Rumen Bacteria Abundance and Fermentation Proﬁle during Subacute Ruminal Acidosis and Its Modulation by Aspergillus oryzae Culture in RUSITEC System

: This study aimed at characterizing changes in rumen bacteria abundance and fermentation proﬁles by artiﬁcial saliva (AS) pH, and at evaluating the Actinobacteria ( p = 0.002) compared to the high AS pH and decreased the abundances of phylum Spirochaetes ( p = 0.032). Compared with the high AS pH, low AS pH increased the abundances of Prevotella ( p = 0.003), Pseudoscardovia ( p = 0.001), Mitsuokella ( p = 0.005), and Dialister ( p = 0.047), and decreased the abundances of Olivibacter ( p = 0.026), Ruminobacter ( p = 0.025), Treponema ( p = 0.037), and Sphaerochaeta ( p = 0.027) at genus level. Under a severe SARA in RUSITEC, supplementation of 2.5% AOC increased OM degradability, the copy numbers of Selenomonas ruminantium and Fibrobacter succinogenes . These ﬁndings indicate that the reduction AS pH at 5.5 caused a strong shift in bacterial composition in rumen. In addition, the addition of AOC in diets increased the growth rate of certain rumen bacteria that digest ﬁber or utilize lactate under SARA condition in RUSITEC system.


Introduction
Subacute rumen acidosis (SARA) has a negative impact in the dairy industry by decreasing day matter intake (DMI), milk production, profitability, and increasing culling rate and death loss [1]. An overall reduction in rumen pH below 5.6 for more than 3 h per day had been defined as SARA for dairy cows [2]. In recent years, major efforts in characterizing the rumen microbial composition and function have been made, along with studying the factors that affect rumen microbiota. Studies have shown that the SARA decreased the richness, diversity, and functionality of microbiota in rumen, but the results vary greatly among studies [3][4][5]. Studies suggested SARA effects are caused by rumen pH and dietary type [6,7]. However, the effect of diet types on rumen bacteria community was different from that of rumen pH. Li et al. demonstrated that there are three groups of bacteria communities in the rumen, including pH-sensitive but substrate insensitive bacteria, pH-insensitive but substrate sensitive, and bacteria that are both pHand substrate-sensitive [5]. It is difficult to test this hypothesis in vivo because of the differences in DMI and the passage rate of rumen content for ruminants varied intake and forage to concentrate diet. The induced-SARA, by reducing the buffer captivity in vitro, could avoid these differences.
Rumen pH is the most known abiotic factor that influences rumen bacteria community, particularly through inhibition pH-sensitive cellulolytic bacteria [5]. Studies on dairy goats, dairy cows, and beef steers indicated that the count of cellulolytic bacteria decreased when animals experienced SARA [4,8]. Nevertheless, some essential factors (e.g., vitamins, additive) can also facilitate and enhance functionality of specific rumen bacteria [9]. The AOC is one of several fungal products and has been widely used as a feed additive in ruminant production [10,11]. The AOC has richer multiple fiber enzymes rather than the single enzymes secreted by the other additive (e.g., xylanase, cellulase). Studies demonstrated that AOC could regulate the rumen bacteria [12] and increase fiber digestion [13]. Earlier studies have investigated the effect of AOC on the degradation of common feeds through rumen fermentation [14,15]. Supplementation of AOC also influences the metabolism of rumen bacteria. For example, adding AOC in diets increased the number of Selenomonas ruminantium and Fibrobacter succinogenes in rumen bacteria in vitro [16]. However, there is little information about the effects of supplementation of AOC on fermentation profiles and the rumen bacteria community under a severe SARA.
Considering that such severe models of SARA were difficult to establish in vivo conditions, due to ethical and health issues, the RUSITEC system was deemed appropriate for such long-term studies [17,18]. Therefore, this study aimed at characterizing changes in rumen bacteria abundance and fermentation profiles by AS pH, and to evaluate a potential modulatory role of AOC in RUSITEC system.

