Primary production needs to transition towards more sustainable systems that reduce environmental impact, mitigate climate change, and ensure healthy food production with limited use of chemical plant protection products, fertilizers, or antibiotics. Rationally designed microbial communities, or engineered microbial consortia, involve the intentional assembly of microorganisms that can underpin more sustainable primary production systems. Rationally designed microbial communities can for example, (i) enhance ecosystem resilience, (ii) improve bioremediation, (iii) enhance industrial processes, or (iv) prevent diseases. In the perspective, we discuss the route towards market applications with a focus on the methodology needed to rationally design microbial communities for applications in the agri-food production systems. Often in silico and in vitro approaches are considered as a continuous process that first consider the in silico genomic and then in vitro condition to develop microbial consortia. However, host-microbe interactions influence both the microbial community assembly and host phenotypes and need to be considered from an early stage when developing microbial communities. As such, we propose that the route towards market application(s) should, from a technical perspective include (i) the host of interest, (ii) a library of both slow and fast-growing species, and (iii) genomic information about functions present in the selected microbial consortia.

Fresh leafy greens like lettuce can be consumed raw and are susceptible to foodborne pathogens if they become contaminated. Recently, the number of reported pathogenic foodborne outbreaks related to leafy greens has increased. Therefore, it is important to try to alleviate the human health burden associated with these outbreaks. Processing of fresh-cut lettuce, including washing, is a step in the supply chain that needs to be well controlled to avoid cross-contamination. Current measures to control the quality of lettuce during washing include the use of chemicals like chlorine; however, questions regarding the safety of chlorine have prompted research for alternative solutions with peracetic acid (PAA). This study evaluates the effectiveness of a PAA (c.a. 75 mg/L) solution on the reduction of a commensal E. coli strain during the washing of fresh-cut lettuce. Experiments were performed at the laboratory scale and validated at the industrial scale. We observed that the use of PAA was not adversely affected by the organic load in the water. The contact time and dose of the PAA showed to be relevant factors, as observed by the approximately 5-log reduction of E. coli in the water. Results showed that once introduced during washing, E. coli remained attached to the lettuce, thus supporting the need to control for pathogenic bacteria earlier in the supply chain (e.g., during primary production) as well as during washing. Moreover, our results showed that the use of PAA during washing did not have an apparent effect on the levels of fluorescent pseudomonads (FP) and total heterotrophic bacteria (THB) in lettuce. Overall, our results at the laboratory and industrial scales confirmed that during the processing of fresh-cut produce, where the accumulation of soil, debris, and other plant exudates can negatively affect washing, the use of a PAA (c.a. 75 mg/L) solution was an effective and safe wash water disinfectant that can potentially be used at the industrial scale.

Controlling water quality is critical in preventing cross-contamination during fresh produce washing. Process wash water (PWW) quality can be controlled by implementing chemical disinfection strategies. The aim of this study was to evaluate the pilot-scale efficacy of chlorine dioxide (ClO2) during processing on the reduction of Escherichia coli in the PWW and on processed fresh-cut 'Lollo Rossa' lettuce. The objective was to have a residual target concentration of either 5 or 3 mg/L ClO2 in the washing tank (3.5 m(3)) before and during 800 kg of lettuce processing (90 min). After 90 min., a nonpathogenic, non-Extended Spectrum Beta-Lactamase (ESBL) E. coli inoculum from an overnight culture broth (37 degrees C) was added to the tank resulting in an approximate final level of 10(6) CFU/mL. PWW and lettuce samples for microbiological and chemical analyses were taken before and after the input and supply halted. ClO2 concentrations quickly decreased after ClO2 input halted, yet a residual concentration of >= 2.5 mg/L and >= 2.1 mg/L ClO2, respectively for 5 and 3 mg/L pilots, was present 12 min after the supply halted. No detectable levels of E. coli (limit of detection 5 log) were determined in the water within 1 min after E. coli was added to the ClO2 containing wash water. Results demonstrated that ClO2 use at the semi-commercial pilot scale was able to reduce the E. coli peak contamination in the PWW. After storage (5 days, 4 degrees C), background microbial communities (i.e., fluorescent Pseudomonads and total heterotrophic bacteria) grew out on lettuce. Overall, ClO2 decreased the potential for cross-contamination between batches compared to when no sanitizer was used. Chlorate levels of the lettuce sampled before entering the wash water ranged from 7.3-11.6 mu g/kg. The chlorate levels of the lettuce sampled after being washed in the ClO2 containing wash water, as well as after rinsing and centrifugation, ranged from 22.8-60.4 mu g/kg; chlorite levels ranged from 1.3-1.6 mg/kg, while perchlorate levels were below the limit of quantification (LOQ, <5 ng/g). In this study, we report the semi-commercial pilot-scale evaluation of ClO2, for its ability to maintain the PWW quality and to prevent cross-contamination in the washing tank during fresh-cut lettuce processing. Furthermore, we provide quantitative values of ClO2 disinfection by-products chlorate and chlorite as well as of perchlorate from PWW and/or lettuce samples.

