Biomonitoring of silage microbial community dynamics under the influence of microbial strains application

. Silage is one of the most common feedstuffs used in modern dairy and beef farms, but silage quality is often measured without assessing the presence of pathogens. However, poorly prepared or contaminated silage can contain pathogens that reduce animal performance, cause bovine disease, and pose a threat to human health. The aim of this study was to analyse changes in the diversity of the silage microbial community during the ensiling process and to evaluate the effectiveness of two strains of promising microorganisms for use as inoculants. As a result of the study, it was shown that epiphytic microbiota has less potential to rapidly create and, most importantly, maintain high acidity conditions. The highest proportion of lactic acid bacteria was found in the variants with the introduction of strains of Lactobacillus plantarum and Bacillus subtilis at almost all stages of ensiling, indicating that the ensiling process was proceeding correctly in these variants. The lowest number of bacteria belonging to the Enterobacteriaceae family was found in the variant with the L. plantarum strain. Some microorganisms were detected in the silage, including mammalian pathogens: representatives of the genera Staphylococcus, Erysipelothrix, and Fusobacterium.


Introduction
Silage of vegetable raw materials is the main biological method of their preservation, based on spontaneous lactic acid fermentation under anaerobic conditions, which has been used for thousands of years to preserve roughage.Normally, preservation is achieved by a rapid decrease in the pH of the medium, which is achieved with the help of lactic acid bacteria.Epiphytic lactic acid bacteria utilise the simple carbohydrates in silage plants and metabolise them to lactic acid and, to a lesser extent, acetic acid, which prevents silage spoilage and allows it to be stored for a long time.Silage production is important in countries with harsh winter seasons, where animals cannot obtain the energy or nutrients, they need from grazing all year round, and in regions with humid climates where it is difficult to make and store hay (Pahlow et al., 2003).When silage processes are functioning properly, a high quality and safe forage for animals is obtained.However, when the processes are disturbed, when the acidification of the medium is not fast enough, pathogenic and opportunistic microorganisms begin to multiply (Vilar et al., 2007;Pedroso et al., 2010;Driehuis et al., 2018).
Silage is one of the most common feedstuffs used in modern dairy and beef farms, but silage quality is often measured without assessing the presence of pathogens and toxins.However, poorly prepared or contaminated silage can contain pathogens (Vilar et al., 2007) that reduce animal performance (Driehuis, 2013), cause bovine diseases (Pedroso et al., 2010) and pose a threat to human health (Ogunade et al., 2016;Driehuis et al., 2018).This is due in part to the fact that the silage ecosystem is artificially created, continuously changing and subjected to anthropogenic press, making it a unique micro-bioecological niche.However, under man-made conditions, regulatory links are weakened and thus, both uncontrolled reproduction of some members of natural microbiota communities and the decline of others are possible (Paola et al., 2011;Li, Driehuis, 2013;Muck, 2018).
Three main phases of fermentation processes can be distinguished in the process of plant material silage succession: aerobic, basic fermentation and quiescence (Merry et al., 1997).In the aerobic fermentation phase, biochemical changes occurring in the stored substrate arise not only due to the activity of microorganisms, but also as a result of the action of plant enzymes.During respiration, which occurs in freshly cut substrate under the action of plant enzymes, most of the energy of oxidised monosaccharides is converted to heat.
When atmospheric oxygen is displaced from the silage substrate, a low redox potential (conditions close to anaerobic) is created, which initiates the beginning of the second phase of silage succession -the phase of basic fermentation.This phase lasts from 1 to 4 weeks, depending on the forage crop genotype and specific silage conditions.In the second phase of silage succession, cardinal shifts in the structure of the microbiocenosis occur: lactic acid bacteria begin to play a dominant role if the technology of planting and storing plant substrate is followed.According to Merry et al. (1997), the increase in the rate of lactic acid bacteria multiplication closely correlates with the rate of pH decrease and lactic acid accumulation.At this stage of silage succession, some facultative and obligate anaerobic microorganisms, such as Enterobacteriaceae, clostridia, bacilli and yeasts, are in certain cases able to compete with lactic acid bacteria for nutrients released from collapsing plant cells and tissues.
The third, final phase (resting phase) is conditioned by low values of pH, redox potential, and reduction of water-soluble carbohydrates in the silage ecosystem.At this stage of silage succession, there is a decrease in lactic acid bacteria in logarithmic progression.Researchers (Buxton et al., 2003) record the presence of some amounts of acid-tolerant yeast species in a weakly active state, as well as endospores of bacilli and clostridia.
At this stage, the processes of change in the physico-chemical composition and nutritional value of the plant substrate are minimised.Theoretically, provided there is no contact of the preserved silage substrate with atmospheric oxygen, the duration of this phase may tend to infinity.
