1.
Introduction
Rice straw represents as a highly available plant structural polysaccharide that can be utilized as feedstuff for ruminants. Every year, the global production of rice straw is 8 × 1011 kg as waste from the rice production process [1,2]. The utilization of rice straw as feedstuff is limited due to the nutritional content of rice straw. Rice straw contains a high level of plant cell wall components such as cellulose, hemicellulose, and lignin [3]. The high content of plant cell wall in rice straw leads to low digestibility when fed to ruminant. Efforts have been made to use rice straw as feed as optimum as possible through several approaches, such as use it while still fresh [4] and understand its degradation pattern in the rumen. Degradation of rice straw in the rumen of dairy cattle took place by a strong contribution of tightly attached bacteria as observed by Illumina sequencing [5]. Another important approach was to observe the chemical structure of feed by spectroscopy-based analysis such as Fourier transform infrared (FTIR) [6,7].
Ensiling or fermentation process of silage has been widely used to preserve the nutrient value of rice straw, which can increase the feeding value of rice straw, increase dry matter (DM), organic matter (OM) digestibility, and dry matter intake (DMI) [8,9]. Ensiling depends on the activity of microorganisms and many works had been conducted to investigate the effect of adding lactic acid bacteria (LAB) inoculant to silage, to ensure that LAB dominates and enhance lactic acid concentration in the fermentation process of silage [10,11]. It is widely accepted that LAB inoculants improved silage quality by increasing lactic acid to preserve nutrients in the silage quickly and inhibit spoilage silage microorganisms such as fungi and clostridial bacteria. At the same time, there is a decrease in pH, ammonia, and organic acids concentration [11,12].
Apart from LAB inoculant, hydrolytic enzymes are also used as additives to increase fermentation quality of silage. The enzyme has two major mode of actions in improving silage quality by 1) releasing of sugar from materials in silage to be used by LAB to produce lactic acid and acetate acid and 2) degrading plant cell walls to reduce fiber content of the silage material [13]. Actinomycetes are widely known as enzymes producers. They are known to contribute in the degradation of soil organic matter by producing enzymes capable of degrading cellulose, hemicellulose, and lignin. Important enzymes produced by Streptomyces species such as cellulase, xylanase, amylase, mannanase, have commercial applications in a variety of industries [14]. The addition of cellulase to rice straw is expected to break down fiber to release simple sugar for LAB to produce lactic acid. Enzyme additives might enhance the quality of low-quality forage and increase rumen digestion of fiber in vitro [15]. Addition of LAB inoculant and enzyme offers promising result to improve fermentation of the silage and rice straw digestibility. However, there are some inconsistent results from previous experiments. This discrepancy is mainly related to the differences of materials used in the study [12]. Therefore, the purpose of this study was to evaluate the effect of addition of LAB inoculant and crude cellulase alone or in combination on the quality of fresh rice straw silage. Evaluation based on the structure of rice straw fiber still rarely done using advanced analytical techniques such as XRD and FTIR. This work is the first stage of an attempt to use rice straw for feed which will be made into a complete feed.
2.
Materials and methods
2.1. Ethics statement
The experimental protocol used in this study was approved by Animal Ethics Committee of the Indonesian Institute of Sciences (approval number 39/klirens/Ⅲ/2021).
2.2. Animals and sampling procedure
The animals used for rumen fluid donor were consisted of 4 male fistulated Ongole cross breed cattle and were placed in individual cages. The rumen fluid was collected through the fistula just before morning feeding at around 8 a.m. local time. The collected rumen fluid was strained through four layers of cheesecloth and kept at 39 ℃ in a thermos flask during transfer to the laboratory.
2.3. Silage additives
Lactiplantibacillus plantarum 1A-2, previously reported as L. plantarum BTCC570 [16] was used as silage inoculant meanwhile Streptomyces sp. 292 was used to produce crude cellulase. Both L. plantarum 1A-2 and Streptomyces sp. 292 were from the collection of Indonesian Culture Collection, under the number of InaCC B253 and InaCC A1140, for L. plantarum 1A-2 and Streptomyces sp. 292, respectively.
