Factors Limit Ruminal Microbial Biomass Yield
While attending the World Dairy Expo this past October, I had the distinct pleasure of spending a few hours listening to Drs. Mary Beth Hall, Mehmet Basalan and Scott Dennis discuss ruminant microbiology.
Their unique perspectives made for a very interesting discussion on the interaction of different protein sources and carbohydrate levels on microbial growth, bacterial glycogen deposition and energy spilling.
After returning home and digging into my James B. Russell Rumen Microbiology textbook, I now have a somewhat better understanding of the concepts they were discussing. I decided to devote this column to sharing my newfound appreciation of the importance of feedstuff associative effects and how synchrony between protein and carbohydrates can affect the efficiency and yield of ruminal microbial biomass.
Ruminal fermentation is a process that converts carbohydrates and other substances to partially oxidized end products, with some of the freed energy trapped as adenosine triphosphate (ATP). The ability of microbes to survive (maintenance) and reproduce is driven by the energy from ATP hydrolysis (Russell, 2002).
When bacteria grow slowly, a large portion of this energy is used just to maintain cells. Maintenance costs are associated with cell motility, turnover of macromolecules such as protein and, most important, establishment of ion gradients across cell membranes.
Continuous culture studies with mixed ruminal bacteria suggest that maintenance energy accounts for 10- 31% of the total energy consumed. When microbial growth occurs, protein synthesis accounts for nearly 65% of the total ATP requirement. Estimates are that the overall efficiency of cell growth is about 12%, with the remaining 88% dissipated as heat (Russell, 2007). This loss of energy as heat is one example of microbial energy spilling (loss).
Intracellular pH regulation can also affect the efficiency of bacterial growth and heat production. Acid-tolerant bacteria (e.g., starch fermenters) allow their intracellular pH to decline, which affects the anabolic rate to a greater extent than the catabolic rate. This imbalance leads to energy spilling and additional heat production (Russell, 2007).
Matching microbial catabolic rates with their anabolic rates can reduce energy spilling and improve the effi ciency of bacterial growth (Russell, 2007). If energy is in excess and microbial growth is limited by other dietary factors (e.g., amino acids), the rate of stationary (resting) cell metabolism can exceed maintenance levels by nearly 18-fold (Russell, 2007).
A study by Russell and Strobel (1990) showed that Streptococcus bovis demonstrated a fermentation rate of 90 mmol of glucose per gram of bacterial protein per hour when incubated in a nitrogen-free medium with an excess of glucose. However, the maintenance rate (as measured under carbon limitation) was only 1.6 mmol of glucose per gram of bacterial protein per hour.
It appears, from this study, that S. bovis had a third avenue of energy expenditure, beyond maintenance and growth, that can be classifi ed as energy spilling. It is known that non-growing S. bovis are capable of "spilling" as much ATP as is utilized by those that are growing rapidly (Russell, 2007).
In the case of S. bovis, energy spilling is due to increased cell membrane ATPase activity and the futile cycle of protons (proton pump) through the cell membrane. When glucose is in excess and the glycolytic rate is faster than the rate at which ATP can be used for growth, fructose 1,6-bis phosphate accumulates. The resulting decrease in intracellular phosphate causes an increase in ATP hydrolysis to pump protons out of the cell (Russell, 2007).
This is another example of energy spilling whereby dietary energy intake is not contributing to increasing microbial biomass production.
Energy spilling is most common when cells are limited for nutrients other than energy (e.g., amino acids), but even rapidly growing cells can spill a significant amount of energy (Russell, 2007). Some theorize that bacteria that spill energy may be better suited for rapid growth when nutrient limitations no longer exist. The only cells that do not seem to spill energy are those limited for energy (Russell, 2002).
|FEEDING TIME: One approach to increasing the efficiency of ruminal microbial biomass production is to minimize the impact of bacterial maintenance expenditures.When cows consume large meals, the rumen operates as a batch culture, with the soluble materials depleted rapidly.
When exogenous energy sources are depleted, some bacteria (not S. bovis) have endogenous sources to sustain their viability (Russell, 2002). This endogenous source is carbohydrate sequestered as glycogen and has essentially the same structure as starch (Hall, 2012b).
