In vitro production of volatile fatty acids by reactivated cellulolytic bacteria and total ruminal bacteria in cellulosic substrate

Sánchez-Santillán, Paulino; Cobos-Peralta, M. Antonio; Sánchez-Santillán, Paulino; Cobos-Peralta, M. Antonio

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Agrociencia

versão On-line ISSN 2521-9766versão impressa ISSN 1405-3195

Agrociencia vol.50 no.5 Texcoco Jul./Ago. 2016

Animal Science

In vitro production of volatile fatty acids by reactivated cellulolytic bacteria and total ruminal bacteria in cellulosic substrate

Paulino Sánchez-Santillán¹^*

M. Antonio Cobos-Peralta²

^¹Unidad Académica de Medicina Veterinaria y Zootecnia No. 2, Universidad Autónoma de Guerrero. Km 197 Carretera Acapulco-Pinotepa Nacional. 41940. Cuajinicuilapa, Guerrero, México. (sanchezsantillanp@gmail.com).

^²Programa de Ganadería, Colegio de Postgraduados, Km 36.5, Carretera México-Texcoco. 56230. Montecillo, Texcoco, estado de México, México.

Abstract:

In vitro methods are suitable for comparing degradation of cellulosic substrates and synthesis of products derived from fermentation, such as volatile fatty acids (VFA). The objectives of this study were to compare the production of VFA of a culture of reactivated cellulolytic bacteria (CBC) with that of total ruminal bacteria (TRB) in cellulosic substrates, and to evaluate the relationship between VFA and in vitro degradation. The culture of cellulolytic bacterial was obtained after four transfers of ruminal fluid to fresh culture media and Whatma paper, and preserved by lyophilization. The inocula were CBC, TRB and one co-culture (50:50) of the two inocula together. The substrates were alfalfa, maize stalks, Bermuda grass and crystalline cellulose. In in vitro degradation, pH, percentage of dry matter (DM) degradation (%DMDEG) and VFA were measured. The experimental design was completely randomized with a 3×4 factorial array; inocula and substrates were factors. The three inocula had heterofermentative activity in the cellulosic substrates. None of the three were different in VFA production (p>0.05) in fermentation of alfalfa, Bermuda grass or maize stalks. However, CBC produced more acetate (p≤0.05) than the TRB in fermentation of alfalfa and Bermuda grass. CBC exhibited synergism in the production of acetate with TRB in crystalline cellulose since the co-culture produced more acetate than TRB (p≤0.05). The co-culture and TRB were not different (p>0.05) in %DMDEG in Bermuda grass, maize stalks and crystalline cellulose. Under these conditions, the culture of reactivated cellulolytic bacteria exhibits heterofermentative activity in cellulosic substrates, and acetate is the main product of fermentation. These bacteria, however, do not improve DM degradation when co-cultured (50:50) with total ruminal bacteria.

Key words: Acetate; in vitro fermentation; in vitro degradation; cellulose; bacteria

Resumen:

Los métodos in vitro son adecuados para comparar la degradación de sustratos celulósicos y la síntesis de productos derivados de la fermentación como los ácidos grasos volátiles. Por lo tanto, los objetivos de este estudio fueron comparar la producción de AGV de un cultivo de bacterias celulolíticas reactivadas (CBC) con bacterias ruminales totales (BRT) en sustratos celulósicos y, además, evaluar la relación entre AGV y la degradación in vitro. El cultivo de bacterias celulolíticas se obtuvo luego de cuatro trasferencias de fluido ruminal fresco en medios de cultivo y papel Whatman, y conservadas mediante liofilización. Los inóculos fueron CBC, BRT y un cocultivo (50:50) entre ambos inóculos. Los sustratos fueron alfalfa, rastrojo de maíz, pasto bermuda y celulosa cristalina. En la degradación in vitro se midió pH, porcentaje de degradación de materia seca (MS) (%DEGMS) y AGV. El diseño experimental fue completamente al azar con un arreglo factorial 3×4 con inóculos y sustratos como factores. Todos los inóculos presentaron actividad heterofermentativa en los sustratos celulósicos. Los tres inóculos no presentaron diferencias en la producción de AGV (p>0.05) en la fermentación de alfalfa, pasto bermuda y rastrojo de maíz, pero el CBC produjo más acetato (p≤0.05) que las BRT al fermentar alfalfa y pasto bermuda. El CBC presentó sinergismo en la producción de acetato con las BRT en celulosa cristalina, ya que el cocultivo produjo más acetato que BRT (p≤0.05). El cocultivo y BRT no presentaron diferencias (p>0.05) en el %DEGMS en pasto bermuda, rastrojo de maíz y celulosa cristalina. Bajo estas condiciones el cultivo de bacterias celulolíticas reactivadas presenta actividad heterofermentativa en sustratos celulósicos y el acetato es el principal producto de la fermentación, pero estas bacterias no mejoran la degradación de la MS al usarse en cocultivo (50:50) con bacterias ruminales totales.