Crude cellulase was prepared under following condition, Streptomyces sp. 292 was inoculated into 50 mL of preculture of ISP-2 modification medium (4 g·L−1 yeast extract, 10 g·L−1 malt extract, 1 g·L−1 glucose and 10 g·L−1 rice straw) and was then aerobically incubated at 37 ℃ for 6 days with shaking at 200 rpm. A 10% of preculture was then transferred to 500 mL fresh medium for crude cellulase production, incubated at 37 ℃ for 6 days in shaking incubator. Supernatant was harvested by centrifugation at 8000 rpm for 10 minutes at 4 ℃. The activity of crude cellulase was measured by using the method of Miller [17] with modifications. Carboxymethylcellulose (CMC) was used as the substrate [18]. Glucose standard was used to plot the standard curve. Measurement of glucose released from CMC was expressed as Unit (U), in which, U = mg of glucose equivalents released min–1·mL–1 crude enzyme. The activity of crude cellulase was 0.248 U·mL–1.
2.4. Silage materials
Rice variety used in this study was Inpari 32, which represent as one of new high yielding rice varieties. Fresh rice straw was collected from Indonesian Center for Rice Research, Sukamandi, West Java. Rice was harvested and threshed traditionally by hand. Rice straw then collected for ensiling. After 1-day wilting at room temperature, collected rice straw was cut into 2–3 cm long manually and mixed to ensure parts of rice straw were distributed equally.
2.5. Silage preparation
Inoculant of L. plantarum 1A-2 was diluted on sterile distilled water to achieve desired application rate at 109 CFU·mL−1. Chopped rice straw (2.5 kg) and rice bran (125 g) represent as basal materials of silage, which then added by silage additives alone or in combination according to the treatment, homogenized and divided into four replicates. The control was sprayed with the same volume of sterile distilled water. Six treatments were used in this study, i.e., 1) rice straw without any treatment as control, 2) rice straw with addition of 0.1% L. plantarum 1A-2 (LAB1), 3) rice straw with addition of 1% crude cellulase (E1), 4) rice straw with addition of 0.1% L. plantarum 1A-2 and 1% crude cellulase (LAB1E1), 5) rice straw with addition of 2% crude cellulase (E2), 6) rice straw with addition of 0.2% L. plantarum 1A-2 and 2% crude cellulase (LAB2E2). Treatment 1–4 and 5–6 with addition of 1% (25 mL) and 2% (50 mL) additives, respectively, according to proportion of L. plantarum 1A-2 and cellulase.
After mixing, the rice straw mixture was put into a vacuum emboss food grade plastic container (30 × 20 cm) as silo in ensiling process which contain 500 g of mixed silage materials. The air in the plastic bag was sucked using a vacuum machine (Yuu Zoo®), then was stored at room temperature for 60 days.
After 60 days of ensiling, silage was opened and measured for temperature, mold percentage, and moisture content. A representative of sample, 10 grams of silage was mixed with 90 mL of sterile distilled water and then mixed in a blender machine for 1 min. The mixture was then strained by using two layers of cheesecloth. Fresh juice was used for pH measurement and the rest was kept in a freezer at −20 ℃ until analysis of NH3-N, lactic acid and short-chain fatty acid (SCFA). The remaining silage from each silo was divided into 2 parts. First part was dried in drying oven at 60 ℃ and was ground to pass through 1 mm sieve for chemical analysis, while the second part was kept in freezer.
