The isolation of rumen enterococci strains along with high potential utilizing cyanide

Cyanogenic glycosides in forage species and the possibility of cyanide (CN) poisoning can have undesirable effects on ruminants. The literature estimates that unknown rumen bacteria with rhodanese activity are key factors in the animal detoxification of cyanogenic glycosides, as they are capable of transforming CN into the less toxic thiocyanate. Therefore, identifying these bacteria will enhance our understanding of how to improve animal health with this natural CN detoxification process. In this study, a rhodanese activity screening assay revealed 6 of 44 candidate rumen bacterial strains isolated from domestic buffalo, dairy cattle, and beef cattle, each with a different colony morphology. These strains were identified as belonging to the species Enterococcus faecium and E. gallinarum by 16S ribosomal DNA sequence analysis. A CN-thiocyanate transformation assay showed that the thiocyanate formation capacity of the strains after a 12 h incubation ranged from 4.42 to 25.49 mg hydrogen CN equivalent/L. In addition, thiocyanate degradation resulted in the production of ammonia nitrogen and acetic acid in different strains. This study showed that certain strains of enterococci substantially contribute to CN metabolism in ruminants. Our results may serve as a starting point for research aimed at improving ruminant production systems in relation to CN metabolism.

Characteristics of rumen CN-detoxifying bacteria.Scanning electron microscopy showed a cell diameter of 1.0-µm for the six enterococci strains (Fig. 3).Biochemical testing revealed that the six enterococci strains were Gram-positive, catalase-negative, starch hydrolysis-negative, and incapable of producing hydrogen sulfide gas (Table 1).They were all facultative anaerobic and fermented sugars (ribose, glucose, lactose, and fructose), as well as amylose.In Lactobacilli de Man, Rogosa, Sharpe (MRS) broth, they all produced lactic acid Rhodanese activity screening in rumen enterococci strains.SCN − , thiocyanate anion; error bar, standard deviation.In this rhodanese assay, the crude enzyme samples (n = 3) from each strain were extracted and incubated with the reaction mixture containing KCN.The negative control group (n = 3) consisted of rhodanese samples that were boiled to inactivate their activity.The rhodanese activity of the isolated strains ranged from 4.35 to 6.60 µmol of SCN -production/min/mg protein.
(from 0.11 to 1.98 mmol/L) and acetic acid (from 5.34 to 7.61 mmol/L).The KKU-BF7 strain even produced a small amount of propionic acid.No strain produced butyric acid or valeric acid in this medium.Ammonia nitrogen production ranged from 0.38 to 3.91 mg/L.Figure 4 shows growth at different pH, temperatures, and CN substrate concentrations in MRS broth after incubation for 24 h.The strains grew well at pH 6.0-9.0 and temperature of 30-40 °C.The KKU-BF7 and KKU-BC8 strains were more resistant to alkaline conditions than the other six strains (p < 0.001).The KKU-BC15 strain grew well at a high KCN concentration (400 mg/L; p < 0.001).
Capacity of strains to detoxify CN. Figure 5a shows the CN-thiocyanate transformation capacities of the strains.This assay was performed without (as a baseline) and with bacteria in 80 mg HCN equivalent/L; the baseline condition showed the amount of CN substrate that remained in the medium.At 0 h of incubation (the starting point), the CN values between baseline and bacteria were similar.At the 12 h endpoint of the CNthiocyanate transformation assay, the estimated amounts of thiocyanate formed by bacteria ranged from 4.42 to 25.49 mg HCN equivalent/L, with the KKU-BF7 strain outperforming the others (p < 0.001).

