Optimizing Ruminal Fermentation with Organic Acids Scott A. Martin Department of Animal and Dairy Science The University of Georgia, Athens 30602-2771 Introduction Only some strains of S. ruminantium (subspecies lactilytica) are able to ferment lactate (Stewart and Much interest has been generated over the Bryant, 1988). past few years aimed at evaluating alternative means to manipulate the gastrointestinal microflora in production Early research showed that S. ruminantium livestock. Motivation for examining these alternatives HD4 requires L-aspartate, CO2, p-aminobenzoic acid, comes from increasing public scrutiny about the use of and biotin for growth in a lactate-salts medium antibiotics in the animal feed industry. However, (Linehan et al., 1978). In addition, the requirement for compared to the amount of information available L-aspartate can be replaced by L-malate or fumarate. detailing the effects of antimicrobial compounds on Because little work had been done in this area since the ruminal fermentation, little research has been conducted report by Linehan et al. (1978), my laboratory initiated to evaluate alternatives to antimicrobial compounds. In studies to evaluate the effects of aspartate, fumarate, the past 10 yr, interest has increased in direct-fed and malate on growth and lactate uptake by S. microbial (DFM) products and research has been ruminantium HD4 (Nisbet and Martin, 1990). Growth conducted to examine the effects of DFM on ruminant of S. ruminantium HD4 in medium that contained DL- performance. While some of these products have lactate was stimulated approximately two-fold by 10 shown promise in favorably altering the ruminal mM L-aspartate, fumarate, or L-malate after 24 h of fermentation and improving animal performance, the incubation (Nisbet and Martin, 1990). Both L-aspartate effects are variable and inconsistent (Martin and Nisbet, and fumarate increased L-lactate uptake over 4-fold, 1992). Recent research has shown that organic acids while L-malate stimulated uptake over 10-fold (Figure stimulate growth of the prominent ruminal bacterium 1). In addition, different concentrations (0.03 to 10 Selenomonas ruminantium, favorably alter the mixed mM) of L-malate stimulated L-lactate uptake by S. ruminal microorganism fermentation, and improve the ruminantium HD4 in a dose response fashion (Table performance of feedlot steers (Nisbet and Martin, 1990, 1). D-Lactate uptake by S. ruminantium HD4 was also 1991, 1993, 1994; Callaway and Martin, 1996; Martin et stimulated by 10 mM L-malate (Nisbet and Martin, al., 1999). Therefore, the objective of this paper is to 1993). provide an overview of this research with organic acids and discuss the potential applications in beef and dairy Sodium concentrations between 25 and 100 cattle. mM stimulated L-lactate uptake by S. ruminantium HD4 in the presence of 10 mM L-malate, whereas Pure Culture Studies uptake in the absence of L-malate was low regardless of the Na+ concentration (Nisbet and Martin, 1994; Figure 2). These results suggest that both L-malate Batch Culture Experiments and Na+ play a role in stimulating L-lactate utilization by S. ruminantium HD4. Sodium is the predominant S. ruminantium is a common gram-negative cation in the rumen and concentrations range between ruminal bacterium that can account for up to 51% of 90 to 150 mM (Durand and Kawashima, 1980). the total viable bacterial counts in the rumen (Caldwell and Bryant, 1966). This bacterium can grow under a A more recent isolate, Selenomonas variety of dietary conditions and ferment many ruminantium H18, also requires organic acids to grow different soluble carbohydrates (Hungate, 1966). on lactate (Strobel and Russell, 1991). However, strain When S. ruminantium is grown in batch culture with H18 differs from strain HD4 in that lactate could be glucose, a homolactic fermentation occurs (Hobson, used to support growth only when Na+ and aspartate 1965). However, after the glucose is depleted from the were added to the medium. Malate or fumarate could medium, S. ruminantium then utilizes the lactate as a replace aspartate, but Na+ was not required (Strobel carbon and energy source (Scheifinger et al., 1975). and Russell, 1991). 11 16 14 12 10 8 6 4 Uptake (nmol/mg protein per min) 2 0 None Aspartate Fumarate Malate Additions (10 mM) Figure 1. Effects of aspartate, fumarate, and malate on lactate uptake by whole cells of Selenomonas ruminantium HD4 (Nisbet and Martin, 1990). Fumarate and malate are four-carbon continuous culture (Evans and Martin, 1997). When S. dicarboxylic acids that are commonly found in ruminantium HD4 was grown on lactate at pH 6.8, the biological tissues because they are intermediates of primary end products were acetate and propionate with the citric acid cycle (Lehninger, 1975). Some strictly all concentrations of lactate. Little succinate or malate anaerobic bacteria use a reductive or reverse citric was produced. Even though not all lactate was utilized, acid cycle known as the succinate-propionate as the lactate concentration was increased there was a pathway to synthesize succinate and(or) propionate corresponding increase in optical density at 600 nm (Gottschalk, 1986). Both malate and fumarate are key (OD600), protein, and carbohydrate. These results intermediates in the succinate-propionate pathway, suggested that lactate was limiting growth. Lactate and S. ruminantium uses this pathway (Gottschalk, utilization ranged between 35 and 50%. 