2.6. Chemical analysis of silage
Ground dry silage sample was used for analysis of dry matter (DM) and organic matter (OM) which were conducted according to AOAC [19] procedure. Crude protein (CP) was determined by using Kjeltec 8400 following FOSS manufacturer's procedure. Crude fiber (CF), neutral detergent fiber (NDF), and acid detergent fiber (ADF) were analyzed according to the FOSS manufacturer's procedure using a Fibertec 2010. Hemicellulose content (%) was calculated using following formulae
The ground dry sample was also used for X-ray diffraction (XRD) and Fourier Transform Infrared (FTIR) analyses. X-ray diffraction (XRD) analysis was conducted by using an XRD diffractometer (X'Pert PRO, Panalytical, The Netherlands). The sample was prepared by grounding to sieve through 100 mesh. XRD patterns were collected with Cu radiation at absorbance wave at 0.154 nm, voltage 40.0 kV, current 30 mA. Data were collected from 10–80° 2 Ø with step size 0.02, and speed at 2°/min. Crystallinity (%) and amorphous (%) were calculated by using the following formula
Scherrer's equation [20] was used for estimating crystallite size:
where λ is the wavelength of the incident X-ray (1.5418 Å), θ the Bragg angle corresponding to the (002) plane, β the full-width at half maximum (FWHM) of the X-ray peak corresponding to the (002) plane, τ is the X-ray crystallite size, and k is a constant with a value of 0.89 [21].
Fourier Transform Infrared (FTIR) Spectrometry was used to analyze functional groups of the control and treated rice straw silage samples. The FTIR spectra of all samples were run on a Perkin-Elmer UATR Two (Waltham, MA, USA) spectrometer and were recorded in the absorption band mode in the range of 4000–400 cm−1, number of scanning 16, resolution 4 cm−1, and data interval 1 cm−1. Total crystalline index (TCI), hydrogen bond index (HBI), lignin-cellulose (L/C) ratio were calculated as the ratio of intensities at particular wave numbers (cm−1) as: 1371/2918; 3333/1320; and 1516/899 respectively [22].
NH3-N was analyzed by adding 10 µL of sample to test tube and added with 1.5 mL of phenol solution and 1.5 mL of NaOCl solution and incubated in a water bath at 100 ℃ for 15 min. The absorbance was measured by using a spectrophotometer at 390 nm and was divided by 17 to determine NH3-N concentration [23]. Lactic acid concentration was analyzed by using a spectrophotometric according to the method by Borshchevskaya et al. [24].
Short chain fatty acids (SCFA) analysis was performed using GC-MS Simadzu-QP2010 SE comprising of AOC-20i+s autosampler and MEGA-WAX MS column (length, 30 m; i.d., 0.25mm; and film thickness, 0.25 μm). Ultrahigh purity helium (99.99%) was the carrier gas with 3 mL/min flow rate. The sample, 1.25 mL, was prepared by mixing with 30 mg 5-Sulfosalicylic acid dehydrate in 1.5 mL tube. Sample was centrifuged at 13000 rpm, 4 ℃ for 10 minutes. The particle-free of sample (1 μL) was taken using a syringe and injected into the injector with a split ratio of 50:1 with the solvent cut time was set at 3 min. The injection, transfer line, and ion source temperatures were all at 250 ℃. The column temperature was at 100 ℃ and held for 9 min followed by an increase to 200 ℃ at a rate of 10 ℃/min for 10 min. The total run time was approximately 29 min. The identities and quantification of SCFA were confirmed with Volatile Free Acid Mix (Supelco, CRM46975) standard.
2.7. In vitro digestibility of rice straw silage
In vitro rumen fermentation to determine digestibility was conducted by the method of Theodorou et al. [25] with modification. Rumen fermentation media for rumen in vitro incubation was a mixture of strained rumen fluid and McDougall buffer [26] in a ratio of 1:2 (rumen fluid: buffer). The buffer was pre-heated to 39 ℃ and flushed with CO2 gas before mixing. Rumen fermentation media was added to 100 mL serum bottle containing 0.5 g ground silage as substrate. The bottle was flushed with CO2 gas for 30 s before sealed in order to get an anaerobic condition. Incubation was carried out in a water bath at 39 ℃ for 48 h. Gas production was recorded at 2, 4, 8, 10, 12, 24 and 48 h incubation using a syringe. After 48 h incubation, samples were centrifuged at 378 × g for 10 min to separate supernatant and residue. Supernatant was analyzed for pH using a pH meter (BP3001 Trans Instruments) and partial SCFA followed the method as described above. The residue was further incubated with 50 mL pepsin-HCl solution (containing 2 g·L−1 pepsin and 17.8 mL·L−1 HCl) at 39 ℃ for 48 h. After 48 h, the residue was separated from the solution by vacuum filtration using WhatmanTM papers no 41 (CAT No.1441-125). Dry matter digestibility (DMD) and organic matter digestibility (OMD) were determined according to Tilley and Terry [27].