Discussion
The genus Enterococcus is ubiquitous in various habitats and currently comprises 38 species 18 .In this study, phylogenic trees of 16S rRNA gene sequences indicated that the rumen isolates are closely related to the species E. faecium and E. gallinarum with bootstrap values ranging from 92 to 99% (Fig. 2a,b).It has previously been shown that the abundance of enterococci in the rumen is low, with ≤ 10 5 colony forming units/mL 19 .Examples of species that can be isolated from rumen are E. casseliflavus, E. gallinarum, E. faecium, E. cecorum, E. mundtii, and E. faecalis [19][20][21][22] .Regarding the rumen ecosystem and fermentation process, an in vitro study demonstrated that the addition of E. faecium can effectively support the function of lactate-utilizing bacteria, thereby promoting the proliferation of rumen microbes and enhancing the synthesis of propionic acid 23 .Some strains of enterococci species can produce beneficial enzymes 24 , bacteriocin and enterocin 25 , and vitamin B12 26 .The rumen enterococci This is the first study to report the CN-utilizing capacity of enterococci species in the rumen.The main CNmetabolizing properties of the isolated strains were determined based on the rhodanese activity of both crude enzyme extracts (Fig. 1) and whole cells (Fig. 5a).The enzyme facilitates the transfer of a sulfur atom from a donor molecule to an acceptor molecule 12 .In the Kyoto Encyclopedia of Genes and Genomes database (https:// www.genome.jp/ entry/ R01931), sulfur metabolism uses thiosulfate and the CN ion to produce sulfite and thiocyanate.The capacity of microorganisms to metabolize CN using rhodanese has been observed in several strains of fungi and bacteria from diverse environments including Fusarium sp., Azotobacter vinelandii, Bacillus brevis, Escherichia coli, Pseudomonas aeruginosa, and Thiobacillus sp. 12 .However, rumen bacteria represent more than 31 genus groups 21 , with approximately 10 10 -10 11 cells/mL and over 200 species, most of which are uncultured using standard techniques 27 .Approximately 80% of ruminal microbiota cannot be cultivated 27 , which naturally requires specific growth factors from the ecosystem of the rumen.Therefore, rumen bacterial species that can metabolize CN are not limited to only certain strains of enterococci.In a non-rumen environment, Gardner and Rawlings 28 found that the levels of rhodanese activity obtained from a combination of bacteria cells present in bio-oxidation plants were 2-2.5 times higher than the activity observed in pure bacteria cultures.The range of rhodanese activity in the current study is in accordance with previous findings 28 .In addition, our results showed that the KKU-BF7 strain can produce a small amount of propionic acid (Table 1).Kim et al. 29 suggested that some strains of E. faecium produce fumarate reductase, which reduces methanogenesis by producing propionic acid with fumarate conversion to succinate.
The results indicated that the bacteria grew within a pH range of 6-9 and a temperature range of 30-40 °C (Fig. 4).This finding suggests that the bacteria can be cultivated under a range of optimal conditions.However, the results showed that only some strains were resistant to a high CN concentration.These findings are similar to those of Prachumchai et al. 16 , which revealed that the numbers of rumen CN-utilizing bacteria decreased by 450 mg KCN/L.The results suggested that the KKU-BF7 strain was generally more active in CN-thiocyanate transformation than the five other strains (Fig. 5a).The ability of enterococci to degrade CN substrates was recently reported by López-Ramírez et al. 30 based on the E. hirae KU175874 strain, which was isolated from gold processing plants.CN detoxification by microbes involves either degradation, transformation, or both biological processes with enzymes including cyanidase, CN dioxygenase, CN monooxygenase, nitrogenase, rhodanese, mercaptopyruvate sulfurtransferase, cyanide hydratase, CN dihydratase, nitrilase, nitrile hydratase, thiocyanate hydrolase, cyanoalanine synthase, and γ-cyano-α-aminobutyric acid synthase 12 .Thiocyanate degradation occurs in many microbial species 31 , which might use the β-carbonic anhydrase family of enzymes 32 , producing sulfur, carbon, and nitrogen substances 12 .According to the relative stability of CN compounds, the CN-thiocyanate transformation assay used in this study could cause distinct losses due to the volatilization of free CN species   the six strains at each