Animals and Procedures
The study was carried out using RUSITEC fermenters (Sanshin, Tokyo, Japan) as described by Kajikawa et al. and Zhao et al. [19,20]. The RUSITEC system included eight fermenters with an effective volume of 800 mL. The inoculum was obtained from four rumen fistulated Hu lambs that received two meals in the form of totally mixed ration (TMR) pellets at 0700 and 1900 h daily. Rumen contents were obtained from the rumen fistula before feeding in the morning, strained through 4 layers of surgical gauze, and separated into liquid and solid fractions. On the first day, each vessel was inoculated with 400 mL of liquid inoculum under CO 2 flux and mixed with 400 mL of AS. Then, two bags (100 µm pore) were deposited in the fermenters, one with feed and the other with solid inoculum (70 g wet weight). After 24 h, solid inoculum bag was substituted with a new feed bag. Subsequently, the feed bag incubated 48 h was substituted with a new feed bag. The fermenters were deposited at 39 • C waters and infused AS at a rate of 600 mL/day.

Experimental Diets and Sampling
The treatment contained high AS pH (pH 6.8) or low AS pH (pH 5.5) following the McDougall's method (Table 1) [21]. In the low AS pH condition, the diets contained either 0% AOC, 1.25% AOC, and 2.5% AOC. Therefore, there are four treatments: (1) high AS pH, 0% AOC (HASP); (2) low AS pH, 0% AOC (AOC0); (3) low AS pH, 1.25% AOC (AOC1); (4) low AS pH, 2.5% AOC (AOC2), respectively. The treatments were randomly assigned to one of eight fermenters and two batches were fermented with an identical procedure. Each treatment had four replications. The experiment was conducted in two independent 13 days, with 9 d for adaption period and 4 d for sample collection. The diets were ground to pass through a 2 mm sieve and supplemented with 16 g of the experimental diets with forage to concentrate ratio of 40:60. The ingredients and chemicals of the experimental diets are presented in Table 2. The pH of all the fermenters was determined at 0800 h, 1400 h, and 2000 h daily throughout the experiment periods.

Sample Collection
To stop fermentation, the ice was placed around the over-flow bottle. 10 mL of rumen fluids were collected before feeding, and the pH in the fermenter was immediately measured with a mobile meter (PHB-4, Shanghai Hongyi instrument limited, Shanghai, China). Five milliliters of rumen fluid were mixed with 1 mL of metaphosphoric acid (25% wt/vol) and preserved at −20 • C to determine the VFA. On day 12 and 13, 10 mL of rumen fluid in the fermenter was collected and preserved at −80 • C for bacteria DNA extraction. On days 10, 11, and 12, the bag with residue from each fermenter was collected and washed with 100 mL of AS. Then, the bag was washed with cold water and preserved to analyze DM, OM, NDF, ADF, and CP.

Analytical Procedures
The DM, ash, and CP were measured according to the Association of Official Analytical Chemists (AOAC) [22]. The DM content was measured at 105 • C in a forced-air oven for 4 h. The ash content was determined in a muffle furnace (PrepASH-340, Precisa, Switzerland) at 550 • C for 6 h. The CP content was determined using a protein analyzer (K9840, Hanon Advanced Technology Group Co., Ltd., Jinan, China) following the method of Kjeldahl, and CP was calculated as N × 6.25. The NDF and ADF analysis were carried out as described by Van Soest et al. [23]. The starch content was determined by a commercial assay kit (Jiancheng Bioengineering Institute, Nanjing, China).
To determine the concentration of individual VFA, thawed rumen fluids were centrifuged at 2500× g for 5 min following the procedure of Liang et al. [24]. The concentrations of VFA were determined using the gas chromatography (TRACE 1300, Thermo Scientific, Milan, Italy) with a silica capillary column (DB-FFAP, 30 m × 0.32 mm × 0.25 µm, Agilent Technologies Co., Ltd., Santa Clara, CA, USA) by the method of Li et al. [25]. The 1% (wt/vol) crotonic acid was used as the internal standard. The temperatures of injector and detector were set at 240 • C. The column temperature was raised from 50 • C to 190 • C at a rate of 25 • C/min, and programmed to increase 200 • C at 10 • C/min and held for 5 min. Finally, the temperature was raised to 220 • C at 10 • C/min and held for 5 min.
The DNA of rumen bacteria was extracted according to the method of Murray et al. and Zhou et al [26,27]. The DNA concentration and purity were determined by an ND-2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The quality of DNA was determined by running aliquot on 1% agarose gel. Primers for all the rumen bacteria were selected from the published literature (Table 3). The qPCR assays were determined using a Bio-Rad CFX96 Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA), and qPCR program was as described by Liang et al. [24]. Table 3. The sequence of primers used to determine the relative abundance of bacteria.

