Understanding risks and optimising anaerobic digestion to minimise pathogen and antimicrobial resistance genes entering the environment.

厌氧消化（AD）利用有机材料通过沼气产生能量，同时也产生营养丰富的固态肥料，非常适合土地应用。然而，病原体传播到土地、进入食物链、直接接触环境或流入水道，可能会对人类（和牲畜）的健康造成风险。这是复杂的抗生素耐药细菌（ARB）进入环境的担忧。抗性基因（ARG）可以在生物反应器和土壤微生物组中广泛转移，包括向病原体转移和从病原体转移。值得关注的病原体包括梭菌，它可以在消化过程中增殖并抵抗巴氏杀菌。关键的挑战是优化反倾销程序，以尽量减少与反倾销应用于土地相关的风险。由于缺乏数据，无法对通过AD进入环境的病原体的风险进行评估，并且关于操纵AD以减少病原体和抗性基因而不影响气体产生的信息也很少。此外，对于原料如何根据其病原体或精氨酸提取物含量进行排序，以及一旦施用到土地上，这些含量如何影响病原体/精氨酸提取物的命运，目前还不清楚。这种理解是至关重要的，因为过程优化和消化后消毒不需要高温巴氏灭菌的能量输入（这在某些情况下也是不切实际的，如小规模系统、发展中国家）可以使通过AD发电更可持续和更广泛可行。1）评估常见AD原料的病原体/ARG含量2）了解原料类型和工艺条件对病原体/ARG持久性的作用3）比较病原体/ARG在传统有机废物改良土壤与亚硝酸盐改良土壤中的持久性。目标将通过一系列实验和文献为基础的步骤来解决：1。原料中的病原体/ARG含量学生将进行文献综述，包括AD不同原料中病原体/ARG存在和浓度的综合数据。这将补充实验工作中，学生将采样和枚举一套病原体/ARG在原料从一系列消化器与主管有现有的联系。2.在AD期间，原料和工艺条件对病原体/ARG持久性的作用学生将建立实验室规模的消化器，通过该消化器进行实验，以在文献综述期间以及通过与现场操作员的讨论确定的行业相关操作范围内操纵工艺条件（例如加载速率/保留时间，含水量，温度）。这些将利用机会来仔细控制原料和条件，以限定用于气体生产和纯度、病原体/ARG减少和农业上有用的植物的生产的最佳操作范围。这将通过与上述（1）中的原料样品相对应的取样状态来补充，并记录工艺条件。病原体/ARG在改良土壤中的持久性学生将建立田间实验，其中将在析因实验中对实验草/作物地块应用草甘膦，还将牲畜粪便/泥浆和无机肥料作为治疗，以及未处理的对照地块。将在一年内对地块进行采样，并进行病原体/ARG、土壤养分/理化特性和土壤微生物组分析。在整个过程中，将使用PCR/多重PCR和培养对病原体/ARG进行样本筛查，然后使用q-PCR进行定量。

Anaerobic digestion (AD) utilises organic materials to produce energy via biogas while also producing nutrient-rich digestate ideal for land application as fertiliser. However, there may be a risk to human (and livestock) health through transmission of pathogens to land and uptake into the food chain, direct environmental contact or run-off into water courses. This is compounded by concerns over antibiotic-resistant bacteria (ARBs) entering the environment. Resistance genes (ARGs) can be transferred widely within the bioreactor and soil microbiome, including to and from pathogens. Pathogens of concern include Clostridia, which can proliferate during digestion and resist pasteurisation. The key challenge is to optimise AD processes to minimise risks associated with application of AD to land. Lack of data has precluded evaluation of risk from pathogens entering the environment through AD and information on manipulating AD to reduce pathogens and resistance genes without compromising gas generation are scarce. Furthermore, there is little clarity on how feedstocks might be ranked in terms of their pathogen or ARG content, nor how that feeds through into the fate of pathogens/ARGs once applied to land. This understanding is critical because process optimisation and post-digestion sanitisation that do not require the energy inputs of high temperature pasteurisation (which is also not practical in some circumstances e.g. small scale systems, developing countries) could make energy generation through AD more sustainable and more widely viable.The project aims to:1) evaluate pathogen/ARG content of common AD feedstocks2) understand the role of feedstock type and process conditions on pathogen/ARG persistence3) compare persistence of pathogens/ARG in traditional organic waste-amended soils vs. digestate-amended soils.The aims will be addressed through a series of experimental and literature-based steps:1. Pathogen/ARG content of feedstocks The student will undertake a literature review including a synthesis of data on pathogen/ARG presence and concentrations in different feedstocks for AD. This will be supplemented with experimental work in which the student will sample and enumerate a suite of pathogens/ARGs in feedstocks from a range of digesters with which the supervisors have existing links. 2. Role of feedstock and process conditions on pathogen/ARG persistence during ADThe student will establish laboratory scale digesters through which to undertake experiments to manipulate process conditions (e.g. loading rates/retention time, water content, temperature) within industry-relevant operating envelopes as identified during the literature review and through discussion with site operators during (1) above. These will exploit the opportunity to control feedstocks and conditions carefully to define optimal envelopes of operation for both gas production and purity, pathogen/ARG reduction and production of an agriculturally useful digestate. This will be supplemented by sampling digestate corresponding to feedstock samples in (1) above, with process conditions recorded.3. Pathogen/ARG persistence in amended soilsThe student will establish field experiments in which digestate will be applied to experimental grass/crop plots in a factorial experiment also incorporating livestock manure/slurry and an inorganic fertiliser as treatments, alongside untreated control plots. Plots will be sampled over the period of a year and pathogens/ARGs, soil nutrients/physico-chemical characteristics and soil microbiome analyses will be undertaken.Throughout, PCR/multiplex PCR and culture will be used for sample screening for pathogens/ARGs followed by q-PCR for quantification.