pH, temperature, or CN substrate concentration was analyzed using one-way ANOVA with three replicates.No strain grew at a pH of 4.0, and the optimal pH range for most strains was 6-9.At a pH of 10.0, abundant growth of KKU-BF7 and KKU-BC8 strains was observed compared with the other four strains (p < 0.001).The bacteria did not grow at 4 °C and 60 °C.The optimal temperature range for most strains was 30-40 °C, and KKU-DC6 and KKU-BC2 strains had an OD > 1.5, which was greater than any other strain (p < 0.001).The results showed that the susceptibility of the KKU-BC15 strain to CN was significantly lower than that of all other strains at high levels of CN (400 mg/L) (p < 0.001).
and biological reactions from the simultaneous presence of the thiocyanate form of CN.Therefore, thiocyanatedegrading pathways can also be tested as additional processes in these bacteria.The results of this study showed that the degradation resulted in ammonia nitrogen and acetic acid production (Fig. 5b).The KKU-BC2 strain produced acetic acid in association with thiocyanate degradation, likely through an acetogenesis pathway.Many enterococci-specific strains, isolated from various environments, have undergone extensive testing to assess their advantageous impacts on the livestock production system, including animals indirectly or directly fed bacteria 23,33 .Based on the findings of this study, future studies should test the therapeutic effects of the KKU-BF7 strain on ruminants receiving CN-containing plants.Compared with an antidote, successful probiotics depend on the synchronization of cyanogenesis and thiocyanate formation, which are fairly complex with several biological factors, especially the type and concentration of cyanogenic glycosides, cyanogenesis rate, mechanism of probiotics, and probiotic growth factors.Majak and Cheng 9 demonstrated that CN levels can peak in the rumen approximately 30 min after consuming plants containing cyanogenic glycosides.The symptoms of CN poisoning in cattle can occur within hours or days 4 .Therefore, there is a possibility that high cyanogenesis under higher ruminal pH conditions or naturally high activity of microbial glucosidase will prevail over all other detoxifying mechanisms 9,10 .To restore optimal health conditions, rumen bacteria and host tissue possess distinct abilities to detoxify CN.A significant population of bacteria can be estimated to facilitate efficient detoxification processes In this assay, a cell-free rumen fluid medium containing KCN substrate was utilized to evaluate the ability of each strain to produce thiocyanate from the CN source.The fermentation process involved analyzing the CN compounds (KCN and thiocyanate) in the medium using a distillation method, and the results are reported in mg of HCN equivalent/L.The assimilation of thiocyanate within the cytoplasm of bacterial cells is thought to occur through their rhodanese activity.To differentiate thiocyanate from the remaining KCN substrate, a baseline reference (negative control without bacteria or a rhodanese source) was used during the incubation period.Compared with bacterial fermentation, the baseline KCN substrate releases free CN (HCN and CN − ) instead of thiocyanate, as it possesses hydrolytic abilities in the medium.In (a), the total CN concentration in the medium was measured at 3, 6, 12, 24, and 48 h of incubation for each strain (n = 3) and baseline (n = 3).After 12 h of incubation, the baseline (plotted in the red line) showed a remarkably low concentration of CN from an initial concentration of 80 mg HCN equivalent/L.This suggests that there was minimal interference from the remaining KCN substrate on thiocyanate production after the strains' rhodanese activity.The statistical difference among the six strains at each time of incubation was analyzed using one-way ANOVA with three replicates.The results indicated that the KKU-BF7 strain exhibited the highest capacity for CN-thiocyanate transformation, with a minimum of 25.49 mg HCN equivalent/L during the 12 h incubation period.This value was significantly greater than that of all other strains (p < 0.001).In (b) the fermentation end-products from each strain at 12, 24, and 48 h of incubation were observed to analyze their correlation with the thiocyanate values using Pearson's correlation coefficient.The results indicate a significant association between the degradation of thiocyanate and the production of ammonia nitrogen (p < 0.05) and/or acetic acid (p < 0.05) in certain strains.

