In vitro production of gas methane by tropical grasses Produccion in vitro de gas metano por gramineas forrajeras tropicales

Objetive. Estimate the production of methane (CH4) by tropical grasses fermented in vitro. Materials and methods. A sample of 20 g dry matter of Cynodon nlemfuensis, Hyparrhenia rufa, Megathyrsus maximus and Digitaria swazilandensis plus 200 ml of culture medium were plated in triplicate flasks sterile stainless steel with CO2 flux, inoculated with 20 ml of ruminal fluid bovine, incubated at 38 °C for 24, 48, 72 and 96 h. Total production of gas, CH4, volatile fatty acids, and pH were evaluated in a completely randomized design with three replicates per treatment and comparison of means with Tukey; the concentration of total and cellulolytic bacteria were analyzed with the Kruskal-Wallis, and the GLM procedure independent data Wilcoxon rank. Results. H. rufa and D. swazilandensis both had the lowest total gas production (p<0.05), while D. swazilandensis had lower production of CH4, increased production of propionic acid (p<0.05) and lower pH 96 hours of incubation (p<0.05). D. swazilandensis showed greater efficiency in energy production due to reduced production of CH4 and increased propionate production. The concentration of total bacteria was similar between treatments (p>0.05), while the concentration of cellulolytic bacteria was lower in C. nlemfuensis y D. swazilandensis when 96 of incubation (p<0.05). Conclusions. The Digitaria swazilandensis, showed favorable conditions to have lower total methane and total gas production.


INTRODUCTION
Ruminants emit between 18 and 25% of the greenhouse gases (GHG), depending on the feeding strategy that has been established, CH 4 is the second largest contributor to this effect (1)(2)(3)(4).Ruminant feed in tropical and subtropical regions is mainly based on the use of forage grasses whose cellulose and hemicellulose content is higher than in temperate climate grasses (5), this higher cell wall content being potentially fermented by cellulolytic bacteria species such as Ruminococcus flavefaciens, Ruminococcus albus and Fibrobacter succinogenes, which transform glucose into acetate and butyrate, whose metabolic pathway produces hydrogen (H 2 ) and carbon dioxide (CO 2 ), which are the main substrates for methanogenic archaea such as Methanobacterium formicicum, Methanobrevibacter ruminantium, Methanomicrobium mobile, Methanosarcina bacteri and Methanosarcina majei (6), where the highest production of CH 4 is produced by this metabolic pathway (6,7).
The production of CO 2 and CH 4 is a necessary process in ruminal biochemistry to obtain energy, this process reduces the accumulation of H 2 and pH reduction, to maintain ruminal ecology under favorable conditions (8).However, this process reduces the efficiency of energy use by the animal by 6.3% for sheep and 6.5% for cattle (9).The use of tropical forage grasses with higher cell content and lower potentially fermentable cell wall content in ruminant feed could allow for greater energy efficiency that contributes to reducing GHG emissions for the purpose of mitigating climate change, Zheng et al (3) y Iñamaga et al (4), reported that feed strategies influenced GHG emissions, also indicate that CO 2 emissions based on the production of fat-corrected milk were higher for high forage feeding strategies.Therefore, the objective of this research was to evaluate the production of total gases (GT) and CH 4 emitted by tropical fodder grasses on in vitro incubation (3,4).Treatments and chemical analysis of grasses.The treatments (pastures) evaluated were T1: Cynodon nlemfuensis; T2: Hyparrhenia rufa; T3: Megathyrsus maximus and T4: Digitaria swazilandensis; with an age of 75 days, during the month of May 2015 (average temperature of 24.81°C, relative humedad of 72.86% and 277 mm of accumulated monthly precipitation at the time of sampling, and 1438.9 mm of precipitation during the year), was obtained from a cattle ranch (El Carmen 9, in Mazatán, Chiapas) located at 14°54´23.93"N and 92°25´37.81"O; 35 meters above sea level.With soil of the Phaeozem (Feosem) type, characterized by a high accumulation of organic matter and by being saturated at the top, the soil is mainly prairie soil, with a móllic epipedion (a relatively thick, dark, humus-rich surface horizon) and without calcium carbonate in the first meter of depth; no fertilization was carried out on the areas of the sampled forage.