2.8. Statistical analysis
The data of silage quality were analyzed according to complete randomized design by using the following mathematical model
where Yij = the predicted output for predictor variable Y; μ = overall mean of the treatment; Ti = i-th treatment effect; and εij = error. The data of the rumen fermentation profile in vitro were analyzed by using ANOVA according to complete block randomized design. Duncan test was used to differentiate between treatment means when the ANOVA test showed a significance difference. The level of significance of an effect was set at p < 0.05. Significant differences were accepted if p < 0.05. Data of gas production were fitted with the Gompertz model [28] which were defined as
where 'P' is volume of gas production (mL) at time t, 't' is incubation period (h), 'b' is asymptotic gas production (mL), 'e' is Euler's number, 'L' is lag time (h), and 'c' is gas production rate constant (mL·h−1). The data were analyzed using IBM SPSS Statistic 23 (SPSS Inc., IL, USA).
3.
Results
3.1. Characteristics of rice straw silage
The temperature of silage during harvest was in the range of 28–30 ℃. Addition of lactic acid bacteria and crude cellulase did not have any effect on the moisture content and mold content of silage during harvest (moisture content p = 0.081; mold p = 0.290). Moisture content of Control, LAB1, E1, LAB1E1, E2, and LAB2E2 were 26.678%, 29.650%, 28.478%, 28.023%, 32.115% and 31.270%, respectively. Meanwhile, mold content of Control, LAB1, E1, LAB1E1, E2 and LAB2E2 were 7.290%, 6.865%, 4.645%, 6.510%, 7.995% and 8.425%, respectively (moisture content p = 0.081; mold p = 0.290). DM and OM content of silage significantly increased (p = 0.02) in LAB1 and LAB1E1, respectively, but not with other treatments. A highly significant effect on silage CP content was observed (p < 0.01), in LAB1 increased in CP content by 9% compared to that of control. Silage CF content significantly decreased by treatment with the highest decrease in LAB1E1. NDF content of rice straw silage was significantly decreased by E1 and LAB1E1 treatments. Meanwhile, ADF content of all treatment were significantly lower than control (p < 0.001). Hemicellulose content of silage was significantly increased by treatment (p = 0.02) with the highest hemicellulose content was LAB2E2 treatment. There was a highly significant reduction of silage pH (p < 0.01) in all treatments except for E1 with the highest reduction in LAB2E2. NH3-N concentration of silage significantly reduced in E2 and LAB2E2 (p < 0.01). Lactic acid content significantly increased in all treatments except for E1 (p < 0.01). The highest lactic acid was observed in LAB2E2 where lactic acid content increased by 300% than control.
The acetic acid content of silage significantly reduced in all treatments (p < 0.01) except for E1. The highest reduction was found in LAB2E2 with a 60% reduction followed by E2 with a 59% reduction than the control. There was also a significant reduction in the propionic acid content of silage (p < 0.01). Propionic acid in LAB1E1 and LAB2E2 was not detected meanwhile there was a 26% and 41% reduction than the control on LAB1 and E2, respectively. There was a highly significant effect of treatment on butanoic acid content of silage. The highest reduction was observed in LAB2E2 with a 75% reduction followed by E2 with a 70% reduction.
The results showed that there is a change in crystallinity of samples (Figure 1). According to the XRD result (Table 2), the crystalline decreased in LAB1, E1, LAB1E1, and E2 but increased in LAB2E2 compared to the control. The lowest crystallite size was observed in LAB2E2 by 10.00 nm. The absorbance ratio A3333/A1320 which is considered as the hydrogen bond Index (HBI), the ratio of A1371/A2918 which is the indication of total crystallinity index (TCI), and the ratio of A1516/A899 which is the indication of lignin per cellulose (L/C) for rice straw silage were calculated and reported in Table 2. Compared to the control, the TCI value decreased in LAB1 and increased in the other treatments with the highest TCI was observed in LAB2E2. The result of this study showed that L/C increased in LAB1 (0.77) but decreased in E1 (0.70), LAB1E1 (0.69), E2 (0.68) and LAB2E2 (0.71) compared to the control (0.74).
