit the experimental results. The kinetic constant, k, of the first-order model increased by 92.8% when stover was pretreated with sulphuric acid compared with control. The transfer function model revealed that the maximum methane production rate, Rm, was obtained for the sulphuric acid treatment, which was 63.5% higher compared to control.

The potential of the oleaginous yeast Rhodotorula paludigena CM33 to produce biolipids.

Sixty-seven yeast strains were isolated from castor beans then their endogenous lipids were stained by Nile Red (NR) fluorescence dye, and flow cytometry was used to obtain a strain with a high relative mean fluorescence intensity (MFI) value. The highest MFI value was obtained for strain CM33, which produced a maximum lipid content of 20.8 % dry cell weight (DCW). Based on the sequence of the ITS-5.8S-ITS rDNA and D1/D2 26S rDNA regions, CM33 showed 99 % identity with Rhodotorula paludigena. The potential of CM33 to assimilate various carbon sources was examined by growth on minimal media using glucose, glycerol, sucrose or xylose. CM33 was grown in glucose-based medium for 96 h and exhibited a maximum lipid content of 23.9 % DCW. Furthermore, when cells were cultured on molasses waste, their biomass, lipid content and lipid concentration reached 16.5 g/L, 37.1 % DCW and 6.1 g/L, respectively. These results demonstrated the potential of R. paludigena CM33 to contribute to a value-added carbon chain by converting renewable waste materials for biolipid production.

Biogas production, waste stabilization efficiency, and hygienization potential of a mesophilic anaerobic plug flow reactor processing swine manure and corn stover.

Swine manure and corn stover are abundant agricultural wastes which contribute to greenhouse gas (GHG) emissions, nutrient runoff leading to eutrophication, and a biosafety risk with respect to improper swine manure handling. Anaerobic co-digestion (AcoD) of swine manure and corn stover can mitigate these negative impacts while producing biogas as a renewable energy source. Semi-continuous mesophilic plug flow reactor (PFR operation) was studied during a step-wise increase in organic loading rate (OLR) over the range of 0.25-4.7 kg volatile solids added (VS) m-3 d-1, which corresponded to total solids content (TS) of 1.5-9.0%. Process stability was observed at all OLR, with the highest total biogas yield and methane content of 0.674 ± 0.06 m-3 kg-1 and 62%, respectively, at 0.25 kg m-3 d-1. As OLR and TS increased, VS reduction decreased and volatile fatty acids (VFA) increased due to shorter hydraulic retention times (HRT). Hygienization potential was assayed using fecal indicator bacteria (FIB), with some groups being reduced (E. coli, fecal coliforms) and others not (Clostridia spp., fecal enterococci). Lignocellulolytic enzyme activity trended upward as OLR was increased, highlighting changes in microbial activity in response to feeding rate.

Hygienization and microbial metabolic adaptation during anaerobic co-digestion of swine manure and corn stover.

This study assessed the effect of different swine manure (SM)/corn stover (CS) mixtures based on total solids (TS) content with respect to hygienization, microbial community dynamics and methane yields on batch anaerobic co-digestion performance. Different ratios of SM and CS with TS content between 0.69 and 6% digested at 75 d revealed SM had the greatest methane yield at 403.9 mL g-1 volatile solids added (VS) and 86.31% VS reduction. BIOLOG AN microplates and lignocellulolytic enzyme assays proved to be rapid tools for characterizing microbial community metabolism as noted by the different carbon source utilization patterns between TS loadings. Hygienization of fecal indicator bacteria groups was achieved with some (E. coli) but not all groups (Clostridia spp.). The results showed that colorimetric biochemical assays and culture-based techniques can rapidly assess microbial community dynamics during co-digestion, and that TS- in the form of lignocellulosic biomass- influences microbial metabolic activities.

Ambient alkaline hydrolysis and anaerobic digestion as a mortality management strategy for whole poultry carcasses.

Livestock mortality management is a critical factor for ensuring biosecurity, minimizing environmental impact, and maintaining public trust in livestock production agriculture. The number of technologies currently used for livestock mortality management is small, including composting, burial, incineration, landfilling, and rendering. Each technology has advantages and disadvantages which make their suitability situational. In this study, ambient alkaline hydrolysis (AAH) using 2, 4, or 8 M potassium hydroxide at ambient temperature and pressure was explored as a disposal method for whole broiler chicken carcasses. Alkaline hydrolysate (pH > 14) resulting from the process was neutralized by mixing with acidic corn silage, and then utilized as a substrate for anaerobic digestion in bench top continuously stirred tank reactors. All AAH treatments solubilized broiler carcasses within 20 days. Corn silage neutralized 2 M hydrolysate using a 2:1 (w/w) mixing ratio, while 4 M hydrolysate required a 4:1 mixing ratio. Anaerobic digestion of neutralized hydrolysate reduced volatile solids by >96% for all treatments. Highest methane yields were observed from the 2 M hydrolysate (607.2 ±â€¯47.9 g mL-1 VS), while biogas production from the 8 M hydrolysate was totally inhibited over a total of 42 days. Ambient alkaline hydrolysis followed by silage neutralization and anaerobic digestion provides a feasible, straightforward technology to manage routine and emergency animal mortalities.