MATERIALS AND METHODS
The samples were dried in a drying oven at 60°C for 24 hours and ground in an ED-5 electric mill equipped with a 1 mm screen.For each of the samples, crude protein (CP) was determined by the Kjeldahl method, as well as ethereal extract (EE) and ash content after incineration of the sample in a muffle at 550° C per 4 h according to AOAC (10).Determination of the neutral detergent fiber (NDF) and acid detergent fiber (ADF) fractions according to the technique described by Van Soet et al (11).
The culture medium (Table 1) used to determine the production of total gases (GT) and methane (CH 4 ), in addition to the degradation of MS, was prepared under sterile conditions and CO 2 flow.The inoculum was fresh rumen fluid (FRF) extracted at 2 h pos-prandium from a 500 kg BW bovine (F1, zebu x swiss) with rumen cannula, which received at libitum (received the first ration at 6:00 am and second at 4:00 pm) a diet based on 85% C. nlemfuensis and 15% of a concentrated feed containing 2.7 Mcal of ME and 14% crude protein.
Al terminar el periodo de incubación se midió la producción de gases totales (GT) en el sistema, mediante el desplazamiento de líquidos a través de una trampa con frascos de Mariotte.El agua desplazada se recolectó en una probeta graduada de 500 ml y con ello se determinó la cantidad de GT por los 20 g de MS fermentada.
Para determinar la cantidad de CH 4 producido en cada tratamiento, en una segunda prueba y bajo las mismas condiciones de cultivo, tiempos y repeticiones, en las trampas de frascos Mariotte se adicionó una solución de NaOH (2N) con pH Production of CH 4 .The in vitro production of GT and CH 4 was determined in triplicate with repetition over time of each treatment (grasses) using bottles (biodigesters) with a capacity of 2.0 L with hermetic seal, where the following mixture was added under aseptic and CO 2 flow conditions: 20 g of MS from each grass (1 g of MS for each 10 mL of medium) according to the Williams technique ( 12) plus 200 ml of culture medium (Table 1) each treatment was inoculated with 20 ml of FRF filtered in cotton gauze, incubated at 38±0.5°C under CO 2 flow for 24, 48, 72 and 96 h in a thermoregulation bath.The initial total bacterial concentration was 1.35 x10 8 CFU ml -1 based on the most probable number technique (MPN, 13) at pH 6.74.At the end of the incubation period, the production of total gases (GT) in the system was measured by moving liquids through a trap with Mariotte flasks.The displaced water was collected in a 500 ml graduated cylinder and thus the amount of GT per 20 g of fermented MS was determined.
To determine the amount of CH 4 produced in each treatment, in a second test and under the same culture conditions, times and repetitions, in the Mariotte flask traps was added a solution of NaOH (2N) with pH of 13.67 according to the technique described by Stolaroff (14); the NaOH solution reacts with CO 2 to form sodium carbonate (Na 2 CO 3 ) and the remaining gases released are a mixture of CH 4 , H 2 , N 2 and hydrogen sulphide (15).The CO 2 trap was coupled to the biodigesters using a Tygon hose (internal Φ 5 mm and a length of 35 cm) that was fitted with a hypodermic needle (31.8 mm) and 10 cm long).In all GT production evaluations, the results of each treatment and its respective repetition were corrected for difference with the gas production of the blank samples (200 ml of culture medium plus 20 ml of FRF).

Production of volatile fatty acids (VFA) and microbiological variables.
At the end of each incubation period 5 ml of culture medium were obtained and centrifuged at 18000 G for 10 min; 2.0 ml of the supernatant was mixed 4:1 with 25% metaphosphoric acid, the vials were shaken in a Vortex and re-centrifuged at 35000 G for two minutes, the concentration of VFA was measured using a Claurus 500 gas chromatograph, using the technique and conditions described by Ley de Coss et al (16).In addition, per incubation period, 0.5 ml of culture medium was obtained from each treatment to estimate the concentration of total bacteria (BT) and cellulolytic bacteria (BC) using the MPN technique and culture media similar to those reported by Ley de Coss et al (17)

RESULTS
The lowest total gas production was in H. rufa and D. swazilandensis, in the latter species it had lower CH 4 production, indicating higher energy production efficiency due to higher propionic acid synthesis.
There was no change in BT concentration; however, in pastures with lower CH 4 synthesis there was lower BC concentration.Table 2 shows the results of the chemical composition of the grasses, showing that the crude protein content of H. rufa was less than 7%, while in C. nlemfuensis, M. maximus and D. swazilandensis it was greater than 9%.The NDF content, the lowest value was H. rufa (63.25%), while D. swazilandensis had the highest content of this compound (71.40%), with an 8.15% difference between the two species, when related to the ADF content that was similar among the four species (42.25 to 43.40%), it can be attributed that the highest content of NDF in D. swazilandensis could be due to the higher content of hemicellulose.