From the observation of chemical structure of rice straw silage by FTIR, (Figure 2) it can be seen that a strong band in 3700–3000 cm−1 corresponds to the O–H stretching band and the peak in the region of 3000–2800 cm−1 corresponds to the C–H stretching [29]. The peak of the band at around 1731 cm−1 was related to the C = O stretching of hemicelluloses [30]. The increase in the intensity of these peaks in all treatments compared to control showed the increasing trend of hemicellulose. The bands at around 1640 cm−1 were present in all samples that corresponds to the conjugated C = O group in the alkyl groups of lignin [31].
Guaiacyl ring-related IR spectra (Aromatic C–O stretching mode for lignin: guaiacyl ring of lignin) were present in all samples at around 1,516 cm−1 [32] and had a lower peak in E1 and E2. Peak around 1422 cm−1 and 1320 cm−1 in all samples were related to the symmetric CH2 bending and wagging [33]. A comparison to the control sample showed that LAB2E2 increased the intensities of the bands at 1371 cm−1 that related to the C-H bending in cellulose [34]. Peak at 1228 cm−1 related to C-O stretching band of ether linkage [35]. The bands at around 1036 cm−1 corresponds to the stretching C-O in cellulose [36,37]. The peak observed in 899 cm−1 corresponds to the β-1, 4 glycosidic bond linkages [38].
3.2. In vitro digestibility of rice straw silage
The effect of silage additives on in vitro rumen fermentation is shown in Table 3. Gas production in the first 4 to 8 h showed a significant difference (p < 0.05) higher than the control and LAB1, except for LAB2E2, which was not significantly different in the first 4 h. E1 showed significantly different results (p < 0.05) with higher gas production from 10 h until the end of fermentation (48 h) compared to the control and LAB1, but not significantly different from other treatments. Kinetics of gas production was obtained from gas production data and fitted using Gompertz equation [28,39]. Lag time (L) of gas production in the control was significantly higher (p < 0.05) than other treatments. Treatment LAB1E1 showed significantly (p < 0.05) the highest total potential gas production (B). Data showed that all treatments increased total potential gas production (B) from the lowest (27.640 mL) in control, to the highest (38.960 mL) resulted from treatment LAB1E1. Different result showed from gas production rate (C) which is not significant among all treatments.
DMD and OMD did not show a significant difference (p > 0.05) from all treatments, but there was a tendency to increase digestibility compared to control. LAB1E1 showed a significantly higher difference (p < 0.05) in the NH3-N content compared to the control and E1, but not significantly different between the other treatments. The pH value of the rumen fermentation was in the normal range of 6.89–6.95 and was not significantly different in all treatments. In vitro rumen fermentation profiles after 24 h incubation is presented in Table 3. Treatments were significant on each SCFA production, in which control produced significantly the lowest (p < 0.05) compared with other treatments. But the rumen A/P ratio was not significantly affected by treatments.
4.
Discussion
4.1. Characteristics of rice straw silage
Ensiling of fresh rice straw with additives is an effective way to preserve its nutrients as well as to improve its digestibility. In this present study, L. plantarum 1-A2, a heterofermentative LAB, and crude cellulase were used as additives to improve the fermentation process of rice straw. Work on addition of LAB inoculant and enzyme have been conducted to improve the digestibility of rice straw. Combination of L. plantarum and cellulase showed the highest lactic acid concentration among all silages over the 7 d of ensiling and also the lowest abundance of Enterobacteriaceae over 30 d of ensiling. The combination of L. plantarum and cellulase further improved fermentation quality compared to silage treated with L. plantarum or cellulase alone, since the combination had a synergistic effect on mixed silage fermentation [40]. Changes on quality, gas kinetics and in vitro digestibility of rice straw silage with Lactobacillus casei TH14, cellulase and molasses additives were also reported [41].
