Fermentation of increasing ratios of grain starch and straw fiber: effects on hydrogen allocation and methanogenesis through in vitro ruminal batch culture

Research ethics

This study was conducted at Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha, China. The procedures of animal experiments were carried out in accordance with the Animal Care and Use Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, China (Approval NO. ISA 2020-0019).

Experimental design

Corn grain and corn straw (Table 1) were served as starch and fiber source, respectively. The seven treatments that were examined differed in the RGS and included 0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0. The experiment was conducted by a completely randomized block design, which included three runs with each treatment containing four fermentation bottles (replicates). Samples were ground to pass through a 1-mm aperture sieve (Daoxujian Instruments Co. Ltd., Shaoxing, China).

In vitro ruminal batch incubation

Rumen fluid was collected from two of three adult male Xiangdong black goats (BW 30.0 ±1.50 kg) with permanent rumen cannula before morning feeding. The three goats were in healthy state and rumen fluid samples were taken until the end of the experiment. The goats were fed a total mixed diet containing corn straw and concentrated mixture (1:1) with the crude protein (CP) content of 137 g/kg dry matter (DM) and the content of neutral detergent fiber (NDF) 380 g/kg DM, free to drink water, and the goat houses are ventilated. The rumen fluid was filtered through four layers of cheesecloth into a pre-warmed insulated bottle and taken to the laboratory.

Approximately 0.6 g of substrate was accurately weighed into a 135-mL fermentation bottle. Then buffered rumen fluid containing 12 mL of rumen fluid and 48 mL of McDougall’s buffer (Cone & Becker, 2012) were added into bottle under a stream of CO₂ at 39.5 °C. Bottles were immediately placed into the automatic incubation system described by Wang et al. (2016b), with venting pressure set at 10.0 kPa. As the incubation bottle was in line with gas chromatograph (GC, Agilent 7890 A, Agilent, Palo Alto, California, USA) via a computer-controlled three way solenoid valve, the released gas was automatically vented into a GC for measuring CH₄ and gH₂ concentrations. Gas production (GP), CH₄ and gH₂ accumulations were calculated using the equation described by Wang et al. (2013a).

In vitro ruminal fermentation was stopped at 48 h. About 2 mL of liquid without visible particles were collected from each bottle and centrifuged at 15, 000 g for 10 min at 4 °C. The supernatant (1.5 mL) was acidified by 0.15 mL of 25% (w/v) metaphosphoric acid, and stored at −20 °C for analysis of VFA and ammonia-N. The pH was measured immediately with a portable pH meter (Starter 300; Ohaus Instruments Co. Ltd., Shanghai, China). Approximately eight mL of samples were collected for measuring MCP after intense shaking of the bottle to ensure that representative portions of liquid and particle fractions. Solid residues were filtered into pre-weighed Gooch filter crucibles, dried at 105 °C to constant weight and weighed to determine degradation of incubated substrates.

Two bottles in each run were used for measuring pH and DM degradation, and the other two bottles were used for obtaining samples for measuring fermentation end-product and MCP. Each run was repeated three times, each on different days, so that each treatment was conducted in triplicate.

Sample analyses

The DM content was determined by drying at 105 °C for 24 h in an oven, and the organic matter (OM) content was determined by ashing at 550 °C for 12 h in a muffle furnace. Gross energy (GE) was measured using an isothermal automatic calorimeter (5EAC8018; Changsha Kaiyuan Instruments Co. Ltd, Changsha, China). The contents of CP (N ×6.25) in feed samples were determined according to procedures of AOAC (1995). The contents of NDF and acid detergent fiber (ADF) in feed samples were determined according to the methods described by Van Soest, Robertson & Lewis (1991) and expressed as inclusive of ash. Heat stable α-amylase was added to for NDF analysis. The starch content was determined after pre-extraction with 80% ethanol (v/v), and glucose released from starch by enzyme hydrolysis was measured using amyloglucosidase (Sigma) according to Karthner & Theurer (1981).

Volatile fatty acid concentration was measured according to the procedure described by Wang et al. (2014), using a GC (Agilent 7890 A, Agilent Inc., Palo Alto, California, USA). Ammonia-N concentration was measured colorimetrically according to Chaney & Marbach (1962). Rumen microorganisms was separated from feed particles according to Makkar et al. (1982) with filtration (four layers of gauze), shaking (125 rpm/min for 1h) and centrifugation (150 × g for 10 min), and microbial nitrogen production was measured by Microplate Reader (Infinite M200 PRO\spark, Tecan Inc., Männedorf, Switzerland) according to Bradford (1976), Using a Coomassie brilliant blue kit (Build a biopharmaceutical research institute, Nanjing, China).

Calculations and statistical analysis

The kinetics of total gas and CH₄ analyzed using the equation provided by Wang, Tang & Tan (2011), which was expressed as follows: ${\displaystyle {GP}_t=VF\frac{1-exp\left(-kt\right)}{1+exp\left(b-kt\right)}}$ where GPt is the accumulated gas/CH₄ production at time t (mL/g); VF is the final asymptotic gas/CH₄ volume (mL/g); k is the fractional rate of gas/CH₄ production (/h); b is the shape parameter of gas/CH₄.