Palabras clave: Acetato; fermentación in vitro; degradación in vitro; celulosa; bacterias

Introduction

Plant carbohydrates are the energy source for ruminants. They are divided into structural and non-structural polysaccharides. Structural polysaccharides are in the cell wall as neutral detergent fiber (NDF), they are made up of cellulose, hemicellulose and lignin (^{Barboza et al., 2009}), and the proportion depends on the species, cell type and development stage (^{Vermerris, 2008}). In the anaerobic environment, microorganisms in the rumen efficiently degrade the polysaccharides from plants. Ruminococcus flavefaciens, Ruminococcus albus and Fibrobacter succinogenes produce endoglucanases, exoglucanases and β-glucosidases, which act synergistically in the degradation of cellulose in the rumen (^{Cai et al., 2010}).

Studies on ruminal fermentation use in vitro assessment of cellulosic substrates to measure degradation of a substrate in a given time, as well as volatile fatty acids (VFA) obtained in fermentation. Ruminal microorganisms are used as inoculum to simulate the conditions in the rumen (^{Dhanoa et al., 2004}; ^{Váradyová et al., 2005}). In vitro methods are suitable for comparing degradation in cellulosic substrates and formation of products derived from fermentation. VFA produced by in vitro fermentation differs depending on experimental conditions (^{Weimer et al., 2011}).

Digestibility of dry matter (DM) from forages and agricultural residues decreases with physiological maturation because of the increase in structural polysaccharides (^{Barboza et al., 2009}). Cellulolytic bacteria can degrade the structural polysaccharides of the cell wall. However, one bacterial species alone does not produce the enzymes necessary for its digestion. For this reason, the bacteria should be associated physiologically to combine the action of the enzymes produced (^{Miron et al., 2001}). Moreover, they must interact with other microorganisms through nutrient interdependence or crossed alimentation (^{Cobos, 2007}). The main product of this degradation is acetate and CO₂ (^{Lynd et al., 2002}).

In the literature reviewed, there are no publications about the addition of cellulolytic bacteria to a population of ruminal microorganisms to observe their effects on fermentation variables. In vitro degradation of DM by cellulolytic bacteria in pure or co-cultures was studied by ^{Min et al. (2006)} and ^{Grilli et al. (2011)}, but it was not evaluated in co-culture with ruminal bacteria. Therefore, the objectives of this study were to compare the production of VFA by a culture of reactivated cellulolytic bacteria (CBC) with that of total ruminal bacteria (TRB) in four cellulosic substrates and to establish the relationship between VFA and in vitro degradation of cellulosic substrates.

Materials and Methods

The study was carried out in the Laboratory of Ruminal Microbiology and Microbial Genetics of the Colegio de Postgraduados, Campus Montecillo, located at Km 36.5, Carretera Mexico-Texcoco, Montecillo, Texcoco, Estado de Mexico.