Primer Name Primer Sequences (5 -3 ) Reference
Fibrobacter succinogenes The variable regions V3-V4 of bacterial 16S rRNA gene were amplified using specific primers (341F: 5 -ACTCCTACGGGAGGCAGCAG-3 and 806R: 5 -GGACTACHVGGGTW-TCTAAT-3 ). The primers were tagged with Illumina adapter, pad, and linker sequences. PCR enrichment was carried out in a 50 µL reaction including 30 ng template, fusion PCR primer, and PCR master mix. The PCR program was as follows: 94 • C for 3 min, followed by 30 cycles of 94 • C for 30 s, 56 • C for 45 s, 72 • C for 45 s, and a final extension at 72 • C for 10 min. The PCR products were purified using AmpureXP beads and eluted by elution buffer. Libraries were assessed using an Agilent 2100 bioanalyzer (Agilent, Palo Alto, CA, USA). The sequence was performed on the platform of lllumina MiSeq (BGI, Shenzhen, China) and generating 2 × 300 bp paired-end reads. Overlapping paired-end sequence of the DNA fragments (minimum overlap of 15 bp, maximum mismatch rate set 0.1) was combined by FLASH (Fast Length Adjustment of Short reads, v1.2.11) to get tags of hypervariable regions [31]. Then, the tags were clustered using the USEARCH software to get operational taxonomic units (OTU) (version v7.0.1090) [32]. The chimeras generated by PCR amplification were filtered by UCHIME (v4.2.40) [33]. Then, The Ribosomal Database Project (RDP) Classifier v.2.2 was used to classify the OTU representative sequences with a minimum confidence threshold of 0.6, and QIIME v1.8.0 was used to train on the Greengenes database v201305 [34]. The OTU abundance statistics table of each sample was obtained by USEARCH_global. Alpha and beta diversity were performed by MOTHUR (v1.31.2) [34] and QIIME software (v1.8.0) at the OTU level [35], respectively. Sample cluster was carried out by QIIME software (v1.8.0) based on UPGMA [34]. The LEfSe software (http://huttenhower.sph.harvard.edu/lefse/ (accessed on 22 June 2022)) was used to determine the difference of rumen bacteria among the AS pH and supplementation AOC. The species was considered as a biomarker when the LDA value was ≥4.

Statistical Analysis
The number of rumen bacteria was expressed as a proportion of total rumen bacterial 16S rRNA according to Chen et al. and Liang et al. [24,36]. Statistical analysis was performed by SPSS version 17.0 (IBM, Armonk, NY, USA). The differences between the high AS pH and low AS pH group were calculated using independent sample T-test. The effect of AOC on nutrients degradability, fermentation variables, and rumen bacteria community were used to the linear and quadratic responses of dietary inclusion levels of AOC. The multiple comparisons of treatment means were performed using Duncan's test. Significance was set at p < 0.05, and a tendency for treatment effect was observed when 0.05 < p ≤ 0.10. Pearson correlation and significance tests was used to analyze the correlation between rumen bacteria at the genus level and VFAs.

Nutrient Degradability and Fermentation Profiles
The effect of AS pH and supplementation of AOC on nutrient degradability and fermentation variables is shown in Table 4. Compared with the high AS pH, low AS pH decreased degradabilities of DM (66.6 vs. 57.6%), OM (66.0 vs. 56.2%), NDF (32.0 vs. 21.1%), ADF (25.5 vs. 15.2%), and CP (64.0 vs. 41.8%). Supplementation of AOC had no effect on the degradabilities of DM, NDF, ADF, and CP under a severe SARA in RUSITEC (p > 0.05), and the OM degradability was greater in AOC2 than that in AOC0 group (p = 0.040). The total concentration of VFA (p = 0.023) and molar proportion of propionate (p = 0.026) were decreased in the low AS pH and tended to increase the molar proportion of butyrate (p = 0.086) and the ratio of acetate to propionate (p = 0.088). The molar proportion of acetate, iso-buyrate, iso-valerate, and n-valerate were not affected by the AS pH (p > 0.05). Similarly, adding AOC in diets had no effect on the molar proportion of individual VFA (p > 0.05).