It is important to understand the underlying factors that lead to shifts in soil microbial communities, not simply for the characterization of these complex biotic systems, but also to understand the impact that changes in microbial community composition may have on terrestrial ecosystems. In this study, the impact of genetically different potato (Solanum tuberosum) plants growing in arable soil on the soil bacterial community composition was investigated. It was hypothesized that the modifications in the tuber starch content of these plants might lead to changes in root exudation, resulting in changes in bacterial composition near the roots. In a crop rotation schedule, including potato-barley-potato, plants of different potato cultivars were grown, including one genetically modified (GM) line that was modified in its tuber starch content. Thus, to establish the normal operating range (NOR) of soil bacterial community make-up in the production system, the variability of the bacterial communities in soil under the subsequent cropping regimes was determined. The results showed that the effect of the GM potato line on soil bacteria fitted within the NOR. The bacterial community structure of the potato rhizosphere samples was mainly affected by plant growth stage. Besides, clone library and PCR-DGGE analyses indicated that the bulk soil community fluctuated over the three growth seasons, concomitant with the crop rotation and fertilization regime. Some bacterial species found in the bulk soil were specific for the plant species under investigation (barley and potato) or plant growth stage. Arthrobacter, Streptomyces, Rhodanobacter and Dokdonella were only found at flowering potato plants in both seasons, whereas Rhodoplanes and Sporosarcina were only observed in the bulk soil with barley.

Washing fresh produce with potable water helps to remove microorganisms, providing about a 1- to 2-log reduction, but this process can also pose an opportunity for cross-contamination of bacteria in the washing tank. The objective of this study was to evaluate the efficacy of three chemical sanitizers, sodium hypochlorite, chlorine dioxide, and a silver-copper solution on the reduction of S. Typhimurium and extended-spectrum beta-lactamase (ESBL) E. coli as well as to evaluate the impact bacterial cell history and water quality had on sanitizer efficacy. This was investigated with three scenarios representing different contamination routes and history of cells: (i) on starved and non-starved cells in potable water, (ii) on starved and non-starved cells in lettuce wash water and on lettuce leaf punches, and (iii) on non-starved cells in organically loaded process wash water (PWW). Sodium hypochlorite (NaClO) and chlorine dioxide (ClO2) were more effective in preventing cross-contamination in the potable water than the silver-copper solution. Starved and non-starved bacterial cells displayed minor differences in their susceptibility to sanitizing agents in the (i) potable water and (ii) lettuce wash water demonstrating that other conditions greater influenced sanitizer efficacy. Particularly, the organic load of the water, wash water temperature, and pathogen attachment and release from the produce were shown to affect a sanitizer's efficacy during washing. Furthermore, results emphasize that chemical sanitizer use should focus more on wash water disinfection, rather than produce decontamination, to prevent pathogenic cross-contamination during processing. Future research should investigate the feasibility of ClO2 application during pilot-scale processing.

Under suboptimal environmental conditions such as low temperatures, many bacteria have an extended lag phase, altered cell structures, and composition such as a less fluid (more rigid) and leaky cytoplasmic membrane. As a result, cells may die, enter into a starvation mode of metabolism or a physiologically viable but non-culturable (VBNC) state. In the latter state, the amount of gene expression per cell is virtually undetectable. In this article, gene expression under (suboptimal) low temperature conditions in non-psychrophilic environmental bacteria is examined. The pros and cons of some of the molecular methodologies for gene expression analysis are also discussed.

Roots are the primary site of interaction between plants and microorganisms. To meet food demands in changing climates, improved yields and stress resistance are increasingly important, stimulating efforts to identify factors that affect plant productivity. The role of bacterial endophytes that reside inside plants remains largely unexplored, because analysis of their specific functions is impeded by difficulties in cultivating most prokaryotes. Here, we present the first metagenomic approach to analyze an endophytic bacterial community resident inside roots of rice, one of the most important staple foods. Metagenome sequences were obtained from endophyte cells extracted from roots of field-grown plants. Putative functions were deduced from protein domains or similarity analyses of protein-encoding gene fragments, and allowed insights into the capacities of endophyte cells. This allowed us to predict traits and metabolic processes important for the endophytic lifestyle, suggesting that the endorhizosphere is an exclusive microhabitat requiring numerous adaptations. Prominent features included flagella, plant-polymer-degrading enzymes, protein secretion systems, iron acquisition and storage, quorum sensing, and detoxification of reactive oxygen species. Surprisingly, endophytes might be involved in the entire nitrogen cycle, as protein domains involved in N2-fixation, denitrification, and nitrification were detected and selected genes expressed. Our data suggest a high potential of the endophyte community for plant-growth promotion, improvement of plant stress resistance, biocontrol against pathogens, and bioremediation, regardless of their culturability.

