Evaluation and optimization of innovative, mycoremediation technologies to remove pathogens and antibiotics from dairy manure

1. IntroductionSoil amendment handling practices can reduce food safety risks to fruits and vegetables and to other crops. Bacterial pathogens can be present in biological soil amendments of animal origin and can be transferred to crops via direct application, wind, or rain. The FDA Food Safety Modernization Act of 2011 (FSMA) mandated Produce Safety Rule Standards for the Growing, Harvesting, Packing, and Holding of Produce for Human Consumption. FDA has deferred its decision on an appropriate time interval between the application of untreated biological soil amendments (including raw manure) and crop harvesting until it conducts a risk assessment and extensive research to strengthen scientific support for any future proposal. Data suggest that bacteria that may contaminate raw agricultural commodities (RACs) may persist in manure for hundreds of days (Sharma and Reynnells, 2017). There is a data gap regarding potential for biocontrol of bacteria in manure. While proper composting can inactivate bacterial pathogens, small-to-mid-sized growers may be reluctant to properly compost biological soil amendments due to lack of space and potential issues with adherence to time and temperature conditions and necessary record keeping. Bacterial pathogens may survive or undergo regrowth in finished compost due to incomplete thermal inactivation during composting or recontamination that can occur (Reynnells et al., 2014). These proposed novel studies will provide a means of treatment for raw manure and the potential for use of manure on land used to grow RACs. Bacterial pathogens including Salmonella and Escherichia coli O157:H7 may exist in raw manure; and irrigation water exposed to contaminated surface water runoff from nearby animal production farms has been linked to foodborne outbreaks associated with contaminated leafy greens (Crohn and Bianchi, 2008). Better methods are needed to ensure that the use of animal manures in agricultural production does not impose a risk to food safety nor contribute to environmental contamination. Recent work in the area of mycoremediation has shown that certain white rot fungi (WRF) are capable of killing bacteria present in manure waste streams (Chirnside, 2016). In addition, these WRF are able to degrade recalcitrant contaminants, like antibiotics and their residues, and have been used to remediate contaminated soils (Golan-Rozen et al., 2015). Therefore, it is feasible that technologies utilizing the WRF can be developed to remove pathogenic bacteria and antibiotics from manure before it is exposed to the environment. This proposed project will provide as stated in Program Area Priority A1331, an economical and adoptable control strategy aimed at reducing the incidence of foodborne hazards during the critical pre-harvest production time.1.1 Long-term Goals and Supporting ObjectivesThe long-term goal of this research project is to develop and implement sustainable, low cost, mycoremediation technologies that can reduce pathogens and contaminants present in agricultural manure waste streams, which have become a considerable threat to public health and overall food safety. This will also allow for the use of biological soil amendments of animal origin to be used in crop production. We have preliminary data to support that the WRF, Pleurotus ostreatus, in bioreactors can reduce E.coli in dairy manure (Chirnside, 2016; Chirnside et al., 2013). As discussed in the following Body of Knowledge section, small bench scale fungal bioreactors were able to reduce the number of E. coli naturally present in aqueous dairy manure. Crude extract from the WRF, Phanerochaete chrysosporium, containing LiP enzyme was able to degrade tetracycline and oxytetracycline, two antibiotics, during in vitro incubation (Wen et al., 2009).The goals of this research are to optimize fungal bioreactor properties, to evaluate the predation/degradation behavior of the WRF, and efficiently degrade pathogenic bacteria and antibiotic contaminants in dairy manure. We propose to achieve these goals by evaluating fungal bioreactor properties in laboratory experiments and coordinating these results with composting experiments utilizing dairy manure inoculated with the fungi under study. We have specifically chosen strains of E. coli and E. coli O157:H7 that are well studied in the literature (Sharma et al., 2016; Reynnells et al., 2014; Tomas-Callejas et al., 2011) and have been shown to survive in biological soil amendments; thereby we will be able to directly correlate our studies to a reduction in food safety risks. Specifically these objectives include:To determine the efficacy of two different white rot fungi, Phanerochaete chrysosporium and Pleurotus ostreatus, grown within fungal bioreactors to remove E. coli (strain TVS 355) from aqueous dairy manure through the assessment of different growth support materials and flow regimes.To evaluate the ability of the white rot fungus grown within a packed-bed fungal bioreactor to remove several pathogenic bacteria present in a buffered growth solution and in aqueous dairy manure.To evaluate the ability of the white rot fungus grown within a packed-bed fungal bioreactor to remove antibiotic residues present in water and in aqueous dairy manure.To determine the fate of E. coli TVS 355 and E. coli O157:H7 and antibiotic residues in dairy manure during composting with two different white rot fungi, Phanerochaete chrysosporium and Pleurotus ostreatus. A third inoculant treatment will consist of a mixture of P. chrysosporium and P. ostreatus.