When the storage facility is opened, atmospheric oxygen penetrates the array of preserved substrate in quantities sufficient to initiate the growth of undesirable microorganisms and, as a consequence, secondary aerobic fermentation.Yeasts and microscopic fungi are most commonly responsible for the occurrence of aerobic instability.An increase in the number of these microorganisms favours a sharp increase in the temperature of the silage substrate and provokes a decrease in the concentration of lactic acid, an increase in pH, and a significant decrease in nutritional value.
The dominance of lactic acid bacteria in the phases of basic fermentation and quiescence, if the technology of planting and storage of plant substrate is observed, is associated with their resistance to pH levels up to 3.0-3.5,which makes them extremely competitive in silage ecosystem conditions.
The composition and abundance of epiphytic bacterial microflora on silage plant material are insufficient for the initiation of lactic acid formation by the lactic acid bacteria present.Natural populations of lactic acid bacteria on plant material are often heterofermentative and sparse (Pahlow et al., 2003).To obtain silages of good quality and high digestibility, it is often necessary to stimulate the ensiling process by the addition of various preparations.In practice, the use of preservatives is recommended, especially for ensiling green forages with low mono-, di-and oligosaccharide content and high protein content and buffer capacity.Preservative application is also recommended in cloudy and rainy weather and for ensiling plants grown on intensively cultivated soils.Expected changes in the ensiling process using microbial additives containing lactic acid bacteria include predominance of these microorganisms in the fermentation process, increased ratio of lactic acid to other fermentation products (e.g.acetic acid, ethanol), faster pH reduction, reduced proteolysis and increased dry matter recovery (Pahlow et al., 2003).
One of the representatives of undesirable microflora that in some cases develop in the silage ecosystem are clostridia.They are Gram-positive microorganisms capable of forming endospores.Most species are obligate anaerobes, but are able to increase their numbers in the presence of atmospheric oxygen at low concentrations.
The activities of proteolytic clostridia produce significant amounts of ammonia, amines, a mixture of organic acids and other products such as alcohols and CO2 in silage (McPherson and Violante, 1966).Acids formed in silage include acetic, propionic, butyric, isobutyric, 2-methyl and 3-methyl butyric acids.The above processes are inevitably associated with losses of feed flavour values ( Van Os, Dulphy, 1996).
Other representatives of undesirable microflora are Enterobacteria family Enterobacteriaceae.These are Gram-negative, often motile organisms.Possession of catalases assigns them to the physiological grouping of facultative anaerobes.Their development in the substrate is strictly subordinate to the presence of carbohydrates (Muck, 2010).The process of glucose fermentation by Enterobacteriaceae occurs with the formation of a mixture of organic acids, primarily acetic acid, but also formic acid, alcohol and in some cases, butanediol.By fermenting carbohydrates, Enterobacteriaceae thereby compete for food sources with lactic acid bacteria and counteract the lowering of pH levels (Pahlow et al., 2003).They are also able to carry out decarboxylation of amino acids.The number of Enterobacteriaceae is a marker indicating the quality level of silage.
Silage starters using microbial strains are the solution to these problems.Additives are not always necessary for good fermentation, but they are particularly useful to enhance fermentation of crops with high buffering capacity, low concentration of water-soluble carbohydrates or high moisture concentration.Additives containing lactic acid bacteria (L.plantarum, Pediococcus acidilacti, P. pentosaceous and Enterococcus faecium) enhance homolactic fermentation by suppressing unwanted bacteria, increasing acidification rates or dominating flora (Kung et al., 2003;Nishino et al., 2007).
The objective of this study was to analyse the change in the diversity of the silage microbial community during the ensiling process and to evaluate the efficacy of two microbial strains promising for use as inoculants.

Methods and Materials
We conducted a model ensilaging of plant matter of hedgehogs of the first harvest, cut in the Leningrad region in the phase of tube emergence at a moisture content of 84.7%±1.3.
The duration of the experiment was 30 days.In the experiment there was a control group and two experimental groups, two bacterial strains were added during ensiling.In the first experimental group -Lactobacillus plantarum strain and in the second -Bacillus subtilis strain.The microorganisms in the silage from harvested hedgerow silage of different storage periods were counted during the ensiling time: 3, 7, 14 and 30 days.The composition of microorganisms was determined using high-throughput sequencing on the Illumina MiSeq platform.DNA isolation of the silage microbial community was performed using the Genomic DNA Purification Kit (Fermentas, Inc., Lithuania) according to the attached instructions.