The addition of LAB inoculant and crude cellulase alone or in combination did not have any effect on the mold content of the silage. Addition of heterofermentative LAB inoculant could lead to a significant reduction of mold content in the silage [12]. Ensiling process in tropical region with high humidity, high temperature, and high oxygen availability is vulnerable to fungal contamination [42]. Heterofermentative LAB such as L. plantarum is known to produce antifungal substances such as acetic acid and propionic acid [43].
An increment of DM content by the addition of combination of LAB inoculant and crude cellulase at 1% showed that it can increase preservation ability from DM loss of silage. DM of silage should have a minimal loss which indicates the efficiency of the ensiling process [44]. CF significantly reduced by LAB inoculant with or without crude cellulase relative to control. The addition of LAB inoculant and crude cellulase alone or in combination might change the composition of fiber of rice straw silage. This is in agreement with previous studies that LAB inoculant and enzyme addition might decrease the fiber content of silage [45,46,47]. Dewar et al. [48] suggested that ensiling process could change plant structural carbohydrate content by three mechanisms such as enzymes, acidolysis, and microbial degradation. Enzymes hydrolyze structural carbohydrates at the initial stage of ensiling, which then allows microorganisms, especially LAB, to use the carbohydrate and converted to lactic acid. Thus, this process would reduce the pH of silage. In the present study, CP content significantly increased by the addition of LAB inoculant alone at the rate of 0.1%. The addition of LAB inoculant inhibits CP loss from the ensiling process. An increase in CP content with the addition of LAB inoculant to silage was reported by Li et al. and Marbun et al. [10,49]. NH3-N concentration was suppressed by the addition of crude cellulase at a level of 2% alone or in combination with LAB inoculant. An increase in NH3-N concentration by the addition of heterofermentative LAB inoculant was reported by Muck et al. [50]. A decrease in pH was observed by the addition of LAB inoculant alone or in combination with crude cellulase, which is related to an increase in lactic acid production. Addition of crude cellulase alone at a level of 1% (E1) might not sufficiently to provide fermentable substrate for LAB to produce lactic acid thus did not have a significant effect on the pH of silage. However, crude cellulase at 2% (E2) support the growth of LAB inoculant to produce lactic acid by 300% and effectively reduced the pH of silage. Crude cellulase used here is low in its activity because it is produced from a wild culture of actinobacteria, however this Streptomyces sp. 292 produced cellulase activity only with very small activity of hemicellulose and mannanase (unpublished results). Heterofermentative LAB is known for its capability to produce lactic acid and acetic acid from the fermentation of pentoses [50]. Lactic acid has the lowest degree of acidity in comparison with other organic acids, such as acetic acid, propionic acid and butyric acid [51]. Thus, higher production of lactic acid might have a greater decrease in the pH of silage [12,52,53]. The addition of LAB inoculant and or without enzyme suppressed acetic, propionic, and butyric acids. E1 and E2 showed a different way in reducing organic acids concentration. Propionic acid was not detected when a combination of LAB inoculant and crude cellulase was added to the silage. This finding confirms that the addition of heterofermentative LAB inoculant reduced pH, acetic acid, butyric acid, and NH3-N [50].
