Renan Stefanini

We examined the microbial populations present in fecal samples of macropods capable of utilizing a mixture of hydrogen and carbon dioxide (70:30) percent. The feces samples were cultured under anaerobic conditions, and production of methane or acetic acids characteristic for methanogenesis and homoacetogenesis was measured. While the feces of adult macropods mainly produced methane from the substrate, the sample from a 2-month-old juvenile kangaroo only produced acetic acid and no methane. The stable highly enriched culture of the joey kangaroo was sequenced to examine the V3 and V4 regions of the 16S rRNA gene. The results showed that over 70% of gene copies belonged to the Clostridia class, with Paraclostridium and Blautia as the most predominant genera. The culture further showed the presence of Actinomyces spp., a genus which has only been identified in the GI tract of macropods in a few studies, and where none, to our knowledge, have been classified as homoacetogenic. The joey kangaroo mixed culture showed a doubling time of 3.54 h and a specific growth rate of 0.199/h, faster than what has been observed for homoacetogenic bacteria in general. IMPORTANCE: Enteric methane emissions from cattle are a significant contributor to greenhouse gas emissions worldwide. Methane emissions not only contribute to climate change but also represent a loss of energy from the animal's diet. However, methanogens play an important role as hydrogen sink to rumen systems; without it, the performance of hydrolytic organisms diminishes. Therefore, effective strategies of methanogen inhibition would be enhanced in conjunction with the addition of alternative hydrogen sinks to the rumen. The significance of our research is to identify homoacetogens present in the GI tract of kangaroos and to present their performance in vitro , demonstrating their capability to serve as alternatives to rumen methanogens.

A homoacetogenic consortium was cultivated from feces from a nursing joey red kangaroo and inoculated into an in vitro ruminal culture. The in vitro ruminal culture was treated with methanogenic inhibitor 2-bromoethanesulfonate (BES), followed by two different homoacetogenic inoculation strategies. Initial observations showed inhibitory effects of BES, with stabilization of the acetic acid concentrations without any increase in concentration, even with the homoacetogenic inoculation. When homoacetogenic bacterial culture was added after the BES addition had ceased, acetic acid production was increased 2.5-fold. Next-generation sequencing showed an increased population of Bacteroidetes after inoculation with the homoacetogenic consortia, along with a slight decrease in diversity. An Almond Shell biochar (AS) addition resulted in a 28% increase in acetic acid concentration if tested directly on the homoacetogenic kangaroo consortia. However, when applied to the rumen culture, it did not enhance acetate production but further promoted other reductive pathways such as methanogenesis and propiogenesis, resulting in increased concentrations of methane and propionic acid, respectively. These findings demonstrate that bioaugmentation with homoacetogenic bacteria can improve acetic acid production of an in vitro rumen culture when methanogenesis has been eliminated. Such advancements can potentially contribute to the optimization of rumen fermentation processes and may have practical implications for improved livestock feed efficiency and methane mitigation strategies.

Homo-acetogens are microbes that have the ability to grow on gaseous substrates such as H₂/CO₂/CO and produce acetic acid as the main product of their metabolism through a metabolic process called reductive acetogenesis. These acetogens are dispersed in nature and are found to grow in various biotopes on land, water and sediments. They are also commonly found in the gastro-intestinal track of herbivores that rely on a symbiotic relationship with microbes in order to breakdown lignocellulosic biomass to provide the animal with nutrients and energy. For this motive, the fermentation scheme that occurs in the rumen has been described equivalent to a consolidated bioprocessing fermentation for the production of bioproducts derived from livestock. This paper reviews current knowledge of homo-acetogenesis and its potential to improve efficiency in the rumen for production of bioproducts by replacing methanogens, the principal H₂-scavengers in the rumen, thus serving as a form of carbon sink by deviating the formation of methane into bioproducts. In this review, we discuss the main strategies employed by the livestock industry to achieve methanogenesis inhibition, and also explore homo-acetogenic microorganisms and evaluate the members for potential traits and characteristics that may favor competitive advantage over methanogenesis, making them prospective candidates for competing with methanogens in ruminant animals.