Evolving understanding of rumen methanogen ecophysiology.

Production of methane by methanogenic archaea, or methanogens, in the rumen of ruminants is a thermodynamic necessity for microbial conversion of feed to volatile fatty acids, which are essential nutrients for the animals. On the other hand, methane is a greenhouse gas and its production causes energy loss for the animal. Accordingly, there are ongoing efforts toward developing effective strategies for mitigating methane emissions from ruminant livestock that require a detailed understanding of the diversity and ecophysiology of rumen methanogens. Rumen methanogens evolved from free-living autotrophic ancestors through genome streamlining involving gene loss and acquisition. The process yielded an oligotrophic lifestyle, and metabolically efficient and ecologically adapted descendants. This specialization poses serious challenges to the efforts of obtaining axenic cultures of rumen methanogens, and consequently, the information on their physiological properties remains in most part inferred from those of their non-rumen representatives. This review presents the current knowledge of rumen methanogens and their metabolic contributions to enteric methane production. It also identifies the respective critical gaps that need to be filled for aiding the efforts to mitigate methane emission from livestock operations and at the same time increasing the productivity in this critical agriculture sector.

Nitrogen transformation processes catalyzed by manure microbiomes in earthen pit and concrete storages on commercial dairy farms.

Storing manure is an essential aspect of nutrient management on dairy farms. It presents the opportunity to use manure efficiently as a fertilizer in crop and pasture production. Typically, the manure storages are constructed as earthen, concrete, or steel-based structures. However, storing manure can potentially emit aerial pollutants to the atmosphere, including nitrogen and greenhouse gases, through microbial and physicochemical processes. We have characterized the composition of the microbiome in two manure storage structures, a clay-lined earthen pit and an aboveground concrete storage tank, on commercial dairy farms, to discern the nitrogen transformation processes, and thereby, inform the development of mitigation practices to preserve the value of manure. First, we analyzed the 16S rRNA-V4 amplicons generated from manure samples collected from several locations and depths (0.3, 1.2, and 2.1-2.75 m below the surface) of the storages, identifying a set of Amplicon Sequence Variant (ASVs) and quantifying their abundances. Then, we inferred the respective metabolic capabilities. These results showed that the manure microbiome composition was more complex and exhibited more location-to-location variation in the earthen pit than in the concrete tank. Further, the inlet and a location with hard surface crust in the earthen pit had unique consortia. The microbiomes in both storages had the potential to generate ammonia but lacked the organisms for oxidizing it to gaseous compounds. However, the microbial conversion of nitrate to gaseous N2, NO, and N2O via denitrification and to stable ammonia via dissimilatory nitrite reduction seemed possible; minor quantities of nitrate was present in manure, potentially originating from oxidative processes occurring on the barn floor. The nitrate-transformation linked ASVs were more prevalent at the near-surface locations and all depths of the inlet. Anammox bacteria and archaeal or bacterial autotrophic nitrifiers were not detected in either storage. Hydrogenotrophic Methanocorpusculum species were the primary methanogens or methane producers, exhibiting higher abundance in the earthen pit. These findings suggested that microbial activities were not the main drivers for nitrogen loss from manure storage, and commonly reported losses are associated with the physicochemical processes. Finally, the microbiomes of stored manure had the potential to emit greenhouse gases such as NO, N2O, and methane.

Nutritional Evaluation of Black Soldier Fly Frass as an Ingredient in Florida Pompano (Trachinotus carolinus L.) Diets.

The aquaculture industry is in need of sustainable fish feed to reduce the use of expensive and environmentally invasive wild-caught fish currently fed to many carnivorous species. The black soldier fly (BSF) has become a popular sustainable alternative protein source; however, the nutritional waste byproduct of BSF, frass, has not been extensively studied as a feed replacement in carnivorous species. This study evaluates the potential of BSF frass on the growth, body composition, and intestinal microbiome of the Florida pompano, Trachinotus carolinus. Four experimental diets were formulated containing different levels of frass, replacing plant-based carbohydrate sources. As a result of this study, the frass did not improve the growth performance, resulting in a lower specific growth rate and higher feed conversion rate. While the frass diets did not alter the body composition, the visceral somatic index (VSI) significantly increased compared to the control diet and the hepatosomatic index (HIS) was lowered. The microbiome analysis showed high variation among the diets, with the control diet having the most distinct consortia, which may have been driven by the increased levels of starch compared to frass diets. This study indicates that BSF frass may not be a suitable feed replacement for carnivorous pompano; however, frass could still potentially be a replacement feed for herbivore or detritivore fish and should be further studied.