XRD, FTIR, and NMR are the most widely used methods to study the crystalline structure of lignocellulose material [54]. XRD and FTIR were used here to determine the crystalline structure of rice straw. The crystallinity of rice straw silage was studied using XRD. The result of this study showed that the addition of LAB inoculant and crude cellulase alone or in combination decreased the crystallinity of rice straw silage except in LAB2E2 treatment. During the ensiling process, the amorphous cellulose and hemicellulose were hydrolyzed by water and fermentation microorganisms, resulting in little damage to the crystal region of the fibers. This result was also supported by the crystallinity index that was estimated based on FTIR spectra. Total crystalline index (TCI), Hydrogen bond index (HBI), and lignin per cellulose (L/C) were used to study the crystalline of cellulose. HBI is closely related to the crystal system and the degree of intermolecular regularity [22]. HBI values were determined as the ratio between peak at 3333 (H-bonded absorption) and 1320 (CH2 rocking vibration). LAB1 did not change the HBI value in rice straw silage but increased when the LAB was combined with crude cellulase. The transformation of rice straw from high crystallinity to low crystallinity was supported by the HBI data. The HBI value increased compared to control, which means that fewer available hydroxyl groups in the cellulose chain can interact by inter- and/or intramolecular hydrogen bonding. Lignin to cellulose (L/C) ratio is a parameter obtained by calculating the ratio of absorption intensities at 1516 and 899 cm−1. The decreasing of L/C in E1 and E2 (crude cellulase alone), LAB1E1, and LAB2E2 (combination with LAB) showed the conversion of crystalline to amorphous cellulose (increase in absorption at 899 cm−1). In this study, the decrease in crystallinity was followed by an increase in hemicellulose. Hemicelluloses are generally easier to degrade enzymatically than cellulose [55]. The total crystalline index (TCI) in all treatments was higher than control with the highest TCI was observed in LAB2E2 treatment. TCI corresponds to the degree of crystallinity of cellulose [56]. The results of XRD and TCI calculations from FTIR spectra showed that the crystallinity of the LAB2E2 treatment increased compared to control. The increase of the crystalline of rice straw in LAB2E2 might be due to the changes in structural properties of cellulose and the release of fermentable sugar from biomass. The ensiled treatment had impacts on increasing the mechanical properties such as internal bond strength and modulus of elasticity [57]. Moreover, the addition of crude cellulase in lignocellulosic biomasses could release the fermentable sugars by altering the cell wall structure. The released fermentable sugars could be used by LAB as indicated by the lowest pH of this treatment among the other treatments.
4.2. In vitro digestibility of rice straw silage
Total gas production is one indicator of the degradation of easily fermentable materials. The addition of additives to rice straw silage resulted in a decrease in total fiber, which is supported by the hemicellulose fiber fraction value, higher amorphous percentage than control, and lower L/C index value. The first 4–8 h of rumen fermentation is the initial process of utilizing the easily digestible parts of feed materials by rumen microbes. Gas production correlated with fiber degradation where LAB and enzyme treatments release simple and more fermentable carbohydrates, available for rumen microorganisms during in vitro incubation [58]. It helps rumen microorganisms to degrade feed substrate and produce gas. Complex carbohydrates in control treatment caused rumen microorganisms need more time to degrade feed substrate which resulted the highest lag time (L) and the lowest total potential gas production (B) compared to the other treatments. Increased potential gas production was reported by Zhao et al. [59] which resulted from enzyme treatments in rice straw silage.
The addition of crude cellulase to silage improves the accessibility of rice straw silage fibers by the rumen microbial community. The addition of LAB inoculant combined with crude cellulase provided an additional role in the utilization of WCS after the enzyme was active at the beginning of the silage process. This activity can be seen from the gas production, which is higher than the control and only in LAB without being given a combination with crude cellulase. The addition of silage inoculant, especially L. plantarum can affect the decrease in total gas production during in vitro fermentation [60]. The crude cellulase treatment affects changes in the composition of the cell wall of rice straw into organic matter that is easily utilized by rumen microbes. The aim of addition of fibrolytic enzymes was to break down the fiber and releasing soluble sugars, to provide a substrate that could be ready to use by microbes [59]. Rice straw silage is the main source of fiber in rumen fermentation, providing opportunities for the role of fibrolytic bacteria in their metabolism such as Bacteroidetes and Fibrobacters [61]. The main degradation products of fibrolytic bacteria are acetic acid, CO2, and hydrogen sinks. The digestibility value in vitro has a positive relationship with VFAs production [62]. These metabolic products tend to increase total SCFA production, especially acetic acid. The combination of silage additives can potentially increase digestibility, gas production, and SCFA during rumen fermentation [59]. This is in line with the results of the study which showed a high trend of DMD and OMD giving results for total VFA and SCFA (Table 3). This phenomenon can be used as the basis for complete feed making with straw, silage as a source of fiber that has a fairly good digestibility value. The pH at the end of rumen fermentation (48 h) from all treatments was not affected by the additives used. These results are in line with the straw silage experiment with the addition of additives at the end of fermentation in the rumen which did not significantly affect the final pH [10].
