The maximum acceptable levels of B. cereus in food vary slightly between countries, but in general concentrations between 3 and 5 log10 CFU g− 1 are considered satisfactory and above 5 log10 CFU g− 1 unsatisfactory . Another important factor to consider/include in our study is that after the nitrification process, preceding the introduction of the biofertilizer in the hydroponic growth system, the levels of B. cereus was monitored to 1 log10 CFU/mL indicating that this process might lower the initial high concentration to acceptable levels. The continuous presence of low levels of B. cereus throughout the challenge tests however indicates that the biofertilizer has a capacity to act as a reservoir for B. cereus spores and this is a critical factor to consider in each risk assessment for this product matrix. The bacteria’s ability to form spores provides an explanation as to how it can be present after hygienization and anaerobic digestion of the biofertilizer, and also to how it can be steadily present in the biofertilizer in the challenge test experiments despite a large amount of the inoculation dying off after a very short time after incubation. The fact that S. enterica and L. monocytogenes do not establish themselves, even seemingly dying off within 48 h after incubation in the biofertilizer, implies that the biofertilizer constitutes a highly inhospitable environment for these food-borne pathogens. In the case of still having viable but non culturable cells, a calorimetric measurement where the biofertilizer was inoculated with the pathogenic bacteria was performed in parallel with the selective plating. As well as the non-supplemented control samples in Section 3.1, these samples showed no signs of microbial activity , which corresponds to the results from the selective plating. The apparent lack of nutrients supporting microbial growth in the biofertilizer could be hindering the establishment of these pathogenic bacteria. This is supported by the findings in the accelerated microbial activity assessments in Section 3.1 where growth was only obtained after supplementation of BHI.
This is also in line with a recentstudy by Fern´ andez-Domínguez et al. who reported that non-biodegradable compounds increased largely after anaerobic digestion. Besides the lack of available nutrients, the chemical composition of the biofertilizer could exert an additional inhibitory effect on the establishment and survival of the food-borne pathogens. The pH of the biofertilizer was measured initially and was between 5.8 and 6.1, thus the pH of the biofertilizer should not be hindering the establishment of the bacteria. A previous chemical analysis of the presence of PPCP’s in the biofertilizer , shows that the samples collected from local Swedish biogas production plants may contain considerable levels,nft system exceeding 100 ng g− 1 of fenbendazole, however this compound is not reported to have any antimicrobial activity . Antimicrobial agent sulfaclozine was detected in low concentration, which might very well have an impact on the establishment of the pathogens. Theobromine, an antimicrobial bitter compound found in cocoa was detected in levels of μg g− 1 , and caffeine was found in similar levels, which also might have an effect in hindering the establishment of the pathogens. Fungicides propiconazole, fludioxonil and imazalil were detected in considerable amounts, ranging from 100 to 900 ng g− 1 . It is possible the presence of these compounds and/or other inhibitory compounds produced by methanogens during anaerobic digestion in the biogas production plants, in combination with the apparent lack of nutrients, make the biofertilizer a non-growth-supporting environment for the food-borne pathogens to survive. Previous studies on the microbial community of anaerobic digestates reveal that the results vary widely depending on the composition and treatment of ingoing substrate, conditions of digestion, and variable region chosen to be sequenced, however, most studies conclude the most dominant phyla to be Firmicutes, Bacteroidetes, and Proteobacteria . The general focus in sequencing of biofertilizers in previous studies has been on plant-growth promoting microbes, with less focus on risks regarding human health in the utilization of biofertilizer for food production. The purpose of this study was thus to consider the information from the microbial community analysis from a food safety perspective. The library preparation of the 16S rRNA gene amplicon sequencing yielded low amounts of DNA for all samples except for the samples of non-nitrified biofertilizer and the starting sample of nitrified biofertilizer .
A negative control was also included in the library preparation. The 25 most abundant genera across all samples can be observed in Fig. 4A. If no genus level classification could be obtained, the lowest assigned taxonomic classification was given. In addition, the phylum level classification was given . The microbial communities of the samples were also analysed with multivariate statistical analysis , demonstrating the similarity in microbial community between the samples, as can be observed in Fig. 4B. The 16S rRNA gene amplicon sequencing revealed that the most abundant genus within all the samples of nitrified biofertilizer from the hydroponic channels was Mycobacterium . This genus could not be detected in the samples of nonnitrified biofertilizer or the inorganic fertilizer. Since it is also present in the Day 0 sample , a transfer or contamination from the plant roots to the biofertilizer can be ruled out. Seeing that the nitrified and non-nitrified biofertilizer differ so drastically in the microbial community composition, as can be observed in the principle component analysis, it is apparent that the nitrification process, including lowering of the pH from 8 to 5.5, aeration, and changes in the composition of nitrogen compounds, affects the microflora present. Mycobacterium are known to be hardy bacteria that have acidic tolerance and resistance to disinfection, and can survive and grow in a low organic carbon environment.Since it is not detected in the non-nitrified biofertilizer it indicates that it emerges in the biofertilizer in some step after nitrification, however a count of 0 in relative abundance might not mean that the genus is absent, but that it is below the limit of detection . Regardless, finding Mycobacterium in a sample such as the biofertilizer is not unreasonable as they have been found to be common in cattle manure and in water distribution systems, and can survive there for long periods of time . It is also not such a surprising result to find this genus in the nitrified biofertilizer considering that species within the genus are denitrifying bacteria, and can rely on nitrate as an energy source during anaerobic conditions . While Mycobacterium tuberculosis and Mycobacterium leprae are well-known pathogenic mycobacteria, several species of environmental mycobacteria can also be opportunistic human pathogens , and a further investigation of species level would be interesting in theaspect of ensuring microbiological safety in the utilization of this biofertilizer for production of food.