Culture medium for cellulolytic bacteria

The culture medium for cellulolytic bacteria contained 30 mL clarified ruminal fluid (fresh ruminal liquid centrifuged at 12 857 g for 10 min and sterilized 15 min at 121 °C and 15 psi), 5 mL mineral solution I [6 g K₂HPO₄ (Sigma) in 1000 mL distilled H₂O], 5 mL mineral solution II [6 g KH₂PO₄ (Sigma) + 6 g (NH₄)₂SO₄ (Merck) + 12 g NaCl (Sigma-Aldrich) + 2.45 g MgSO₄ (Sigma) + 1.6 g CaCl-2H₂O (Sigma) in 1000 mL distilled H₂O], 0.1 mL resarzurin at 0.1 % (Sigma-Aldrich), 0.2 g soy peptone (Merck), 0.1 g yeast extract (Sigma), 2 mL cysteine sulfide solution[2.5 g L-cysteine (Sigma) in 15 mL 2 N NaOH (Meyer) + 2.5 g Na₂S-9H₂O (Meyer), gauged in 100 mL distilled H₂O5], 5 mL 8% solution of Na₂CO₃ (Baker) and 52.6 mL distilled H₂O. The culture medium was sterilized at 121 °C and 15 psi for 15 min in an autoclave (Tuttnauver™ 2540F, Israel) (^{Cobos and Yokoyama, 1995}).

Culture of cellulolytic bacteria

The ruminal fluid was obtained from a Jersey cow fitted with a ruminal cannula. The cow grazed on pastures of alfalfa (Medicago sativa) before collection of ruminal fluid sample, which was centrifuged at 1157 g in a centrifuge (Eppendorf^® 5804, Germany) at 25 °C for 3 min. In a Labconco™ (USA) biosafety bell with a Class II purifier equipped with ultraviolet rays, the supernatant was recovered and used as the inoculum. Nine mL of sterile culture medium was added to three sterile 18×150 mm tubes containing a strip of Whatman™ paper (3×30 mm) and 0.05 g crystalline cellulose (Sigma). In an incubator (Riossa™ EO-71, Mexico), the three tubes were incubated at 39 °C for 24 h to verify their sterility. They were then inoculated with 1 mL of the inoculum and incubated at 39 °C for 7 d until the Whatman™ paper degraded. To obtain only bacteria with the capacity to degrade cellulosic matter, 1 mL of inoculated medium was transferred to another sterile tube and incubated at 39 °C until the Whatman paper was degraded (7 d). Four transfers were performed to obtain a culture of cellulolytic bacteria capable of degrading the cellulose that makes up Whatman paper.

In sterile glass serological vials (50 mL) containing a strip of Whatman paper (3×30 mm) and 0.1 g of crystalline cellulose (Sigma), 27 mL of sterile culture medium were deposited under a constant flow of CO₂, and incubated 72 h at 39 °C to verify sterility. The culture medium in the vials were inoculated with 3 mL of the product obtained from the fourth transfer of the cellulolytic bacterial culture and incubated at 39 °C until degradation of the Whatman paper (10 d). Cellulolytic bacteria require a preservative for lyophilization, and activated carbon satisfies this function. Therefore, 0.1 g activated carbon (Hycel) was added to each vial, which was incubated at 39 °C for 2 h. The vials were frozen in a roller freezer (Labconco^® Shell Freezer, USA) to a temperature of ‒38 °C. Then, in a lyophilizer (Labconco^® Freezone 6 L, USA), the bacteria were lyophilized 24 h in automatic mode (‒50 °C and 13.5 Pa pressure).

Inocula and substrates

Three inocula were used. 1) TRB=total ruminal bacteria obtained from ruminal fluid of a Jersey cow fitted with a ruminal cannula; the cow had grazed on alfalfa pastures before collection of ruminal fluid. The ruminal fluid was centrifuged at 1157 g, 25 °C for 3 min, to precipitate protozoa and fibrous particles. 2) CBC=reactivated cellulolytic bacterial culture. In sterile glass serological vials (50 mL) containing a strip of Whatman paper (3×30 mm) and 0.02 g crystalline cellulose (Sigma), 30 mL of sterile culture medium was deposited under a constant flow of CO₂. The culture medium in the vials was inoculated with 0.05 g of lyophilized CBC under a constant flow of CO₂ and incubated 10 d at 39 °C until the Whatman paper degraded. 3) Co-culture = TRB and CBC (50:50); in both inocula, the concentration of total bacterial was determined by direct count in a Petroff-Hauser chamber (Hausser #39000, Electron Microscopy Sciences, USA) and the formula: bacterial concentration=(average direct count) (dilution factor) 2×10⁷) to equate the concentration of TRB with that of CBC (^{Ley de-Coss et al., 2013}).