Time post incubation (h)
within the rumen 16 .On the other hand, host tissue utilizes different pathways to detoxify and eliminate CN from the body 13 .Aminlari and Gilanpour 13 reported that the epithelium of rumen, omasum, and reticulum are the richest sources of rhodanese in sheep and cattle.Thus, while rumen bacteria contribute significantly to CN utilization, host tissues also play a role in overall detoxification processes.
In conclusion, this is the first study to demonstrate that specific strains of enterococci play a role in the process of detoxifying CN in the rumen by increasing the synthesis of thiocyanate.Different strains of enterococci degrade thiocyanate differently, with some producing ammonia nitrogen and others producing acetic acid.These results will help fill a significant research gap in the field of nutritional ecology of ruminants.The use of bacteria as a probiotic therapeutic for ruminants receiving CN-containing plants requires more studies.

Methods.
All experiments were approved by the Animal Care and Use Committee of Khon Kaen University (Khon Kaen, Thailand), based on the Ethics of Animal Experimentation of the National Research Council, Thailand (Record No. IACUC-KKU-45/64).All analytical methods were carried out in accordance with relevant guidelines and regulations.The study was carried out in compliance with the ARRIVE guidelines.All reagents were of high purity grade.All KCN solutions were prepared with sterile deionized water and used immediately.
Animal care and rumen fluid collection.The rumen fluid strains were obtained from mature non-pregnant female swamp buffalo (Bubalus bubalis), beef cattle (Thai-native × Brahman, Bos indicus), and Holstein dairy cows (Bos taurus) at Khon Kaen University in November 2020.Animals grazed on grasslands mainly containing Guinea grass, Ruzi grass, Signal grass, Verano, Leucaena, and Centro.In these grasslands, the donor animals were inadvertently subjected to small amounts of CN due to the presence of particular forage species and weeds known to generate CN such as Signal grass, Ruzi grass, and Giant sensitive plant.According to the findings of Euswas et al. 34 , the grasses contain less than 200 ppm CN, which is within safe limits for grazing.Napier grass, rice straw, and mineral bricks were supplemented in the barns.In addition, a concentrate consisting of cassava chips, rice bran, oil palm meal, soybean meal, cornmeal, urea, sulfur, salt, and premixed vitamins was provided daily at about 0.5-1.0%body weight.The animals were given free access to drinking water.Two heads of fresh rumen fluid (200 mL/head) per animal species were collected using a stomach tube device before their morning feeding.The rumen fluid samples from each animal species were pooled, filtered through four layers of sheet cloth into a prewarmed thermos (39 °C), and immediately transported to the laboratory.
Isolation, purification, and preparation of rumen-derived bacteria.The desired bacteria were enriched using a closed fermenter system (DURAN ® 250 mL glass screwcap bottle; Merck KGaA, Darmstadt, Germany) supplemented with KCN solution 35 .The incubation temperature was set at 39 °C with shaking at 120 rpm/min.The rumen fluid (10 mL) was first enriched for 4 days in nutrient broth (90 mL) and supplemented with 1 mL of a 10 g/L KCN solution.Then the enriched strains (10 mL) were inoculated into new nutrient broth (90 mL) supplemented with 1 mL of 20 g/L KCN solution and fermented for 4 days.This inoculation of nutrient broth with enriched bacteria was repeated three times, each time with a higher concentration of KCN solution (30-50 g/L).Next, the enriched strain (10 mL) was transferred to a mineral-glucose medium (90 mL) supplemented with 1 mL of 10 g/L KCN solution and fermented for 4 days.This step was repeated twice, each time with a higher concentration of KCN solution (20-30 g/L).
The final strain enrichment from the mineral-glucose medium (1 mL) was serially diluted with 9 mL NaCl solution (8.5 g/L) from 10 0 to 10 −5 to isolate 50-100 colonies/plate.An aliquot of each dilution (100 µL) was spread on three plates of nutrient agar and anaerobically incubated at 39 °C for 48 h (Memmert IF160; Memmert GmbH + Co. KG, Schwabach, Germany).The isolates were identified using stereomicroscopy at 40 × magnification and distinguished by colony appearance.The strain numbers were counted within animal species (buffalo had 8 strains, beef cattle had 21 strains, and dairy cows had 15 strains).The strains were purified twice by streaking on nutrient agar, and cryopreserved at − 80 °C.
For use in assays, the strains were defrosted at 4 °C and inoculated in nutrient broth (100 mL) supplemented with 1 mL KCN solution (10 g/L).Other than a 48 h incubation, the culturing and isolation conditions were the same.Each 30 mL of regrowth bacteria was centrifuged for 5 min at 4200g and 4 °C, washed twice, and resuspended in 10 mL of NaCl solution.The absorbance of the final pellet was adjusted to an optical density at 600 nm (OD 600 ) of 1.0 using a spectrophotometer (UV/VIS Spectrometer; PG Instruments Ltd., London, UK).At OD 600 = 1.0, the bacterial cell density in this study was 10 8 colony forming units/mL.

Rhodanese activity screening.
The rhodanese activity present in each strain was screened in triplicate using a modified rhodanese assay as previously described 28,36 .The strain sample (10 mL at OD 600 = 1.0) was pipetted into a 15 mL screwcap tube.After centrifugation for 10 min at 4200g and 4 °C, the supernatant was discarded and the cell pellet was resuspended in 10 mL TE-NaCl buffer, pH 7.6 28 .The sample underwent three sonication cycles on ice (60 s with 30 s cooling intervals, Transonic Digital S; Elma Schmidbauer GmbH, Singen, Germany).After centrifugation for 10 min at 4200g and 4 °C, the crude bacterial enzyme was pipetted (2.0 mL) into a 15 mL screwcap tube containing a reaction mixture (1.0 mL of 67 mM K 2 HPO 4 and 1 mL of 100 mM Na 2 S 2 O 3 in 200 g/L NaCl solvent).Then the sample was incubated at 39 °C for 10 min, supplemented Figure1.Rhodanese activity screening in rumen enterococci strains.SCN − , thiocyanate anion; error bar, standard deviation.In this rhodanese assay, the crude enzyme samples (n = 3) from each strain were extracted and incubated with the reaction mixture containing KCN.The negative control group (n = 3) consisted of rhodanese samples that were boiled to inactivate their activity.The rhodanese activity of the isolated strains ranged from 4.35 to 6.60 µmol of SCN -production/min/mg protein.

