Product accumulation and its control during microbial carbohydrate Fermentations by Rumen- and other mixed microbial consortia

This abstract compiles the abstracts of the individual thesis chapters. Anaerobic acid stage fermentation of carbohydrates can generate a variety of desired or undesired end-products. Besides physical parameters such as pH and temperature, the types of carbohydrate being fermented influences the fermentation end-products. The results of the current study indicate that under uncontrolled pH, microbial mixed cultures from activated sludge and anaerobic digester sludge anaerobically produced ethanol from glucose while producing lactic acid from starch conversion. This trend was confirmed by batch and chemostat trials. After shifting from glucose to starch feed or vice versa the chemostat enrichment culture responded by shifting from ethanol to lactic acid or the reverse. Results also showed that only 25% of starch was converted to the lactic acid. The low conversion yield could be explained by the low pH in the broth that is known to become inhibitory already at a pH of below 5. As maltose is an intermediate sugar derived from the digestion of starch, it was tested as a fermentation substrate and compared to glucose fermentation. Results showed that independent of the inoculum source maltose supported lactic fermentation while glucose led to ethanolic fermentation. The trend was confirmed in batch as well as chemostat culture. Under uncontrolled pH, fermentation of maltose ceased with the production of small amounts of lactic acid and acetate as the main metabolites, while fermentation of glucose continued and produced ethanol as the main end-product. Further investigation with other disaccharides (lactose and sucrose) showed that lactose was fermented to lactic acid and acetate as the main metabolites while sucrose formed ethanol as the major fermentation end-product. Comparative experiments showed that mixed microbial consortia and chemostat enrichments reproducibly produced lactate from maltose fermentation and ethanol from glucose fermentation. However, when using rumen bacteria as the inoculum, lactic acid was the key fermentation end-product, suggesting that rumen microflora comprises larger populations of lactic acid producing bacteria. Lactic acid accumulation in the rumen is a well-known problem known as rumen acidosis and was investigated further in this study. Acute ruminal acidosis can occur when pH in the rumen drops below 5.0. This condition can completely disrupt rumen microbiota leading to the mortality of ruminants. The results of the current study showed that acidosis could occur within 6 hours of incubation in the rumen culture fermenting sugars or starch. Along with the formation of lactic acid, acetic acid alone or in mixture with ethanol was found to be the reason for high acid build-up in the rumen. Acidosis resulted even with only 20% of a normal daily feed load for all soluble and non-soluble carbohydrates. DNA-based microbial analysis revealed that Prevotella was the dominant species present in the rumen fluid, which is considered as the main lactic acid producing bacteria. The phenomenon of acidosis comprises at least two components, a dramatic drop in pH and the over-production of lactic acid. Two approaches can be used to ameliorate the effects, the addition of pH buffering or neutralizing species, or the addition of active microbial cultures that can degrade lactate at a rate that reduces lactate accumulation. The former can be harmful to the animal when the buffering is locally too concentrated due to a lack of uniform distribution; the latter can be costly, if produced as a pure strain. The results of the current study showed that from rumen cultures, mixed microbial lactic acid utilizing bacteria (LUB) could be enriched and subsequently used as a controlling agent against in vitro acidosis. The addition of liquid cultures of LUB enrichments to a rumen culture that was producing lactic acid from fermentation of starch resulted in a reduction of lactic acid, and produced VFA with acetate and propionate as the main metabolites. Breeding lactate utilizing bacteria (LUB) from rumen fluid that can be used as probiotics is potentially useful for the cattle industry. This is because probiotics can be used as a supplement to prevent lactic acidosis when ruminants are fed with readily fermentable carbohydrates. The results of the current study showed that the addition of concentrated cell suspensions of LUB as probiotics to rumen cultures fermenting corn starch successfully prevented lactic acid accumulation, and could also be used as a therapy by converting lactic acid accumulated in rumen cultures into volatile fatty acids (VFA) with acetate and propionate as the main end-products. The combination of concentrated cell suspensions of LUB with buffer could prevent lactic acid accumulation, and rapidly degrade lactic acid without the use of excessive amounts of LUB probiotics. Metagenomic sequencing analysis showed the key LUB that was enriched to include Bacteroides, Acinetobacter, Oscillospira, Clostridium, Dysgonomonas and Pseudomonas.