Rumen Bacteria
The results of AS pH and supplementation of AOC on the copy number of rumen bacteria is presented in Table 5. Compared with the high AS pH, low AS pH had the higher copy numbers of Selenomonas ruminantium (p < 0.001) and Prevotella brevis (p < 0.001) but decreased the copy number of Ruminococcus albus (p < 0.001). The copy numbers of Butyrivibrio fibrisolvens and Fibrobacter succinogenes were similar between the high AS pH and low AS pH treatment (p > 0.05). The AOC2 group tended to increase the mounts of Selenomonas ruminantium (p = 0.058) and Fibrobacter succinogene (p = 0.089) compared with the AOC0 group.

Alpha Diversity and Rumen Bacteria at the Phylum
The effect of AS pH and supplementation of AOC on alpha diversity and rumen bacteria at the phylum level are shown in Table 6. Low AS pH decreased the indexes of Chao1, ACE, and Shannon compared with high AS pH (p < 0.01) and had no difference on Simpson index (p = 0.389). However, adding AOC in diets had no effect on alpha diversity (p > 0.05). The abundance of Firmicutes (p = 0.065) tended to be greater in low AS pH group than high AS pH group, and the abundance of Proteobacteria tended to be lower (p = 0.063). Low AS pH also increased the abundance of Actinobacteria (p = 0.002) compared with the high AS pH, while decreased the abundance of Spirochaetes (p = 0.032). Nevertheless, the alpha diversity and rumen bacteria at the phylum level did not differ among supplementation of AOC groups under a severe SARA in RUSITEC (p > 0.05).

Beta Diversity
The beta diversity of rumen bacteria within different treatment for each fraction were calculated and visualized through the two-dimensional PCoA analysis based on weight_unifrac. A significant difference between the bacterial communities in the AS pH and supplementation of AOC treatment was noted (Figure 1). Both principal components accounted for 66.62% (PC1) and 16.07% (PC2) of the explained variance.

Rumen Bacteria at the Genus
The effect of AS pH and supplementation of AOC on rumen bacteria at the genus in RUSITEC is shown in Table 7. Compared with the high AS pH, low AS pH increased the abundances of Prevotella (p = 0.003), Pseudoscardovia (p = 0.001), Mitsuokella (p = 0.005), and Dialister (p = 0.047), and decreased the abundances of Olivibacter (p = 0.026), Ruminobacter (p = 0.025), Treponema (p = 0.037), and Sphaerochaeta (p = 0.027). Low AS pH tended to increase the abundance of Lactobacillus (p = 0.055). Under a severe SARA in RUSITEC, adding AOC in diets tended to increase the abundance of Megasphaera (p = 0.094).

Rumen Bacteria at the Genus
The effect of AS pH and supplementation of AOC on rumen bacteria at the genus in RUSITEC is shown in Table 7. Compared with the high AS pH, low AS pH increased the abundances of Prevotella (p = 0.003), Pseudoscardovia (p = 0.001), Mitsuokella (p = 0.005), and Dialister (p = 0.047), and decreased the abundances of Olivibacter (p = 0.026), Ruminobacter (p = 0.025), Treponema (p = 0.037), and Sphaerochaeta (p = 0.027). Low AS pH tended to increase the abundance of Lactobacillus (p = 0.055). Under a severe SARA in RUSITEC, adding AOC in diets tended to increase the abundance of Megasphaera (p = 0.094).

LefSe Analysis
Using LefSe analysis, we compared the difference of bacteria taxa in the addition of AOC under a severe SARA at the phylum and genus level (Figure 2). The abundances of Actinobacteria, Bifidobacteriaceae, and Bifidobacteriales were enriched in the low AS pH group compared with the high AS pH group. Under a severe SARA in RUSITEC, supplementation of 1.25% AOC in diets had a higher abundance of Prevotella, Bacteroidales, and Bacteroidla, and the abundances of Fimicutes and Megasphaera were more abundant in the addition of 2.5% AOC group.

