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

厌氧消化（AD）利用有机物质通过沼气产生能量，同时也产生营养丰富的消化物，是土地施肥的理想选择。然而，通过病原体传播到土地和进入食物链、直接与环境接触或流入水道，可能对人类（和牲畜）的健康构成风险。对抗生素耐药细菌（ARBs）进入环境的担忧加剧了这种情况。抗性基因（ARGs）可以在生物反应器和土壤微生物组内广泛转移，包括向病原体转移和从病原体转移。令人担忧的病原体包括梭状芽孢杆菌，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AD流程，以最大限度地降低与AD在陆地上的应用相关的风险。由于缺乏数据，无法对病原体通过AD进入环境的风险进行评估，而关于在不影响气体生成的情况下操纵AD减少病原体和抗性基因的信息也很少。此外，对于原料如何根据其病原体或ARG含量进行排名，以及一旦应用于土地，病原体/ARG的命运如何，也几乎没有明确的规定。这种理解是至关重要的，因为不需要高温巴氏消毒的能量输入的过程优化和消化后消毒（这在某些情况下也不实际，例如小规模系统，发展中国家）可以使通过AD发电更具可持续性和更广泛的可行性。该项目旨在：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)比较传统有机废物改性土壤和消化不良改性土壤中病原体/ARG持久性的差异。这些目标将通过一系列实验和基于文献的步骤来解决：该学生将进行文献综述，包括对AD不同原料中病原体/ARG的存在和浓度的综合数据。这将辅以实验工作，在实验工作中，学生将对来自一系列消化器的原料中的一套病原体/ARGs进行取样和枚举，导师与这些消化器有现有的联系。2. 学生将建立实验室规模的消化器，通过该消化器进行实验，以在文献综述中确定的行业相关操作范围内操纵工艺条件（例如加载率/保留时间、含水量、温度），并在上述(1)中与现场操作员进行讨论。这些将利用机会仔细控制原料和条件，以确定最佳的操作包络，以实现气体产量和纯度、病原体/ARG减少和农业有用消化液的生产。这将由与上述(1)中原料样品相对应的消化液取样补充，并记录工艺条件。病原体/ARG在改良土壤中的持续性学生将建立田间实验，在一个因子实验中将消化液应用于实验草地/作物地块，同时结合畜禽粪便/泥浆和无机肥料作为处理，以及未经处理的对照地块。将在一年的时间内对地块进行采样，并进行病原体/ARGs、土壤养分/理化特征和土壤微生物组分析。在整个过程中，PCR/多重PCR和培养将用于病原体/ARGs的样本筛选，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.