Amplification for subsequent NGS sequencing was performed using a Verity DNA Amplifier (Life Technologies, Inc., USA) using eubacterial primers (IDT), 343F (5'-CTCCTACTACGGRRSGCAGCAG-3') and 806R (5'-GGACTACNVGGGTWTCTAAT-3'), flanking the V1V3 region of the 16S rRNA gene.Metagenomic sequencing was performed on a MiSeq genomic sequencer (Illumina, Inc., USA) with MiSeq Reagent Kit v3 (Illumina, Inc., USA).The maximum length of the sequences obtained was 2 x 300 nt.Chimeric sequences were excluded from the analysis using the programme "USEARCH 7.0" (http://drive5.com/usearch/).Processing of the obtained 2 x 300 nt reids was performed using the bioinformatics platform "CLC Bio GW 7.0" (Qiagen, The Netherlands) and included overlapping, quality filtering (QV>15), and primer trimming.Taxonomic affiliation of microorganisms to the genus was determined using the RDP Classifier programme.Statistical processing of data and construction of diagrams were carried out using Microsoft Excel and Past.

Results and Discussion
As a result of the study using the NGS sequencing method, it was shown (Fig. 1) how different are the microbial silage communities obtained using microorganism strains that create more favourable conditions for the development of lactic acid bacteria and the production of high-quality and safe silage.depending on the introduction of microbial strains.The analysis showed that the composition of silage microbiocenosis was characterised by a rather rich generic diversity contrary to traditional (Lin et al., 1992) ideas.About 70 genera of bacteria were identified in the epiphytic microflora of D. glomerata, and about 100 genera in silage, depending on the stage of the technological process of silage production and additives.
The composition of epiphytic microflora of D. glomerata included mainly representatives of the phylum Proteobacteria (94.08%): bacteria of the family Enterobacteriaceae, genus Pseudomonas, and order Burkholderiales.At the same time, bacteria of the family Enterobacteriaceae were dominated by bacteria of the genus Serratia and Pantoea (Fig. 2).
In the structure of silage microbiological community in the process of ripening, representatives of the phylum Firmicutes dominated (up to 91.54%), in which the main share was made by lactic acid bacteria of the order Lactobacillales (up to 91.21%).The highest proportion of Lactobacillales was found in the variants with the introduction of L. plantarum and B. subtilis strains at almost all stages of ensiling, indicating that the ensiling process in these variants proceeded correctly.
On the 3rd-7th day of storage, representatives of the genus Enterococcus predominated in the control variant without the introduction of strains, and bacteria of the genus Lactococcus predominated in the variant with the introduction of microbial populations.The exception is the variant with the introduction of L. plantarum on the 3rd day of ensiling, where representatives of the genus Lactobacillus were dominant.Probably, on the 3rd day of ensiling there was active multiplication of the introduced strain, which indirectly indicates a fast acidification rate and the expected favourable outcome of ensiling in this variant.On 14-30 days of ensiling, representatives of the genus Lactobacillus were dominant in almost all variants.The number of bacteria of the Enterobacteriaceae family in the silage microbial community ranged from 0.01 to 26.49%, whereas in the composition of the epiphytic microflora of hedgehog assemblage their content was much higher -67.58%.It is known that this family includes many species pathogenic to mammals.In the microbioecosystem of hedgerow silage, in addition to non-culturable representatives, 15 genera of bacteria of the Enterobacteriaceae family were identified, including the genera Klebsiella, Salmonella, Yersinia, etc., among which there are dangerous pathogens of various diseases of mammals.Enterobacteriaceae deaminate and decarboxylate amino acids in silages and reduce NO 3 , thereby increasing the production of ammonia and biogenic amines (Pahlow et al., 2003).Enterobacteriaceae also compete with lactic acid bacteria for nutrients during fermentation (Pahlow et al.,.2003).However, their growth and viability are reduced when pH is lowered (Heron et al., 1993).
In the initial phases (3-7 days) of the ensiling process, in all variants of the experiment, the content of bacteria of the Enterobacteriaceae family was the highest -20.95-56.63%, in the second phase of ensiling (14-30 days) their number decreased to 4.23-32.22%.This is probably due to the fact that the lower threshold for the development of bacteria of the Enterobacteriaceae family is pH 4.5.The lowest number of bacteria belonging to this taxon was found in the variant with the introduction of L. plantarum strain.The fact of presence of bacteria of the Enterobacteriaceae family in silage has been noted by a number of researchers for quite a long time.