It is important to understand the underlying factors that lead to shifts in soil microbial communities, not simply for the characterization of these complex biotic systems, but also to understand the impact that changes in microbial community composition may have on terrestrial ecosystems. In this study, the impact of genetically different potato (Solanum tuberosum) plants growing in arable soil on the soil bacterial community composition was investigated. It was hypothesized that the modifications in the tuber starch content of these plants might lead to changes in root exudation, resulting in changes in bacterial composition near the roots. In a crop rotation schedule, including potato-barley-potato, plants of different potato cultivars were grown, including one genetically modified (GM) line that was modified in its tuber starch content. Thus, to establish the normal operating range (NOR) of soil bacterial community make-up in the production system, the variability of the bacterial communities in soil under the subsequent cropping regimes was determined. The results showed that the effect of the GM potato line on soil bacteria fitted within the NOR. The bacterial community structure of the potato rhizosphere samples was mainly affected by plant growth stage. Besides, clone library and PCR-DGGE analyses indicated that the bulk soil community fluctuated over the three growth seasons, concomitant with the crop rotation and fertilization regime. Some bacterial species found in the bulk soil were specific for the plant species under investigation (barley and potato) or plant growth stage. Arthrobacter, Streptomyces, Rhodanobacter and Dokdonella were only found at flowering potato plants in both seasons, whereas Rhodoplanes and Sporosarcina were only observed in the bulk soil with barley.

A. Sessitsch,1 P. Hardoim,2,3 J. Döring,4 A. Weilharter,1 A. Krause,4 T. Woyke,5 B. Mitter,1 L. Hauberg-Lotte,4 F. Friedrich,4 M. Rahalkar,4 T. Hurek,4 A. Sarkar,4 L. Bodrossy,1 L. van Overbeek,3 D. Brar,6 J. D. van Elsas,2 and B. Reinhold-Hurek4 AIT Austrian Institute of Technology GmbH, Bioresources Unit, Tulln, Austria; University of Groningen, Department of Microbial Ecology, Haren, The Netherlands; Plant Research International, 6708PB Wageningen, The Netherlands; University of Bremen, Department of Microbe-Plant Interactions, Bremen, Germany; Department of Energy (DOE) Joint Genome Institute, Walnut Creek, CA, U.S.A.; International Rice Research Center (IRRI), Los Baños, Philippines

AIMS:The survival capability of pathogens like Escherichia coli O157:H7 in manure-amended soil is considered to be an important factor for the likelihood of crop contamination. The aim of this study was to reveal the effects of the diversity and composition of soil bacterial community structure on the survival time (ttd) and stability (irregularity, defined as the intensity of irregular dynamic changes in a population over time) of an introduced E. coli O157:H7 gfp-strain were investigated for 36 different soils by means of bacterial PCR-DGGE fingerprints. METHODS AND RESULTS:Bacterial PCR-DGGE fingerprints made with DNA extracts from the different soils using bacterial 16S-rRNA-gene-based primers were grouped by cluster analysis into two clusters consisting of six and 29 soils and one single soil at a cross-correlation level of 16% among samples per cluster. Average irregularity values for E. coli O157:H7 survival in the same soils differed significantly between clusters (P = 0.05), whereas no significant difference was found for the corresponding average ttd values (P = 0.20). The irregularity was higher for cluster 1, which consisted primarily of soils that had received liquid manure and artificial fertilizer and had a significant higher bacterial diversity and evenness values (P < 0.001). CONCLUSIONS:Bacterial PCR-DGGE fingerprints of 36 manure-amended soils revealed two clusters which differed significantly in the stability (irregularity) of E. coli O157 decline. The cluster with the higher irregularity was characterized by higher bacterial diversity and evenness. SIGNIFICANCE AND IMPACT OF THE STUDY:The consequence of a high temporal irregularity is a lower accuracy of predictions of population behaviour, which results in higher levels of uncertainty associated with the estimates of model parameters when modelling the behaviour of E. coli O157:H7 in the framework of risk assessments. Soil community structure parameters like species diversity and evenness can be indicative for the reliability of predictive models describing the fate of pathogens in (agricultural) soil ecosystems.