Composting for Biocontained Cattle Mortality Disposal and Associated Greenhouse Gas and Leachate Emissions.

Composting can be an effective means of biodegrading livestock mortalities in emergency disposal situations, such as disease outbreaks. Within the past decade, our knowledge detailing composting has increased substantially. However, research data linking the environmental impact of composting to atmospheric and terrestrial systems are limited. We investigated composting efficacy, greenhouse gas emissions, and leachate properties from two static compost piles, each containing 16 cattle mortalities, built with either beef manure (BM) or wood shavings (WS) as envelope material. Wood shavings achieved a greater maximum temperature than BM (60 vs. 50°C) and maintained higher temperatures over 200 d ( < 0.001). Greenhouse gas emissions were evaluated using a static chamber and gas chromatography. Emissions of NO ( < 0.001), CH ( < 0.01), and CO ( < 0.05) were lower from WS than BM, resulting in 3-fold lower total CO equivalent emissions. After 250 d of composting, piles were relocated, and soil cores were taken (i) from beneath the piles, (ii) adjacent to the piles where leachate had accumulated, and (iii) in a control zone without compost exposure. Elevated concentrations of ammonium ( < 0.05) and chloride ( < 0.05) were found in soil beneath both BM and WS. Microbial DNA profiles suggested that leachate from BM compost increased bacterial diversity in soil, maintaining a biological soil impact after pile removal. Degradation of bovine mitochondrial DNA fragments was monitored by polymerase chain reaction. Limited migration of genetic bovine material from compost into soil was observed. Based on the mortalities decomposition and leachate contents, both BM and WS are suitable envelope materials for composting.

Application of protein misfolding cyclic amplification to detection of prions in anaerobic digestate.

The exceptional physio-chemical resistance of prions to established decontamination procedures poses a challenge to assessing the suitability of applied inactivation methods. Prion detection is limited by the sensitivity level of Western blotting or by the cost and time factors of bioassays. In addition, prion detection assays can be limited by either the unique or complex nature of matrices associated with environmental samples. To investigate anaerobic digestion (AD) as a practical and economical approach for potential conversion of specified risk materials (SRM) into value added products (i.e., renewable energy), challenges associated with detection of prions in a complex matrix need to be overcome to determine potential inactivation. Protein misfolding cyclic amplification (PMCA) assay, with subsequent Western blot visualization, was used to detect prions within the AD matrix. Anaerobic digestate initially inhibited the PMCA reaction and/or Western blot detection. However, at concentrations of ≤1% of anaerobic digestate, 263K scrapie prions could be amplified and semi-quantitatively detected. Infectious 263K prions were also proven to be bioavailable in the presence of high concentrations of digestate (10-90%). Development of the PMCA application to digestate provides extremely valuable insight into the potential degradation and/or fate of prions in complex biological matrices without requiring expensive and time-consuming bioassays.

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.

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 (PrP(TSE)) for 24 h in three replicate studies with PrP(TSE) in protein extracts being detected by western blot, IDEXX and Bio-Rad diagnostic tests. Recombinant prion protein (PrP(C)) bound to roots, but was not detected in the stem or leaves. Protease-digested CWD prions (PrP(TSE)) in elk brain homogenate interacted with root tissue, but were not detected in the stem. This suggests wheat was unable to transport sufficient PrP(TSE) from the roots to the stem to be detectable by the methods employed. Undigested PrP(TSE) 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.

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).

Emergency euthanasia of cattle challenged with Escherichia coli O157:H7 - a case study for evaluating the response to an infectious disease outbreak.

In the event of an infectious disease outbreak in cattle, carcasses must be disposed of in a rapid and contained manner. This brief communication details injection of a barbiturate to euthanize cattle inoculated with Escherichia coli O157:H7 followed by carcass composting in a manner that prevents the spread of infectious agents.

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 (PrP(TSE)) are inactivated. This study investigated the degradation of SRM and the fate of scrapie prions (PrP(Sc)) 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, PrP(Sc) was detectable in manure with 1-2 log(10) sensitivity, but was not observable after 14 or 28 days of composting. This may have been due to either biological degradation of PrP(Sc) or the formation of complexes with compost components that precluded its detection.

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.

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 degrees 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 approximately 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 (approximately 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.

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.