Dominant remodelling of cattle rumen microbiome by Schedonorus arundinaceus (tall fescue) KY-31 carrying a fungal endophyte.

Tall fescue KY-31 is an important primary forage for beef cattle. It carries a fungal endophyte that produces ergovaline, the main cause of tall fescue toxicosis that leads to major revenue loss for livestock producers. The MaxQ, an engineered cultivar, hosts an ergovaline nonproducing strain of the fungus and consequently is nontoxic. However, it is less attractive economically. It is not known how rumen microbiome processes these two forages towards nutrient generation and ergovaline transformation. We have analysed the rumen microbiome compositions of cattle that grazed MaxQ with an intervening KY-31 grazing period using the 16S rRNA-V4 element as an identifier and found that KY-31 remodelled the microbiome substantially, encompassing both cellulolytic and saccharolytic functions. The effect was not evident at the whole microbiome levels but was identified by analysing the sessile and planktonic fractions separately. A move from MaxQ to KY-31 lowered the Firmicutes abundance in the sessile fraction and increased it in planktonic part and caused an opposite effect for Bacteroidetes, although the total abundances of these dominant rumen organisms remained unchanged. The abundances of Fibrobacter , which degrades less degradable fibres, and certain cellulolytic Firmicutes such as Pseudobutyrivibrio and Butyrivibrio 2, dropped in the sessile fraction, and these losses were apparently compensated by increased occurrences of Eubacterium and specific Ruminococcaceae and Lachnospiraceae . A return to MaxQ restored the original Firmicutes and Bacteroidetes distributions. However, several KY-31 induced changes, such as the low abundance of Fibrobacter and Butyrivibrio two remained in place, and their substitutes maintained significant presence. The rumen microbiome was distinct from previously reported faecal microbiomes. In summary, KY-31 and MaxQ were digested in the cattle rumen with distinct consortia and the KY-31-specific features were dominant. The study also identified candidate ergovaline transforming bacteria. It highlighted the importance of analysing sessile and planktonic fractions separately.

Clinicopathological Associations of K-RAS and N-RAS Mutations in Indonesian Colorectal Cancer Cohort.

BACKGROUND: K-RAS and recently N-RAS gene mutation testing are mandatory requirements prior to anti-epidermal growth factor receptor (EGFR) monoclonal antibody treatment of metastatic CRC. Mutation prevalence and distribution in Indonesian colorectal cancer (CRC) are not known. METHODS: Combined methods of PCR high-resolution melt (HRM), restriction fragment length polymorphism (RFLP), and direct DNA sequencing were used to genotype exons 2, 3, and 4 of both K-RAS and N-RAS genes for routine clinical testing of CRC patients. Descriptive analytical review of 595 consecutive CRC patients (years 2013 to 2016) was performed to find associations between gene mutations and clinicopathologic features. RESULTS: This retrospective study revealed overall K-RAS gene mutation in exon 2 (codon 12 and 13) rates being 34.9%. Women (42.5%), stages I and II (43.4%), and well and moderate differentiations (37.7%) had higher frequency of K-RAS exon 2 mutations than men (29%, p = 0.006), stages (III and IV 31.9%, p = 0.05), and poor differentiation (11.8%, p = 0.002), respectively. At later period (2015-2016), 121 of 595 patients were genotyped for the remaining exons 3 and 4 of K-RAS as well as exons 2, 3, and 4 of N-RAS mutations resulting in overall RAS mutation prevalence of 41%. Mucinous histology had highest frequency of N-RAS mutation. CONCLUSIONS: Combination of PCR HRM with either RFLP or direct DNA sequencing was useful to detect K-RAS exon 2 and extended RAS mutations, respectively. Frequency of all RAS mutations in stage IV Indonesian (41%) was similar among Asians (41-49%), which tend to be lower than western (55%) CRC.