LefSe Analysis
Using LefSe analysis, we compared the difference of bacteria taxa in the addition of AOC under a severe SARA at the phylum and genus level ( Figure 2). The abundances of Actinobacteria, Bifidobacteriaceae, and Bifidobacteriales were enriched in the low AS pH group compared with the high AS pH group. Under a severe SARA in RUSITEC, supplementation of 1.25% AOC in diets had a higher abundance of Prevotella, Bacteroidales, and Bacteroidla, and the abundances of Fimicutes and Megasphaera were more abundant in the addition of 2.5% AOC group.

Discussion
Low AS pH with reducing buffer capacity decreased the degradabilities of DM, OM, NDF, ADF, and CP, which were similar with those of Calsamiiglia et al. and Cerrato-Sánchez et al. [37,38]. The decreased degradabilites of NDF and ADF in low AS pH group were likely the result of reducing the fibrolytic population in the fermenters. Calsamiiglia et al. identified that the fibrolytic bacteria could survive transitory at low rumen pH [37]. Nevertheless, in this experiment, the rumen bacteria in the fermenters were exposed to a low pH of 5.5 for a long time, which inhibited the growth of fibrolytic bacteria and reduced their activity. In contrast, low pH can facilitate the growth and activity of amylolytic bacteria [8]. This result was consistent with our findings-that the amylolytic bacteria of Selenomonas ruminantium and Prevotella brevis were increased in the low AS pH.
We found adding AOC in diets tended to increase the copy numbers Selenomonas ruminantium and Fibrobacter succinogenes under a severe SARA in RUSITEC. Similarly, Beharka et al.
found the filtrate AO increased the growth rates of Selenomonas ruminantium and Fibrobacter succinogenes [16]. Nisbet et al. demonstrated that soluble components in AOC and another culture filtrate stimulated lactate uptake by Selenomonas ruminantium [39]. The comprehensive enzymes in the AOC stimulated the growth of microorganisms by converting nutrients into micromolecular nutrients for microorganisms. These results demonstrated that the AOC provides factors that are needed for growth by Fibrobacter succinogenes and Selenomonas ruminantium.
In the present study, the reduction in CP degradation in low AS pH group was consistent with Hoover [40]. A possible explanation is that the reduction in protein degradation is related to the reduction in the digestibility of fiber. The undigested fiber within feed would reduce the access of bacteria and enzymes to the protein, therefore reducing the degradation of protein in feed [38].
The Aspergillus oryzae (AO) secretes cellulases and proteases, which should increase the digestibility of feed [41]. In the present study, the addition of 2.5% AOC in diets increased the degradation of OM, but had no difference on DM, NDF, ADF, and CP degradation under a severe SARA in RUSITEC. The main reason for this is that the AOC increased the number of Selenomonas ruminantium utilizing the carbohydrates. Previous studies also obtained similar results. Manslield et al. found supplementation of AOC had no effect on the digestion of DM, OM, NDF, and ADF in vitro [14]. Oellermann et al. reported that the digestibility of CP, NDF, and ADF did not affect supplementation of AOC in diets for cattle [15]. The inconsistent results among studies could be due to the different fungi secreting different types of cellulase enzymes.
The total concentration of VFA was decreased from 104.8 mmol/L to 67.6 mmol/L in the low AS pH group compared with the high AS pH group, which was consistent with the results of OM digestion. The molar proportion of propionate was decreased in the low AS pH group, but the molar proportion of acetate was not affected, which resulted in increasing the ratio of acetate to propionate. These results are similar to an earlier study in our lab [42]. The difference in the molar proportion of propionate may be relate to the reduction of starch digestion. Low AS pH group tended to increase the molar proportion of butyrate in our study, which was consistent with the results of other studies [43,44]. Previous studies showed that the concentration of butyrate was only affected by the changes of pH and not affected by the diet composition in vitro [6]. The higher molar proportion of butyrate in the low AS pH group could be explained the increased abundance of Prevotella in the fermenters.