Pathogenic strains of E. coli can persist during silage making when the acidification rate is low (Ogunade et al., 2017).Chen et al. (2005) used an E. coli strain that was labelled with a green fluorescent protein and was resistant to kanamycin to inoculate wheat and maize forage and reported that the strain survived longer in silage from wilted wheat because the pH decreased more slowly than in silage from un-wilted straight-cut wheat.Ogunade et al, (2016) demonstrated that compared to untreated samples, inoculation of alfalfa with Lactobacillus plantarum or L. buchneri increased the rate of pH reduction, resulting in earlier inhibition (7 days versus 16 days) and eventual elimination of the pathogenic E. coli strain O157:H7 that was added during ensiling.The slow rate of pH decline in the control alfalfa silage was attributed to high buffering capacity, low concentration of water-soluble carbohydrates, or both.In a similar trial using corn silage, which has a much lower buffering capacity than alfalfa, the pH dropped below 4.0 within 3 days of fermentation and the pathogen was removed from silage that was or was not inoculated with L. plantarum or L. buchneri (Ogunade et al., 2017).
Silage quality is degraded by the presence of Clostridium spp.and when consumed, such silage jeopardises animal health and productivity.Minimising its occurrence reduces economic and animal welfare risks (Goldsztejn et al., 2020).Non-pathogenic bacteria of the genus Clostridium were detected in silage samples in negligible amounts.
Minor (less than 1%) taxa in silage were representatives of the family Bacillaceae, phylum Actinobacteria, genus Pseudomonas and order Burkholderiales.Whereas in the composition of epiphytic microflora, representatives of the genus Pseudomonas and order Burkholderiales were found in significant numbers: 23.4 and 3.02%, respectively.Probably, low redox potential and decreased pH level had an inhibitory effect on these microorganisms.
In addition, as the results of our studies showed, symbionts of higher plants -bacteria of the order Rhizobiales -capable of degrading a wide range of compounds: proteins, carbohydrates, etc., are able to survive in silage.The survival of these microorganisms in silage is not surprising since it is known that some strains of bacteria of the order Rhizobiales can show resistance to low pH values (Somasegaran and Hoben, 1994).Previously, Ni et al. (Ni et al., 2016) also detected bacteria of the order Rhizobiales under silage succession conditions, which was not possible using classical microbiological methods.In addition, the researchers found that the epiphytic microorganisms Agrobacterium sp., Methylobacterium sp. and Sphingomonas sp.continued to be viable in the silage.
In addition, some genotypes of unidentified bacteria were detected in silage, as well as microorganisms, among which mammalian pathogens are often found: representatives of the genera Staphylococcus, Erysipelothrix and Fusobacterium.It should be noted that a representative of the genus Fusobacterium is the bacterium F. necrophorum, a dangerous pathogen of mammals.F. necrophorum is a dangerous pathogen of ruminants, causing necrobacteriosis of rumen mucosa, hoof tissues, liver damage, endometritis, and mastitis (Josefsen et al., 2018).These microorganisms are resistant to the low rumen pH values typical of cows consuming concentrate-rich diets.A case of an infectious disease outbreak associated with supplementary feeding of semi-domesticated reindeer with silage contaminated with Fusobacterium spp.has been described (Tryland et al., 2019).Some representatives of the genus Erysipelothrix are causative agents of rye and pose a threat to humans.These microorganisms are known to be able to remain viable in frozen animal tissues for several months (Upapan, 2015).

Conclusions
Preventing pathogen contamination of silage requires the identification of critical control points that are associated with contamination and multiplication of silage pathogens.Contamination of silage with pathogens can occur before, during, or after ensiling, and it is essential that adequate control measures are used at each of these stages to prevent contamination.Methods to promote rapid homolactic fermentation should be used before and during ensiling, as rapid pH reduction is critical to inhibit Clostridium spp.and Enterobacteriaceae, which cause proteolysis and secondary oil fermentation, as well as the development of pathogenic microbiota.As a result of this study, it was shown that the epiphytic microbiota has less potential to rapidly establish and, more importantly, maintain high acidity conditions.The highest proportion of lactic acid bacteria was found in the variants with the introduction of strains of L. plantarum and B. subtilis in almost all stages of ensilation, indicating that the ensilation process was proceeding correctly in these variants.The lowest number of bacteria belonging to the Enterobacteriaceae family was found in the variant with the introduction of the L. plantarum strain.Thus, the communities of microbial silage obtained with the help of microorganism strains created more favourable conditions for the development of lactic acid bacteria and, consequently, for the production of high quality and safe silage.

Fig. 1 .
Fig. 1.Scatter plot showing the similarity between samples, comparing the relative content of all taxa observed in the silage samples Calculation of biodiversity indices showed that on 3 and 7 days of silage storage the structure of silage microbiocoenosis was most homogeneous on 14 and 30 days of storage in variants with L. plantarum and B. subtilis introduction.The structure of bacterial community of silage from D. glomerata changed significantly in the process of successional changes occurring during silage maturation, as well as