1986). The fact that dicarboxylic acids, especially malate and fumarate, stimulate lactate utilization is S. ruminantium HD4 was unable to grow consistent with the presence of this pathway in this (culture washout) on 6 mM lactate at an extracellular ruminal anaerobe. pH of 5.5 (Evans and Martin, 1997). Growth did occur on 30 and 54 mM lactate at this pH and acetate and Continuous Culture Experiments propionate were the main end products that were produced. Little malate or succinate accumulated. Bacterial protein and OD increased as lactate In general, the dilution rate within the rumen is 600 concentration increased and there was a decrease in between 0.05 and 0.10 h-1 (Hungate, 1966). Most studies cellular carbohydrate. When 8 mM malate was added aimed at evaluating lactate utilization by S. ruminantium to the growth medium, strain HD4 was able to grow on have been conducted in batch culture, and few 6 mM lactate at pH 5.5 and 80% of the lactate was experiments have been performed in continuous culture. utilized. Acetate, propionate, and succinate were the Therefore, experiments were conducted to examine the primary fermentation products produced with all three effects of extracellular pH, lactate concentration, and malate addition on growth of S. ruminantium HD4 in 12 Table 1. Effect of increasing concentrations of L-malate on L-lactate uptake by Selenomonas ruminantium (Nisbet and Martin, 1991). L-Malate, mM Specific activitya 0 0.8 + 0.04 0.03 1.4 + 0.03* 0.06 2.7 + 0.13* 0.12 3.2 + 0.13* 0.48 5.6 + 0.13* 2.5 8.5 + 0.22* 5.0 10.4 + 1.40* 10.0 12.2 + 0.09* aNanomoles per milligram of protein per minute, mean + SD. *P < 0.05. lactate concentrations in the presence of malate. may be possible to improve the ability of S. Malate addition increased the amount of lactate utilized ruminantium HD4 to utilize lactate at pH 6.0. and OD600, as well as the concentrations of protein and cellular carbohydrate synthesized by strain HD4. Mixed Culture Studies Lactate utilization ranged between 77 and 80% in the presence of malate compared to 40 and 70% in the absence of malate. Malate utilization ranged between Based on the stimulation of lactate utilization 51 and 64%. Similar effects were seen when strain HD4 by dicarboxylic acids in S. ruminantium and because was grown at a dilution rate of 0.10 h-1 (Evans and information was limited detailing the effects of organic Martin, 1997). acids on ruminal fermentation, experiments were conducted to evaluate the effects of L-aspartate, When domestic ruminants (beef and dairy fumarate, and DL-malate on the in vitro mixed ruminal cattle) are fed diets high in rapidly fermentable microorganism fermentation (Martin and Streeter, 1995; carbohydrates (i.e., cereal grains), lactate can Callaway and Martin, 1996). Fermentation of cracked accumulate and decrease ruminal pH (Slyter, 1976; corn in the presence of 8 or 12 mM DL-malate resulted Russell and Hino, 1985; Russell and Strobel, 1989). in an increase in final pH and propionate concentration Lactate concentrations as high as 29 mM have been (Martin and Streeter, 1995). Total VFA tended to observed with these types of diets (Counotte et al., increase, while final concentrations of L-lactate 1981). If lactate concentrations remain elevated, numerically decreased. In the case of soluble starch, 8 ruminal pH will drop below 6.0 and this leads to a and 12 mM DL-malate caused a decrease in CH4 variety of microbial and physiological problems concentration. When only ruminal fluid (no added (decreased fiber digestion, decreased digesta turnover, anaerobic medium) was used as the inoculum rather decreased salivation, rumen ulceration, founder, death) than 20% ruminal fluid medium, similar results for final (Slyter, 1976; Russell and Hino, 1985). pH, propionate, L-lactate, and total VFA were observed for soluble starch and corn incubations treated with Based on our continuous culture results, it DL-malate. These changes in fermentation products appears that malate enhances the ability of strain HD4 are analogous to ionophore effects (Russell and to grow on all three lactate concentrations at an Strobel, 1989). extracellular pH of 5.5 (Evans and Martin, 1997). These results are consistent with the observation that malate To determine whether organic acids plus treatment increased final pH and decreased lactate monensin have an additive effect on ruminal concentrations in mixed ruminal microorganism fermentation, the effects of organic acid (L-aspartate, fermentations of cracked corn and soluble starch fumarate, or DL-malate) and monensin treatment on the (Martin and Streeter, 1995; Callaway and Martin, 1996).