The substrates were maize stalks (Zea mays), alfalfa with 35 d of growth, Bermuda grass (Cynodon dactylon) with 45 days of growth, and crystalline cellulose (Sigma). The maize stalks, alfalfa and Bermuda grass were ground, sifted through a 1 mm mesh, and finally washed with running water to eliminate microparticles (<25 𝛍m) and taken to constant weight.

In vitro degradation of dry matter

The 18×150 mm tubes with 0.05 g of a substrate were sterilized at 121 °C and 15 psi for 15 min. Then, 9 mL of sterile culture medium was added under a flow of CO₂ and incubated at 39 °C for 72 h to verify sterility. The tubes inoculated with 1 mL TRB, CBC or co-culture were incubated at 39 °C for 72 h. After incubation, pH was measured with a potentiometer (Orion model 250A, Brazil; calibration: pH 7 and 4). Capacity for in vitro DM degradation (%DMDEG) was calculated as the difference between initial and residual matter after fermentation (^{Getachew et al., 2004}).

Concentration of volatile fatty acids (VFA)

After 72 h of incubation, 1 mL of the culture medium was mixed with 25 % metaphosphoric acid (ratio 4:1) in a microcentrifuge tube (Hettich™ EBA 21, Germany). The tubes were centrifuged at 18 800 g for 10 min. The supernatant was placed in chromatograph vials (1.5 mL, Perkin Elmer, USA). VFA concentration was determined in a gas chromatograph (Perkin Elmer, model Claurus 500, USA) equipped with a flame ionization detector and a 15 m × 0.32 mm capillary column (Elite FFAP, Perkin-Elmer^®); nitrogen was used as the carrier gas with a flow of 4 mL min^-1 and H₂ and O₂ to generate a flame with a flow of 45 and 450 mL min^-1. The oven, injector and column temperatures were 120, 250 and 250 °C. One 𝛍L of sample was injected. In this way, three peaks were obtained at retention times of 2.16, 2.59 and 3.11 for acetate, propionate and butyrate (^{Cobos et al., 2007}).

Statistical design and analysis

The experimental design was completily randomized with a 3×4 factorial arrangement (five independent samples). The factors were inocula (TRB, CBC and co-culture) and substrates (maize stalks, Bermuda grass, alfalfa and crystalline cellulose). The data on %DMDEG, VFA and pH were analyzed with the GLM procedure (^{SAS Institute Inc., 2011}). The averages were fit by least squares for comparison with the Tukey test (p≤0.05). The relationships between variables were analyzed with the Pearson correlation (p≤0.05) (^{SAS Institute Inc., 2011}).

Results and Discussion

The reactivated cellulolytic bacterial culture (CBC), total ruminal bacterial (TRB) and the co-culture produced acetate, propionate and butyrate by fermentation of the carbohydrates present in the cellulosic substrates through heterofermentative activity (Table 1). This heterofermentative activity took place because of the interactions among ruminal microorganisms in the degradation of cellulosic biomass (^{Zhang et al., 2015}): alimentary interdependence and cross alimentation (^{Cobos, 2007}). These results agree with those published by ^{Mateo-Sánchez et al. (2002)} regarding differences in heterofermentation using sawdust as the substrate for Bacteroides stercorys in mixed culture with coccobacilli.

Table 1: Volatile fatty acid production (mM L^-1) of a reactivated cellulolytic bacterial culture (CBC), total ruminal bacteria (BRT) and a co-culture CBC-BRT (50:50) in different cellulosic substrates.

CBA produced more acetate (p≤0.05) than TRB when fermenting with alfalfa and Bermuda grass (Table 1), due to the action of the cellulolytic bacteria, which are fermenters of acetate (^{Zhang et al., 2015}). However, CBC and TRB in crystalline cellulose and maize stalks were not different (p>0.05) since the products of in vitro fermentation depend on the type of inoculum and the type of substrate (^{Weimer et al., 2011}).