The total concentration of VFA and molar proportions of acetate, propionate, and butyrate were similar among the treatments under a severe SARA in a RUSITEC system. Newbold et al. reported the addition of AOC in the high barely diets for sheep decreased the molar proportion of propionate [45]. However, Mansfiled et al. found that addition of AO did not affect the molar proportion of individual VFA in vitro [14]. The inconsistent results among studies were likely due to difference in experiment treatment, effective dosage of AOC, and experiment animals. The reason in the present study was that the activity of AOC was inhibited with a severe SARA in the fermenters.
As expected, low AS pH significantly decreased the alpha diversity and richness, including the indexes of Chao1, Ace, and Shannon compared with the high AS pH. This result was in accordance with Brede et al. [46]. The PCoA analysis based on uniFrac distance analysis showed that bacterial community clustered separately [47], which indicated that the overall community was changed by the AS pH and supplementation of AOC. The Actinobacteria, Bifidobacteriaceae, and Bifidobacteriales were enriched in the low AS pH group. Under a severe SARA in RUSITEC, supplementation of 1.25% AOC in diets showed a higher abundance Prevotella, while the abundances of Fimicutes and Megasphaera were higher in the addition of 2.5% AOC. The results indicated that supplementation of AOC increased the growth rate of certain rumen bacteria that digest starch (Prevotella) or utilize lactate (Fimicutes and Megasphaera) under a severe SARA.
In the present study, the three main phylum Bacteroidetes, Firmicutes, and Proteobacteria were altered by the low AS pH. The abundance of Bacteroidetes did not differ among the treatments according to Brede et al. and Guo et al. [42,46], who found the abundance of Bacteroidetes was not altered by the SARA challenge or low AS pH at 6.0 in RUSITEC system. However, these results were in contrast to some previous studies, which reported a decrease in the abundance of the phylum Bacteroidetes during SARA challenges [3,7]. Bacteroidetes are the most abundant Gram-negative bacteria found in the anaerobic communities of the rumen, and low pH led to the death and lysis of Gram-negative bacteria [48]. Bacteroidetes were not altered in our study by a severe SARA in RUSITEC and may be related to the diet type. Firmicutes is the largest bacterial phylum and contains more than 200 genera, which are mainly comprised of Grampositive bacteria [49]. Mao et al. reported that the Firmicutes was more abundant in cattle fed SARA diets compared with the control diets [3]. Guo et al. found that the abundance of Firmicutes was not affected by AS pH at 6.02 in RUSITEC [42]. In this experiment, decreasing AS pH at 5.5 increased the abundance of Firmicutes. This result indicated that Firmicutes have different responses to the vary rumen pH. Low AS pH group significantly decreased the abundance of Proteobacteria from 14.6% to 0.96% compared with the high AS pH group, which was an agreement with Mao et al. [3], who found that feeding SARA diets in cattle decreased the abundance of Proteobacteria. Nevertheless, this is in contrast with findings of Guo et al. [42], who found that the abundance of Proteobacteria was greater in low AS pH at 6.02 in RUSITEC system. Combined with other studies, the results indicated that Proteobacteria was more sensitive to rumen pH; the growth and activity of Proteobacteria was inhibited by low AS pH 5.5, while it increased in low AS pH 6.02. In the current study, we found the abundance of Spirochaetes was decreased in low AS pH. In contrast with earlier studies, Petri et al. found that the higher abundance of Spirochaetes was observed in SARA cows [50]. The results indicated that the abundance of Spirochaetes could be affected by the combination of rumen pH and diets type. The Gram-positive Actinobacteria are colonized and actively influenced the digestive system of ruminants [51]. We found the abundance of Actinobacteria increased from 1.51% to 9.51% in low AS pH group compared with the high AS pH group. Similarly, the SARA challenge increased the relative abundance of Actinobacteria in the rumen fluid. However, the information is limited on the ecology and biology of Actinobacteria [51]. The results indicated that Actinobacteria could survive and increase under a SARA condition and low AS pH stimulated their growth and activity.