Glycogen storage increases with increasing levels of available carbohydrate. While glycogen can be used as an energy source during times of increased cellular demand for ATP, it is not a particularly efficient process for storage of carbohydrate because glycogen costs one ATP per hexose, which is 25-50% of total ATP from the fermentation of a hexose (Hall, 2012b).
Researchers at The Ohio State University (Hackmann et al., 2012) investigated whether mixed rumen microbes direct excess carbohydrate toward glycogen synthesis, energy spilling or both. Cultures were maintained under anaerobic conditions (39°C) and contained prokaryotes, protozoa and fungi. Cultures were dosed with 5 mmol or 20 mmol of glucose. Heat production, free glucose, cell protein and glycogen were measured.
For cultures dosed with 5 mmol of glucose, endogenous metabolism and glycogen synthesis accounted for all of the heat production, with no energy spilling detected. For cultures dosed with 20 mmol of glucose, endogenous metabolism and glycogen synthesis accounted for all of the heat production during early incubation time points. Energy spilling was detectable by approximately 30 minutes, and in one culture, it eventually accounted for as much as 30% of heat production.
After glucose was exhausted, cultures dosed with 20 mmol of glucose degraded glycogen rapidly and continued to spill energy. This implies that spilling may be mediated by rapid glycogen degradation.
As demonstrated in pure culture studies, mixed cultures can also spill energy. However, mixed cultures tested in the Hackmann et al. study responded mainly to small excesses in carbohydrate predominantly by glycogen synthesis rather than energy spilling.
Role of protein
Dietary protein appears to influence ruminal organic acid production by influencing whether carbohydrate is fermented immediately, stored as glycogen or wasted via energy spilling pathways (Hall, 2012b).
In vitro studies (Argyle and Baldwin, 1989) have shown that when the supply of amino acids and peptides was increased, microbial biomass yield increased linearly at each level of carbohydrate tested (Hall, 2012b). When bacteria are forced to use ammonia, they grow more slowly than if amino nitrogen is available, resulting in a lower anabolic growth rate than the catabolic rate. This imbalance leads to excess ATP that is then dissipated by energy spilling (Russell, 2007).
Microbial glycogen storage has been shown to increase as rapidly available carbohydrate levels increase (Prins and Van Hoven, 1977). Conversely, glycogen levels have been shown to decrease as dietary protein is increased (McAllan and Smith, 1974), presumably because the synchrony of nutrients allows energy to be directed to microbial growth rather than glycogen storage.
An in vivo study with lactating cows fed grass/clover silage and various supplements supported the glycogen storage concept by demonstrating a net ruminal synthesis of starch (microbial glycogen) that was passed to the small intestine (Larson et al., 2009).
A study with fresh-cut grass fertilized with urea to provide a high or low nitrogen content showed an unexpected effect in that the higher-nitrogen forage drove more total volatile fatty acid production compared to carbohydrate supplementation treatments (Carruthers and Neil, 1997).
Aldrich et al. (1993) showed that cows given more dietary rumen degradable protein (RDP) tended to have greater ruminal concentrations of organic acids despite the lack of difference among protein treatments in ruminal organic matter digestion, which could have explained the increased organic acid production (Hall, 2012b).
These are examples of studies showing a protein effect on what is usually considered a carbohydrate-driven response (Hall, 2012b).
Hall (2012a) presented data at the 2012 American Dairy Science Assn. meeting on the ruminal effects of corn sources and ruminal degradability of dietary protein (i.e., RDP). Treatments were dry-ground corn or high-moisture corn and +RDP (added protein from soybean meal) or -RDP (heat-treated expeller soybean product partially substituted for soybean meal). Diets were formulated to be isonitrogenous and similar in starch and neutral detergent fiber.
The +RDP treatment had a significant effect on increasing organic acid concentrations and lowering ruminal pH, leading Hall to question if this was due to more immediate fermentation of starch rather than storage as microbial glycogen. Unfortunately, the presence of dietary starch precluded the measurement of glycogen. Hall concluded that RDP effects need further exploration.
These studies also raise the possibility that dietary RDP levels may need to be adjusted throughout the feeding season in response to the increase in starch digestibility observed over time in ensiled corn silage and high-moisture grains (Feedstuffs, June 11, 2007).
