[WATER] Competitive exclusion as a means to reduce E. coli regrowth in digested sludge

将污泥回收用于农业被认为是欧洲最实际的环境选择。然而，由于担心病原体转移到农产品的风险是一个主要问题，实践中存在许多挑战。近年来，有报道称，离心后的大肠杆菌数量显著增加。这被称为大肠杆菌再生现象，令人严重关注，因为大肠杆菌被用作污泥衍生产品的微生物质量的指标，如果不符合质量标准，将导致更高的处置成本和消费者对该行业的信心丧失。有人提出，经过离心脱水的污泥中大肠杆菌数量的明显突然增加是由活的但不可培养的细胞重新激活引起的。随后在脱水后的蛋糕储存的最初几天内，大肠杆菌的快速增长很可能是由于营养物质的现成供应和缺乏微生物对这些营养物质的竞争所致。这项研究中要检验的假设是，通过应用竞争排斥原理，即通过大肠杆菌与将被引入脱水污泥中的快速生长的非致病细菌之间的竞争，可以减少储存污泥中的大肠杆菌的再生长。多年来，竞争排斥原则作为一种感染控制手段在食品工业中得到了成功的应用。在这项研究中，我们的目标是开发不同微生物群落组成的接种剂，并评价它们抑制消化污泥中大肠杆菌生长的能力。目的1：检验竞争排斥概念为检验研究的基本前提，将设计一个可控、可重复的台式实验，对污泥样品进行灭菌，然后再接种大肠杆菌和竞争微生物。在这项实验中，大肠杆菌将受到不同程度竞争可用资源的接种物的挑战。在大肠杆菌没有竞争或竞争有限的地方，我们预计种群数量会增加。在大肠杆菌不得不与多样化和适应良好的社区争夺可用资源的情况下，我们预计人口增长将放缓或消失。目标2：优化竞争排除产品一旦竞争排除概念被成功测试，竞争排除产品将被优化。我们将调查：(I)挑战接种的最佳来源是什么--例如，来自土壤或来自消化的蛋糕本身。(Ii)相互竞争的人口需要多大？种子数量是否足以与大肠杆菌竞争？或者，竞争排除产品是否需要达到一定的规模才能竞争？(3)竞争性排除产品的微生物群落结构的关键特征有多重要。将通过磷脂脂肪酸(PLFA)表型分析来测量微生物群落的结构。目标3：一旦在小试规模上得到证实，培养工作将使用中试规模的发酵罐进行放大。如果这项试点工作的结果是有希望的，将有机会在联合公用事业公司的设施中全面试验这一过程。

The recycling of sludge to agriculture is regarded as the Best Practical Environmental Option in Europe. However, there are many challenges with the practice with concern about the risk of pathogen transfer to farm produce being a major issue. In recent years, it has been reported that numbers of Escherichia coli (E. coli) increase significantly following centrifugation. This is known as the E. coli regrowth phenomenon and it is of serious concern because E. coli is used as an indicator of the microbiological quality of sludge-derived products and compliance failure of the quality standards would result in higher disposal costs and loss of consumer confidence in the industry. It has been proposed that the apparent sudden increase in E. coli numbers in sludge subject to centrifugal dewatering is caused by reactivation of viable but nonculturable cells. Subsequent rapid growth of E. coli in the first few days of cake storage following dewatering is likely to be caused by the combination of a ready supply of nutrients and a lack of microbial competition for those nutrients. The hypothesis to be tested in this research is that the regrowth of E coli in stored sludge can be reduced by the application of the competitive exclusion principle; i.e. by competition between E. coli and fast-growing non-pathogenic bacteria that will be introduced to the dewatered sludge. The principle of competitive exclusion has been used successfully as a means of infection control for many years in the food industry. In this research we aim to develop inocula of differing microbial community composition and evaluate their ability to suppress the growth of E. coli in digested sludge. Objective 1: Test the competitive exclusion concept To test the basic premise of the research, a controlled and replicated bench-scale experiment will be designed in which sludge samples are sterilised and then re-inoculated with E. coli and competing microorganisms. In this experiment the E. coli will be challenged with inocula which present differing degrees of competition for available resources. Where the E. coli are presented with no or limited competition we would expect an increase in population. Where E.coli have to compete with a diverse and well adapted community for available resources we would expect population growth to be slower or absent. Objective 2: Optimise the competitive exclusion product Once the competitive exclusion concept has been successfully tested a competitive exclusion product will be optimised. We will investigate: (i) What is the best source of the challenge inoculum - for example from soils or from digested cake itself. (ii) How big does the competing population need to be? Is a seed population sufficient to compete with E. coli? Or is it necessary for the competitive exclusion product to reach a certain size before it is capable of competing? (iii) How important are key characteristics of microbial community structure of the competitive exclusion product. Measurements of the structure of the microbial community will be made by means of phospholipid fatty acid (PLFA) phenotypic profiling. Objective 3: Scale-up Once proven on bench scale, the culture work will be scaled up using a pilot-scale fermenter. Should the results of this pilot-scale work be promising there will be the opportunity to trial the process at full scale at a United Utilities facility.