Total production of gases and CH 4.
In all the fermented pastures, the highest proportion of gases (Table 3) was obtained in the period from 48 to 72 h, which indicates that in this period the highest activity of the bacteria to degrade the substrate was obtained.When considering the total accumulated gas production per g -1 of dry matter fermented (DMf), it was lower for H. rufa and D. swazilandensis (p<0.05).Producción total de gases y CH 4. En todos los pastos fermentados, la mayor proporción de gases (Tabla 3) se tuvo en el periodo de 48 a 72 h, lo que indica que en este periodo se tuvo la mayor actividad de las bacterias para degradar el substrato.Al considerar la producción total de gas acumulado por g -1 de materia seca fermentada (MSf), fue menor para H. rufa y D. swazilandensis (p<0.05).
Al igual que la producción GT, la mayor proporción en la producción de CH 4 (Tabla 4) ocurrió en el periodo de 48 a 72 h, pero la producción In the same way as GT production, the largest proportion in the production of CH 4 (Table 4) occurred in the period from 48 to 72 h, but the total accumulated production of CH 4 was similar between C. nlemfuensis, H. rufa and D. swazilandensis (p>0.05) as well as between C. nlemfuensis, H. rufa and M. maximus (p>0.05), while there was a difference between M. maximus and D. swazilandensis with lower production (p<0.05).In relation to the percentage of CH 4 of total gas production, for the grasses H. rufa, M. maximus and D. swazilandensis represented 76.5%, while for C. nlemfuensis it was 73.9%, which indicates that the highest proportion of gas produced during fermentation corresponds to this GHG.
The total production of VFA and acetic acid production was similar in the grasses evaluated (p>0.05), while D. swazilandensis had higher production of propionic (p<0.05) and butyric acids (p<0.05).The acetic: propionic ratio showed that during the fermentation of D. swazilandensis the energy loss was lower and was related to the lower production of CH 4 obtained (    There was no difference in BT concentration among treatments (p>0.05) during the entire incubation period and the maximum concentration, in all treatments, was 10 9 cells ml-1 of culture medium.Regarding the concentration of cellulolytic bacteria, at 24 h of incubation, the highest concentration was observed in C. nlemfuensis (p<0.05), at 48 and 72 hours there was no difference among treatments (p>0.05); while at 96 hours it was lower (p<0.05) in C. nlemfuensis and D. swazilandensis (Table 7).

DISCUSSION
Generally, grasses have a low crude protein content, with a lower nitrogen content that limits microbial activity in the rumen (20), Avellaneda et al (21), report values of 6.37 and 71.96% crude protein and NDF, respectively in Guinea grass (Panicum maximum var Mombasa),  (24), which generates a different profile in the production of VFA, producing a higher proportion of propionic acid and therefore less CH 4 .On the other hand, the ruminal fermentation of forages with a higher content of cell wall does not cause a significant decrease in pH, because the greater amount of glucose released is fermented by acetate, in this case, the released H 2 can be used as a substrate by methanogenic archaea, which is associated with higher production of CH 4 (3), as in the case of H. rufa and C. nlemfuensis, while with forages that cause low rumen pH, methanogenesis is decreased as in the case of M. maximus whose pH was less than 6.5 since 72 hours of incubation and D. swazilandensis since 24 hours.
One of the important factors affecting the production of CH 4 is the ratio of produced VFA, specifically the acetic: propionic ratio, which regulates the production and availability of H 2 and subsequent production of CH 4 ; this ratio can vary from 0.9 to 4 and energy utilization is more efficient if the ratio is close to 1.0 (25) In conclusion, the tropical grasses analyzed show a high cell wall concentration, which limits their digestibility and reduces their quality as fodder; however, Digitaria swazilandensis showed a lower total production of methane and total gases, possibly due to a higher concentration of propionic acid, lower concentration of cellulolytic bacteria, a pH and a lower acetic:propionic ratio, being the most efficient in energy use.

Area of study.
The study was developed in the Laboratory of Animal Science of the Faculty of Agricultural Sciences, Campus IV of the University Autonomous University of Chiapas located in Huehuetán, Chiapas, Mexico and the Ruminal Microbiology and Microbial Genetics Laboratory of the Postgraduate School Livestock Program, Montecillos Campus, Texcoco, Mexico.

Table 3 .
Total gas production of tropical grasses C. nlemfuensis, H. rufa, M. maximus and D. swazilandensis on in vitro incubation.

Table 4 .
CH 4 production by period and total accumulated CH 4 of tropical grasses C.

swazilandensis SEM 1
a, b, c Means with different letters in the same row are different (p<0.05)1Standarderror of mean.

Table 5 .
Production of volatile fatty acids from tropical grasses C. nlemfuensis, H. rufa, M. maximus and D. swazilandensis in vitro incubation.

Table 6 ,
shows the pH of the medium during 96 h of fermentation.D. swazilandensis and M. maximus had the lowest pH at 24, 72 and 96 h of incubation, even less than 6 at 96 h .

Table 6 .
pH of the culture medium in which the tropical grasses C. nlemfuensis, H. rufa, M. maximus and D. swazilandensis were fermented in vitro.
a, b, c Means with different letters in the same row are different (p<0.05)1Standarderror of mean.

Table 7 .
Concentration of total and cellulolytic bacteria in the culture medium in in vitro incubation.

swazilandensis SEM 1
a, b, c Means with different letters in the same row are different (p<0.05)1Standarderror of mean.