The three inocula were not different (p>0.05) in VFA production in the substrates alfalfa, Bermuda grass and maize stalks (Table 2) because cellulolytic bacteria affect only the proportion of acetate in the fermentation of cellulosic substrates (^{Zhang et al., 2015}). Cellulolytic bacteria require neutral pH to function correctly (^{Barboza et al., 2009}; ^{Anrique, 2010}) and are sensitive to pH. In pH below 6.0, cellulolytic bacteria are inhibited (^{Chen et al., 2011}). In our study pH 6.86 to 7.14 (Table 2); therefore pH ranged from did not affect the behavior of the inocula on the evaluated substrates.

Table 2: In vitro degradation of cellulosic substrates inoculated with reactivated cellulolytic bacteria (CBC), total ruminal bacteria (BRT) and co-culture CBC-BRT (50:50).

♦ Average values with different letters in a column are statistically different (p≤0.05); EEM: standard error of the mean. BRT: total ruminal bacteria, concentration 5×10⁹ bacteria mL^-1 (the concentration was equaled to that of CBC); CBC: cellulolytic bacterial consortium, concentration 8.7×10⁸ bacteria mL^-1; Cocultivo: co-culture of BRT and CBC (50:50). %DMDEG, percentage of in vitro degradation of dry matter after 72 h; pH, pH measured after 72 h of incubation; AGV, volatile fatty acids (mM L^-1) after 72 h of incubation.

The TRB in the evaluated substrates fermented more propionate (p≤0.05) than CBC (Table 1) because of the presence of amylolytic bacteria that ferment propionate (^{Anrique, 2010}). Butyrate production of the inocula was less than 4 mM L^-1 in the four substrates (Table 1) because it requires substrates such as fructose or galactose for higher production (^{Oba, 2011}). In our study, structural carbohydrates were fermented.

CBC had the lowest values of in vitro %DEGMS in Bermuda grass, maize stalks and crystalline cellulose. In contrast, CBC and the co-culture in alfalfa had higher values (p≤0.05) than TRB. In crystalline cellulose, maize stalks and Bermuda grass, the co-culture was not different (p>0.05) from TRB in in vitro degradation (Table 2). These results are higher than those published by ^{Juárez et al. (2009)} and ^{Grilli et al. (2011)}, who reported lower in vitro degradation in pangola grass inoculated with ruminal bacteria (^{Juárez et al., 2009}) and alfalfa inoculated with F. succinogenes (^{Grilli et al., 2011}). This is due to the fact that the type of inoculum, conformation of the microbial population and donor species of the inoculum (^{Mould et al., 2005}) are determinant in in vitro degradation of cellulosic substrates.

pH correlated negatively with total VFA and acetate (Table 3). When VFA production increased, pH became acid. This agrees with ^{Relling and Mattioli (2003)} in that, because of its acid feature, the higher the production of VFA, the lower the ruminal pH. Total VFA production correlated positively with %DMDEG, thus, when total VFA production increased, %DMDEG also increased. These results agree with ^{Getachew et al. (2004)}, who point out that the correlation between in vitro degradation and VFA production is positive. Production of acetate increases when that of propionate decreases, based on the negative correlation of these variables (Table 3). This is due to the type of microorganisms present during fermentation of cellulosic substrates (^{Anrique, 2010}).

Table 3: Coefficients of correlation between variables of the in vitro degradation test with cellulosic substrates inoculated with a culture of reactivated cellulolytic bacteria (CBC), total ruminal bacteria (BRT) or a CBC-BRT co-culture (50:50).

♦ ^† Significant coefficient (p≤0.05); ^❡ non-significant coefficient (p>0.05); %DEGMS: percentage of in vitro dry matter degradation after 72 h; pH, measured after 72 h of incubation; AGV: volatile fatty acids (mM L^-1) after 72 h of incubation.

Conclusions

The culture of reactivated cellulolytic bacteria during fermentation in cellulosic substrates exhibited heterofermentative activity, in which acetate was the main product of fermentation, compared with that of total ruminal bacteria. Addition of a culture of reactivated cellulolytic bacteria to ruminal bacteria did not produce synergism in in vitro dry matter degradation of cellulosic substrates. It did, however, improve production of acetate.

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Received: September 2015; Accepted: April 2016

^* Author for correspondence: sanchezsantillanp@gmail.com

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