The Bottom Line
One approach to increasing the efficiency of ruminal microbial biomass production is to minimize the impact of bacterial maintenance expenditures. When cows consume large meals, the rumen operates as a batch culture, with the soluble materials depleted rapidly. Thereafter, bacterial growth is dependent on the slower degradation rates of insoluble materials.
Fiber-digesting bacterial species have a lower maintenance energy requirement than non-fiber carbohydrate (NFC)- digesting bacteria. However, fiber-digesting bacteria grow more slowly than NFC-digesting bacteria, and rapid growth of NFC-digesting populations minimizes their overall maintenance expenditures and heat production. The potential problem with this approach is the maladies that result from high total volatile fatty acid loads contributing to subclinical acidosis (Russell, 2007).
When energy is not limiting, or when other key nutrients for cellular growth such as amino acids are lacking, some rumen bacteria deposit glycogen as an energy storage mechanism. However, this pathway does have an energy cost compared to when carbohydrates are directly metabolized via glycolysis. There is a need for a better understanding of how ruminal passage rates affect the fate of microbial glycogen: whether it is fermented and recycled in the rumen or passed on to intestinal digestion (Hall, 2012b).
Laboratory methods, such as Fermentrics, that allow for the direct measurement of microbial biomass can prove extremely helpful in understanding the associative effects of dietary ingredients. Ingredient associative effects are likely a significant contributing factor to why some diets perform as predicted while others don't seem to elicit the expected response.
It may be that these associative effects are affecting whether ATP is being utilized efficiently for microbial growth, stored as glycogen for later use or wasted via energy spilling due to lack of synchrony of dietary nutrients.
Aldrich, J.M., L.D. Muller, G.A. Varga and L.C. Griel Jr. 1993. Nonstructural and carbohydrate effects on rumen fermentation, nutrient flow and performance of dairy cows. J. Dairy Sci. 76:1091-1105.
Argyle, J.L., and R.L. Baldwin. 1989. Effects of amino acids and peptides on rumen microbial growth yields. J. Dairy Sci. 72:2017-2027.
Carruthers, V.R., and P.G. Neil. 1997. Milk production and ruminal metabolites from cows offered two pasture diets supplemented with non-structural carbohydrate. N.Z. J. Agric. Res. 40:513-521.
Hackmann, T.J., K.L. Backus and J.L. Firkins. 2012. Mixed rumen microbes respond to excess carbohydrate by synthesizing glycogen and spilling energy. J. Dairy Sci. 95 (suppl. 2):LB5 (abstr.).
Hall, M.B. 2012a. Corn source and dietary protein degradability: Effects on ruminal measures and proposed mechanism for degradable protein effects. J. Dairy Sci. 95 (suppl. 2):W323 (abstr.).
Hall, M.B. 2012b. Protein and carbohydrate interactions in the rumen - Protein does what? Proceedings of 33rd Western Nutrition Conference. Sept. 19-20. Winnipeg, Man.
Larson, M.P., P. Lund, M.R. Weisberg and T. Hvelplund. 2009. Digestion site of starch in cereals and legumes in lactating dairy cows. Anim. Feed Sci. Technol. 153:236-248.
McAllan, A.B., and R.H. Smith. 1974. Carbohydrate metabolism in the ruminant: Bacterial carbohydrates formed in the rumen and their contribution to digesta entering the duodenum. Br. J. Nutr. 31:77-88.
Prins, R.A., and W. Van Hoven. 1977. Carbohydrate fermentation by the rumen ciliate Isotricha prostoma. Protistologica 13:549-556.
Russell, J.B. 2002. Rumen microbiology and its role in ruminant nutrition. Cornell University.
Russell, J.B. 2007. Can the heat of ruminal fermentation be manipulated to decrease heat stress? Proceedings of 22nd Annual Southwest Nutrition & Management Conference. Feb. 22-23. Tempe, Ariz.
Russell, J.B., and H.J. Strobel. 1990. ATPase-dependent energy spilling by the ruminal bacterium, Streptococcus bovis. Arch. Microbiol. 153:378-383.
This article was originally published in December 2012 Feedstuffs issue, and is reproduced with their permission.