ion Te at ch nt n e o l m o r g e y F Ahring et al., Ferment Technol 2018, 7:1 Fermentation Technology DOI: 10.4172/2167-7972.1000152 ISSN: 2167-7972 Research Article Open Access Fermentation of Cellulose with a Mixed Microbial Rumen Culture with and without Methanogenesis Birgitte Kiaer Ahring1* , Nanditha Murali2 and Keerthi Srinivas1 1Bioproducts Sciences and Engineering Laboratory, Washington State University, Tri-Cities, Richland, WA -99354, USA 2Gene and Linda Voiland School of Chemical Engineering and Bioengineering and Biological Systems Engineering, Washington State University, Pullman, WA-99163, USA *Corresponding author: Birgitte Kiaer Ahring, Bioproducts Sciences and Engineering Laboratory, Washington State University, Tri-cities 2710 Crimson Way Richland, WA 99354, USA, Tel: +01-(509)-372-7682; Fax: 01-(509)-372-7690; E-mail: [email protected] Received date: May 26, 2018; Accepted date: June 06, 2018; Published date: June 14, 2018 Copyright: © 2018 Ahring BK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Ruminal fermentation has been well studied and includes cellulolytic microorganisms to hydrolyze cellulose to monomers, acidogenic microbes including cellulolytic microorganism to convert the monomers to volatile fatty acids (VFA), hydrogen and carbon dioxide and methanogens to convert the acetic acid, hydrogen and carbon dioxide to methane. Notably, methane production in ruminants causes energy loss for the animal and emitted methane contributes significantly to greenhouse gases in the atmosphere. The present study focuses on selectively inhibiting of the methanogens using 2–bromoethanesulfonate (BES) and its effect on ruminal fermentation in an anaerobic rumen bioreactor model system. It was found that BES inhibited methane production (99.7%) and that addition of BES decreased the total VFA productivity from 3 g/L/day to 1.3 g/L/day. Our study also found that addition of BES not only inhibited the methanogens, but also had an impact on the non-methanogenic bacteria as well, resulting in a decrease in the acetic acid productivity from 1.8 g/L/day, in a reactor without BES to 0.8 g/L/day in reactor with BES added. Endoglucanase assay revealed that addition of BES further inhibits cellulolytic microbes, resulting in a decrease in endoglucanase concentration in the reactor supplemented with BES. A notable increase in hydrogen partial pressure was seen in the reactor with BES (from 1.7% to 29.8%). Keywords: Rumen; Methanogenesis; BES; Volatile fatty acids; particular, is converted to glucose by the action of cellulolytic bacteria Hydrogen; Carbon dioxide through excretion of cellulase enzymes. Enzyme activities including, cellulases, hemicellulases, xylanases, etc. [10] have been confirmed in Introduction the rumen and ruminal cellulase activity is principally located on the bacterial cell surface [11]. The acidogenic and actogenic bacteria utilize Cellulose, the most abundantly available polymer, found in both the monosaccharides from the first hydrolysis step to produce acetic, plant and animal cells as a structural material, is a branched polymer propionic, butyric acids, hydrogen (H2) and carbon dioxide (CO2). The with β-D-glycosidic linkages [1]. Avicel is commercially-available VFA including acetic acid, propionic acid and butyric acids are microcrystalline cellulose produced from acid hydrolysis of absorbed by the intestinal epithelia in the rumen and are the main amorphous cellulose to remove the fibrous hinges [2]. One of the key source of energy for the animal [12]. players in cellulosic degradation is the cellulolytic microorganisms [3], which can biologically convert cellulosic materials to volatile fatty Ruminant gut consists of almost 25% of Firmicutes, including acids (VFA) and gases such as hydrogen and carbon dioxide [4]. The Ruminococcus, Butyrivibrio, Eubacterium and Pseudobutyrivibrio different cellulolytic microorganisms, those that are naturally species, which are responsible for cellulose hydrolysis in the rumen occurring in environment, such as the rumen has shown to be [13]. Interestingly, human gut microbiota has been characterized and it markedly more efficient in degrading cellulose to produce VFAs [4,5]. was found that gut of healthy adults consisted of Firmicutes and In addition to its superior cellulose degrading capabilities, studies have Bacteroidetes with significant amounts of Actinobacteria and indicated that the rumen is also a rich source of different enzymes with Proteobacteria [14]. Additionally, the ruminant gut also contains 60% different activities which will eliminate the need for separate enzymatic Bacteriodes including Bacteroidales and Prevotella, which degrades hydrolysis as often found in bio refineries [6]. Furthermore, hemicellulose and pectin [15]. Hess et al., 2011 [16] found that rumen fermentation using a mixed microbial rumen consortia, being non- fluid consisted of 63% β–1,4–endoglucanases, 86% β–glucosidases and sterile, can further significantly decrease process costs [6,7]. 