The profile of products formed during rumen fermentation has implications for animal productivity and the environment. The most important products of the anaerobic microbial fermentation of carbohydrates in the alimentary tract of ruminants are SCFA, which cover 80% of the animals' demand for gross energy [63,64]. In this study, total SCFA significantly increased in all treatments. It is well known that total SCFA production is positively correlated with digestibility. Although DMD in this study was not significantly increased, higher gas production can be assumed as higher digestibility. Interestingly, treatments of LAB inoculant and crude cellulase significantly increased acetic, propionic, iso-butyric, butyric, iso-valeric, and valeric acids from rumen fermentation. Guo et al. [65] reported only significantly higher acetic and propionic acids with the addition of L. plantarum as silage inoculum compared with non-inoculated silage. The addition of L. plantarum was also reported by Zhao et al. [59] to increased acetic acid, but in contrast, decreased propionic and butyric acids. The increase of fermentation products by the addition of LAB was caused by a decrease of structural carbohydrate contents, which provides more soluble components retained in additive-treated silages.
The addition of crude cellulase produced a lower fermentation product compared with LAB1, but still higher than control. However, LAB1E1 produced a higher fermentation product compared to control or LAB1. In contrast, Dönmez et al. [66] reported a significant decrease of total SCFA, acetic acid, propionic acid, and butyric acid by enzyme addition in silage. The effect of enzyme additives on the silage rumen fermentation profile has not been widely studied. Cell wall degrading enzymes, such as cellulases, has been used to increase WSC availability to LAB [67] presumably due to degradation of NDF. Colombatto et al. [68] found that additions of enzymes reduced pH, NDF, and ADF contents of maize silage. A different result was reported by Sun et al. [69] which showed that the addition of cellulase reduced losses of NDF, but increased gas production on maize stover silage. Khota et al. [70] used LAB and cellulase as additives on sorghum silage. The addition of LAB, enzyme and both LAB and enzyme did not show significant changed in in vitro rumen digestibility. The effect of fibrolytic enzyme activity on silage fermentation depending on both the temperature and pH conditions [71]. The highest fermentation product from all treatments was found from LAB2E2 which applied double dosage for LAB and enzyme addition. It seems that there is an interaction of LAB and enzyme, to provide higher WSC due to mechanisms of cellulase treatment that can be used by LAB during ensiling. More compounds are available for rumen bacteria to ferment in the rumen. Although LAB and enzyme addition increased SCFA production, in this study A/P ratio was not significantly affected by treatments. It means that the rumen fermentation pattern was not shifted from acetic acid production to propionic acid production.
5.
Conclusions
Addition of L. plantarum 1A-2 inoculant alone or in combination with crude cellulase improved silage quality. Individual crude enzyme addition was effective to improve silage quality at a level of 2%. Combination of LAB2E2 gave the highest effect on silage quality and produced the highest fermentation products. The increase of the crystalline of LAB2E2 might be due to the use of fermentable sugars released from rice straw biomass by the additives used. There is significant effect on increasing the main SCFA products from in vitro rumen fermentation, which provide potential energy source for the animal. This report described new information on the use of rice straw of new high yielding rice variety for silage. Further study on the population of microorganisms involved during ensilage is needed.
Acknowledgements
This study was supported by National Innovation System Research Incentive (INSINAS) program (Contract No. 9/INS/PPK/E4/2021), Ministry of Research and Technology, Republic Indonesia. The authors express gratitude to the Indonesian Research Center for Rice for providing the rice straw materials, and to the Advanced Characterization Laboratory Cibinong–Integrated Laboratory of Bioproduct, and Characterization Biotechnology Laboratory, through E-Layanan Sains, National Research and Innovation Agency, for facilities, scientific and technical support.
Conflict of interest
All authors declare there is no conflict of interest.
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