In the non-nitrified biofertilizer the most abundant genera from the16S rRNA gene amplicon sequencing were Pseudomonas, Leuconostoc and Sporosarcina. Pseudomonas and Sporosarcina are naturally found in soil , and Leuconostoc is normally found widespread throughout the environment, in fermented foods and in plant matter.The microflora of the nitrified biofertilizer also varied between the samples taken at the same timepoint but from different channels; in comparison the samples of the inorganic fertilizer are much more clustered and vary less between samples. This behaviour can be connected to the variance shown between samples from the viable count analysis in Section 3.1, where the microflora differed in different replicates of the same kind of sample, and also from the discovery that some members of the microbial community exert antagonistic behaviour towards others. In the inorganic fertilizer, the most abundant genera were Lactobacillus, Enterococcus, Serratia, and Pseudomonas. The high relative abundance of OTUs detected in the negative control in the samples is a result of the low DNA yield of the samples , rendering the sequenced background more prominent. It was concluded that these genera cannot be distinguished to originate from the sample or the sequenced background. The genera can originate in the sequenced background as the ingredients of the PCR reaction of the sequencing may contain bacterial DNA, which is a common occurrence. . It was furthermore not expected to have high yields of DNA in the inorganic fertilizer. In the inorganic fertilizer the most abundant genera were Lactobacillus, although this was also the most abundant in the negative control and is believed to be sequenced background, Enterococcus, also present in negative control but in generally lower abundances, Serratia, also present in negative control, and Pseudomonas present in negative control but in very low relative abundances. The low DNA yield from the biofertilizer samples was a quite unanticipated result as the biofertilizer was expected to have a rich microflora as a result of the anaerobic digestion. If the nitrification process that the biofertilizer undergoes was the culprit for the reduction in natural microflora, it would at least have been expected to find some genera of nitrifying bacteria in the 16S rRNA gene amplicon analysis. As this was not the case one explanation is that the apparent lack of factors for growth, as is fortified by the findings in the microbial activity assessment of the biofertilizer in Section 3.1, has simply reduced the types of microorganisms that can survive to very hardy bacteria such as Mycobacterium or spore formers that can endure in the low-carbon environment that the biofertilizer constitutes. In a study of the microbial community of soil, hydroponic gutter it is found that the low DNA yield is in fact a result of poor growth rather than an inadequate DNA extraction .
It is also reported that low DNA content might introduce bias in 16S rRNA gene amplicon sequencing analysis, which is an important parameter to keep in mind when drawing conclusions regarding the composition of the microbial community . Food security has been one of the main concerns of the countries that depend on imported food, for instance, the Middle East countries like Qatar and United Arab Emirates. Full dependence on fresh food supplies from other cities or overseas has negative effects on food security. Shortages of fresh vegetables in some cities during the Covid-19 lockdown period have forced the government to reconsider its sources of fresh vegetables. Hydroponic systems are recognized as being among the main technical approaches to providing sustainable food and reducing pressure on agricultural land by shifting food production to urban environments. For instance, the state of Qatar imported approximately 90% of its food until 2017 and has been encouraging the firms to develop hydroponic farms to satisfy the country’s food demand. Hydroponic systems provide water-efficient food production but they are not an energy-efficient solution. This is because they require electricity for heating and cooling, ventilation, irrigation, LED lighting, and other horticultural practices to maintain the hydroponic farm operations in controlled environments. The huge energy consumption of the hydroponic system not only leads to an increase in operating costs but also environmental pollution. The industries are required to reduce the carbon emissions to achieve the 2 ◦C global warming target by adopting clean energy. In order to enhance sustainability when addressing the aforementioned challenges, governments must seek innovative solutions to achieving sustainable supplies of fresh food and energy supply. The photovoltaics presents a promising technology that can provide a portion of the clean energy needed to meet the huge electricity requirements of hydroponic farms. Fig. 1 illustrates a hydroponic system with solar energy generation. First, a timer is set such that irrigation starts at specific time intervals. At the appropriate time, the system starts performing irrigation using a reservoir that contains nutrient-rich water. The water-nutrient solution is pumped into the bottom of the growth tray where plant roots are held. The plant roots absorb some of the water-nutrient solutions and the rest is returned to the reservoir, where it can be used in the next irrigation process. Therefore, there is a recirculating system. These hydroponic systems are intended for use in closed buildings that are automatically irrigated, ventilated, cooled and heated, and illuminated. The energy required to run the various crop illumination equipment is supplied through a dual system in which regular grid energy is supplemented by solar power generation.