87% cellobiohydrolases and recent studies have indicated that Bacteriodes and Bacteriodales are predominantly producers of β– Rumen microorganisms comprises of three groups of bacteria: glucanases and xylanases [17]. In addition to cellulolytic bacteria, fermentative bacteria that degrade cellulose anaerobically to produce ruminant gut also consists of acidogenic bacteria including monosaccharides; acidogenic and acetogenic bacteria that convert Acetitomaculum, Propionibacterium, Peptostreptococcus, and these monosaccharides to volatile fatty acids (VFA), and methanogenic Chlostridium, which convert the ‘sugars’ to volatile fatty acids (VFAs) Archae, which further hydrogen and carbon dioxide [8]. The and methanogens including mainly Methanobacter, fermentative bacteria hydrolyze complex compounds like Methanobrevibacter and Methanococcus, which convert formic acid, polysaccharides, proteins and lipids into simpler compounds like monosaccharides, amino acids and fatty acids [9]. The cellulose, in Ferment Technol, an open access journal Volume 7 • Issue 1 • 1000152 ISSN:2167-7972 Citation: Ahring BK, Murali N, Srinivas K (2018) Fermentation of Cellulose with a Mixed Microbial Rumen Culture with and without Methanogenesis. Ferment Technol 7: 152. doi:10.4172/2167-7972.1000152 Page 2 of 7 hydrogen and carbon dioxide to methane [18]. In some instances fermentation products including headspace gases were monitored members of the genus Methanosarcina has further been identified [19]. along with cellulase enzyme activity in the bioreactor. Methanogenesis is an energetically-favorable final step in ruminants (Figure 1) and is vital in the rumen for continuous removal of Materials and Methods hydrogen, production of ATP [20], which results in a stable rumen microbiome. However, methanogensis in the rumen results in almost Inoculum 10-15% energy loss inside the animal as methane just leaves the system Fresh rumen fluid was collected from a slaughterhouse facility in and ends in the atmosphere without any benefits for the animal in the Richland WA [29]. The rumen fluid was transported to the lab in form of meat. Methane emissions significantly contribute to global tightly-sealed bottles and was degassed under 80/20% w/w N -CO warming issues and methane emissions from cattle in the United States 2 2 mixture and stored at -20°C in anaerobic jars until further use. 10% alone contribute to 21% of the global greenhouse gases [21]. Hence, rumen fluid was used as inoculum for all the experiments. extensive studies have been done on inhibiting methanogenesis during rumen fermentation. As methanogenesis functions as an important Chemicals process for keeping stability of the system, increasing the H2 concentration can potentially have negative effect for the overall Sodium hydroxide (5 N), sulfuric acid (4 mM), yeast extract, rumen fermentation. sodium 2-bromoethanesulfonate, cellulase enzyme from Trichoderma reesei and Avicel Ph-101 were obtained from Sigma Aldrich, (St. Louis, MO, USA). Oxarc® Inc. (Pasco, WA, USA) provided the nitrogen carbon dioxide gas mix. Azo-CMC substrate for the cellulase assay was obtained from Megazyme (Bray, County Wicklow, IR). Fermentation Figure 1: Thermodynamic differences between methanogenesis and acetogenesis showing that methanogenesis is energetically more VFA fermentation at different pH favorable (McAllister & Newbold, 2008); ΔGo’ refers to the free Batch experiments were conducted at different pH, including pH energy of formation. 5.5, pH 6.0, pH 6.5 and pH 7.0, all at 37°C. This was done to evaluate the effect of pH on the fermentation products produced. Avicel Ph–101 (2.5%) was used as substrate in all the experiments. The fermentation Studies have shown that inhibition of methanogenesis shifts the media consisted of 2.5% avicel and 0.1% of yeast extract. The hydraulic ruminal fermentation towards propionic acid and an increased butyric retention time (HRT) for the fermentations was 6 days and the acid production [22] or acetogenesis [23]. Studies have also shown that fermentations were carried out for 24 days (4 HRT). Daily gas analysis mitigation of methane emissions can be done via chemical inhibitors, was done to measure headspace gases and High Performance Liquid like ionophores or organic acids, or methane analogs like 2- Chromatography (HPLC) was done to