The kinetics of gH₂ production was analyzed using the equation provided by Wang et al. (2013b), which was expressed as follows: ${\displaystyle V_{H2t}=\frac{V{F}_{H2}\left\{1-\mathit{exp}\left[-k_{H2}\left(t-la{g}_{H2}\right)\right]\right\}\left\{1+c_{H2}\mathit{exp}\left[-{\mu }_{H2}\left(t-la{g}_{H2}\right)\right]\right\}}{1+exp\left[b_{H2}-k_{H2}\left(t-la{g}_{H2}\right)\right]}}$ where V_H2t is the accumulated gH₂ production at time t (mL/g); VF_H2 is the final asymptotic gH₂ volume (mL/g), b_H2 and c_H2 are shape parameters of gH₂ curve without dimension, k_H2 is the fractional rate of gH₂ production (/h), µ_H2 is the fractional rate of gH₂ utilization (/h), and lag_H2 is discrete lag time (h).

The stoichiometric equations developed by Wang et al. (2014) was used to calculate the estimated net [H] production (P_NH2, mM) and estimated [H] production relative to the amount of total VFA produced (R_NH2, moL/100 mol of VFA), which was expressed as follows:

P_NH2 = 2(Ace + But + Isobut) − (Pro + Val + Isoval)

R_NH2 = 100 P_NH2/(Ace + But + Isobut + Pro + Val + Isoval)

where ace, but, pro, val, isobut and isoval were concentration (mM) of acetate, propionate, valerate, isobutyrate, and isovalerate respectively.

In vitro DM degradation (DMD) was calculated using the equation provided by Zhang et al. (2018), which was expressed as follows: ${\displaystyle DMD\left(g/kgofDM\right)=\left[1-W_1\times \left(V_1/V_2\right)/W_2\right]\times 1,000}$ where W₁ is the DM weight of the residue after 48 h of incubation; W₂ is the DM weight of substrate before incubation; V₁ is the volume of buffered rumen fluid in the bottle before sampling (i.e., 60 mL); V₂ is the volume of buffered rumen fluid in the bottle after sampling (i.e., 56 mL).

The data were analyzed using general linear model (GLM) with SPSS 26.0 (Chicago, IL, USA), and are presented as mean and SEM. The analytic model included treatment (n = 7) as fixed effect and run (n = 3) as random effect, and were analyzed for linear or quadratic responses to ratios of corn grain to corn straw using orthogonal contrasts. Statistical significance was considered at P ≤ 0.05 with 0.05 < P ≤ 0.10 considered as a tendency.

Impacts of the ratios of corn grain to corn straw (RGS) on gas production

Elevating RGS increased DMD (P_linear < 0.001) and altered the kinetic of gas production, with an increase in 48-h gas production, final asymptotic gas production, and fractional rate of gas production (P_linear < 0.001; P_quadratic < 0.01) (Fig. 1A and Table 2). Increasing RGS altered the kinetics of CH₄ accumulation, with an increase in 48-h CH₄ production (P_linear and P_quadratic < 0.001), final asymptotic CH₄ production (P_quadratic < 0.001), and fractional rate of CH₄ production (P_linear < 0.001; P_quadratic = 0.007), and a reduction in 48-h CH₄ production relative to DM degraded (P_linear and P_quadratic < 0.001) (Fig. 1B and Table 3). With the increase of RGS, the 48-h gH₂ production (P_linear = 0.011; P_quadratic = 0.003), final asymptotic gH₂ production (P_linear = 0.03; P_quadratic = 0.002), and the 48-h gH₂ production relative to DM degraded (P_linear < 0.001) were decreased, whereas the fractional rate of gH₂ utilization was increased (P_linear = 0.035) (Fig. 1C and Table 3).

Impacts of the ratios of corn grain to corn straw (RGS) on rumen fermentation

Elevating RGS decreased pH (P_linear < 0.001) and increased total VFA concentration (P_linear < 0.001). Improving RGS decreased acetate (P_linear < 0.001), butyrate (P_quadratic = 0.002), isobutyrate (P_quadratic = 0.02) molar percentage and acetate to propionate ratio (P_linear < 0.001). Improving RGS increased propionate, valerate and isovalerate molar percentage (P_linear < 0.001; P_quadratic < 0.01) (Table 4). With the increase of RGS, the estimated net [H] production (P_linear < 0.001; P_quadratic = 0.005) increased, whereas the estimated net [H] production relative to DM degraded (P_linear < 0.001) and total VFA produced (P_linear < 0.001; P_quadratic = 0.004) were decreased. Increasing RGS decreased molar percentage of [H] utilized for CH₄ (P_quadratic = 0.003) and gH₂ (P_linear < 0.001) production. Furthermore, the MCP (P_linear < 0.001; P_quadratic < 0.001) and ammonia-N concentrations (P_quadratic = 0.004) increased with the increase in RGS (Table 5).