Figure 2 .
Figure 2. Phylogenetic trees of E. faecium (a) and E. gallinarum (b) type strains based on 16S rRNA gene sequences.The strains studied in our experiment are marked (red circle) and referred to the NCBI accession number.The nucleotide sequences were used to construct the highest log likelihood trees (− 14,901.92for E. faecium and − 11,697.18for E. gallinarum) with Lactobacillus plantarum WCFS1 (KC429782) as an outgroup.The trees were constructed using MEGA X, employing the Maximum Likelihood method and Tamura-Nei model, with 10,000 bootstrap iterations.The percentage of trees in which the associated taxa clustered together is shown next to the branches.Bars indicated sequence divergences.

Figure 3 .
Figure 3. Scanning electron microscopy (SEM) at 150,000× magnifications of rumen enterococci strains.The image displays the cell morphology of pure strains.These bacteria have an ovoid shape with a cell diameter of approximately 1.0 µm.

Figure 4 .
Figure 4. Growth at different pH, temperatures, and CN substrate concentrations in MRS broth after incubation for 24 h.Bar, standard error of the mean; ***, p < 0.001; ns, p > 0.05.The statistical difference among the six strains at each pH, temperature, or CN substrate concentration was analyzed using one-way ANOVA with three replicates.No strain grew at a pH of 4.0, and the optimal pH range for most strains was 6-9.At a pH of 10.0, abundant growth of KKU-BF7 and KKU-BC8 strains was observed compared with the other four strains (p < 0.001).The bacteria did not grow at 4 °C and 60 °C.The optimal temperature range for most strains was 30-40 °C, and KKU-DC6 and KKU-BC2 strains had an OD > 1.5, which was greater than any other strain (p < 0.001).The results showed that the susceptibility of the KKU-BC15 strain to CN was significantly lower than that of all other strains at high levels of CN (400 mg/L) (p < 0.001).

Figure 5 .
Figure 5. CN-thiocyanate transformation assay of rumen enterococci strains using an in vitro rumen fermentation technique.(a) Total CN concentration during the 48 h fermentation.Error bar, standard error of the mean; **, p < 0.01; ***, p < 0.001.(b) Pearson's correlation heatmap of thiocyanate concentration and fermentation end-products for 12-48 h of incubation.The color scheme indicates the strength of correlation of thiocyanate degradation and fermentation end-products in each strain (n = 9).*, p < 0.05; OD 600 , optical density at 600 nm; VFAs, volatile fatty acids.In this assay, a cell-free rumen fluid medium containing KCN substrate was utilized to evaluate the ability of each strain to produce thiocyanate from the CN source.The fermentation process involved analyzing the CN compounds (KCN and thiocyanate) in the medium using a distillation method, and the results are reported in mg of HCN equivalent/L.The assimilation of thiocyanate within the cytoplasm of bacterial cells is thought to occur through their rhodanese activity.To differentiate thiocyanate from the remaining KCN substrate, a baseline reference (negative control without bacteria or a rhodanese source) was used during the incubation period.Compared with bacterial fermentation, the baseline KCN substrate releases free CN (HCN and CN − ) instead of thiocyanate, as it possesses hydrolytic abilities in the medium.In (a), the total CN concentration in the medium was measured at 3, 6, 12, 24, and 48 h of incubation for each strain (n = 3) and baseline (n = 3).After 12 h of incubation, the baseline (plotted in the red line) showed a remarkably low concentration of CN from an initial concentration of 80 mg HCN equivalent/L.This suggests that there was minimal interference from the remaining KCN substrate on thiocyanate production after the strains' rhodanese activity.The statistical difference among the six strains at each time of incubation was analyzed using one-way ANOVA with three replicates.The results indicated that the KKU-BF7 strain exhibited the highest capacity for CN-thiocyanate transformation, with a minimum of 25.49 mg HCN equivalent/L during the 12 h incubation period.This value was significantly greater than that of all other strains (p < 0.001).In (b) the fermentation end-products from each strain at 12, 24, and 48 h of incubation were observed to analyze their correlation with the thiocyanate values using Pearson's correlation coefficient.The results indicate a significant association between the degradation of thiocyanate and the production of ammonia nitrogen (p < 0.05) and/or acetic acid (p < 0.05) in certain strains.

Table 1 .
Characterization of rumen enterococci strains.OD 600 , optical density at 600 nm; − , negative test; + , positive test; np, not produced.Three analytical repetitions of each measurement were done for each strain.