This study detected a significant difference in the population structure at the genus level between the high AS pH and low AS pH group. Correlation analysis revealed the potential relationship between rumen bacteria and individual VFA. Prevotella (Bacteroidetes) has been reported to be the most predominant rumen genus. It produces succinate and acetate [52], and plays an important role in the degradation cellulose, protein, and starch. In the present study, low AS pH increased the abundance of Prevotella. The result was consistent with Bekele et al. [52], and the change in the abundance of Prevotella was associated with the rumen pH [50]. Pseudobutyrivibrio (Firmicutes) is dominated in anaerobic gastroin-testinal microbiomes, particularly in the rumen [53]. The rumen genus of Pseudobutyrivibrio is an important degrader and utilizer of lignocellulosic plant material [54]. Similarly, the abundance of Pseudoscardovia increased from 1.13% to 8.26% in the low AS pH compared with the high AS pH. Correlation analysis showed that Pseudoscardovia was negatively associated with TVFA and propionate, and positively associated with butyrate and the ratio of acetate to propionate. These results suggested that low pH facilized the growth and activity of Pseudoscardovia, which also utilized the dietary energy and degraded lignocellulosic plant material under a severe SARA. Low AS pH increased the abundance of Megasphaera (Firmicutes) from 2.13% to 6.70% at the genus level compared with the high AS pH in this research. The genome analysis and physiological characterization of these Megasphaera isolates highlighted their ability to produce short chain fatty acids via propionate, acetate, and butyrate [55]. Considering the metabolic features of Megasphaera explained earlier and the effect of different environmental factors, it is possible that the acid condition in the fermenters was suitable for growth, and increased the abundance of Megasphaera in this experiment. However, low AS pH decreased the abundance of Olivibacter (Bacteroidetes) (22.11% vs. 0.01%) compared with the high AS pH. Olivibacter existed almost solely in the high AS pH, which indicated that this genus was constrained by low rumen pH. The application of Lactobacillus (Firmicutes) strains as silage inoculants has been widely studied in the field of ruminant nutrition (Sarong et al., 2020) [56]. In the current study, the relative abundance of Lactobacillus was greater in the fermenters when the AS pH was decreased at 5.5. These results were similar with findings by Guo et al. [42], who reported the abundance of Lactobacillus was increased when the AS pH decreased to 6.02. This result was likely due to rapid growth at low pH in Lactobacillus. In the present study, the abundance of Ruminobacter (Proteobacteria) was decreased in the low AS pH compared with the high AS pH. These results were consistent with Guo et al. [42], who also found that reduction of rumen pH at 6.02 decreased the abundances of Ruminobacter in RUSITEC system. Correlation analysis showed that Ruminobacter was negatively associated with butyrate and the ratio of acetate to propionate, while being positively associated with propionate and TVFA. Ruminobacter is mainly known for amylolytic activities. The decreased abundance of Ruminobacter in low AS pH group indicated that the degradation rate of starch might decline. Some rumen bacteria always co-occur with metabolites, and this result has important significance for regulating animal nutrition and health by microbiome intervention [57].

Conclusions
In conclusion, decreasing rumen pH at 5.5 significantly decreased the nutrients degradability. For the rumen fermentation, the reduction of pH at 5.5 increased the molar proportion of butyrate and ratio of acetate to propionate, while it decreased the total concentration of VFA and the molar proportion of propionate. Low AS pH increased the abundances of phylum Firmicutes, Proteobacteria, and Actinobacteria, while also decreasing the abundance of Spirochaetes. Compared with the high AS pH, low AS pH also increased the abundances of Prevotella, Pseudoscardovia, Mitsuokella, and Dialister, while decreasing the abundances of Olivibacter, Ruminobacter, Treponema, and Sphaerochaeta at the genus. These findings indicated that the reduction AS pH at 5.5 caused a strong shift in bacterial composition in the rumen. In addition, the addition of AOC in diets increased the growth rate of certain rumen bacteria that digest fiber or utilize lactate under SARA conditions in RUSITEC system.

Informed Consent Statement: Not applicable.
Data Availability Statement: The raw sequences used in this study were deposited in the Sequence Read Archive (SRA) of NCBI, and the accession number is PRJNA851265.