Evelyn M. Doyle

Microbiome analysis of dairy cows fed pasture or total mixed ration diets.

Understanding rumen microbial ecology is essential for the development of feed systems designed to improve livestock productivity, health and for methane mitigation strategies from cattle. Although rumen microbial communities have been studied previously, few studies have applied next-generation sequencing technologies to that ecosystem. The aim of this study was to characterize changes in microbial community structure arising from feeding dairy cows two widely used diets: pasture and total mixed ration (TMR). Bacterial, archaeal and protozoal communities were characterized by terminal restriction fragment length polymorphism of the amplified SSU rRNA gene and statistical analysis showed that bacterial and archaeal communities were significantly affected by diet, whereas no effect was observed for the protozoal community. Deep amplicon sequencing of the 16S rRNA gene revealed significant differences in the bacterial communities between the diets and between rumen solid and liquid content. At the family level, some important groups of rumen bacteria were clearly associated with specific diets, including the higher abundance of the Fibrobacteraceae in TMR solid samples and members of the propionate-producing Veillonelaceae in pasture samples. This study will be relevant to the study of rumen microbial ecology and livestock feed management.

Comparative metatranscriptomics reveals widespread community responses during phenanthrene degradation in soil

Soil microbial community response to phenanthrene was evaluated by metatranscriptomics. A marked increase in transcripts involved in aromatic compound metabolism, respiration and stress responses, and concurrent decreases in virulence, carbohydrate, DNA metabolism and phosphorus metabolism transcripts was revealed. Phenanthrene addition led to a 1.8-fold to 33-fold increase in the abundance of dioxygenase, stress response and detoxification transcripts, whereas those of general metabolism were little affected. Heavy metal P-type ATPases and thioredoxin transcripts were more abundant in the phenanthrene-amended soil, and this is the first time these proteins have been associated with polycyclic aromatic hydrocarbon (PAH) stress in microorganisms. Annotation with custom databases constructed with bacterial or fungal PAH metabolism protein sequences showed that increases in PAH-degradatory gene expression occurred for all gene groups investigated. Taxonomic determination of mRNA transcripts showed widespread changes in the bacteria, archaea and fungi, and the actinobacteria were responsible for most of the de novo expression of transcripts associated with dioxygenases, stress response and detoxification genes. This is the first report of an experimental metatranscriptomic study detailing microbial community responses to a pollutant in soil, and offers information on novel in situ effects of PAHs on soil microbes that can be explored further.

The alkaline amylase of the alkalophilic Bacillus sp. IMD 370

A novel extracellular amylase of alkalophilic Bacillus sp. IMD 370 was purified to homogeneity and displayed maxima for activity at pH 10.0 and 40°C. It had an isoelectric point of 4.9 and a relative molecular mass of 159,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was inhibited (47%) by ethylenediaminetetraacetic acid (1 mM). The α-amylase of Bacillus sp. IMD 370 was quite distinct by virtue of its mechanism of action and its inability to fit into the conventional classification scheme of amylolytic enzymes. It had a mode of action intermediate between an endo-acting enzyme, α-amylase, and a typical exo-acting enzyme, amyloglucosidase. The sugars produced on hydrolysis of starch had the α-configuration, but no oligosaccharide higher than maltotetraose was obtained at any stage during starch hydrolysis.

Microbial PAH degradation.

Since the Industrial Revolution, human activity has created a legacy of environmental contamination widespread through industrialized economies (Baveye et al., 1999). As modern economies move to postindustrial economic activity, and heavy industry retreats, more sites affected with by-products from industrial processes are revealed, especially as environmental protection, legislation, and monitoring become more effective, and also as ‘‘brown’’ land is released for other uses (Philp et al., 2005).

Microbial community changes during the bioremediation of creosote‐contaminated soil

Aims:  To investigate the effects of aeration on the ex situ biodegradation of polycyclic aromatic hydrocarbons (PAHs) in creosote‐contaminated soil and its effect on the microbial community present.

Production studies on the alkaline amylases of three alkalophilic Bacillus spp.

SummaryBacillus alcalophilus subsp. halodurans ATCC 21591, Bacillus sp. NCIB 11203 and Bacillus sp. IMD370 are obligate alkalophiles and produce alkaline α-amylases with maxima for activity at pH 10.0. All three organisms yielded maximum amylase activity in a medium containing starch as a carbon source and yeatex as a nitrogen source with an initial pH 10.0.

The raw starch-degrading alkaline amylase ofBacillus sp IMD 370

The amylase ofBacillus sp IMD 370 is the first report of an alkaline amylase with the ability to digest raw starch. The amylase could degrade raw corn and rice starches more effectively than raw potato starch. It showed no adsorb-ability to any type of raw starch at any pH value tested. The enzyme digested raw corn starch to glucose, maltose, maltotriose and maltotetraose. The maximum pH for raw starch hydrolysis was pH 8.0 compared to pH 10.0 for soluble starch hydrolysis. The metal chelator, ethylenediaminetetraacetic acid, strongly inhibited raw starch-digestion and its effect was reversed by the addition of divalent cations. Degradation of raw starch was stimulated six-fold in the presence of β-cyclodextrin (17.5 mM).

The high maltose-producing α-amylase of the thermophilic actinomycete, Thermomonospora curvata

The α-amylase of Thermomonospora curvata catalyses the formation of very high levels of maltose from starch (73%, w/w) without the attendant production of glucose. The enzyme was produced extracellularly in high yield during batch fermentation in a 5-1 fermentor. Purification was achieved by ammonium sulphate fractionation, Superose-12 gel filtration and DEAE-Sephacel ionexchange chromatography. The enzyme exhibited maxima for activity at pH 6.0 and 65°C, had a relative molecular mass of 60900–62000 and an isoelecric point at 6.2. The exceptionally high levels of maltose produced and the unique action pattern exhibited on starch and related substrates indicate a very unusual maltogenic system. The predominance of maltose as the final end-product may be explained by the participation of reactions other than simple hydrolysis and the preferential cleavage of maltotriose from higher maltooligosaccharides. The enzyme exhibits very low affinity for maltotriose (Km=7.7 × 10−3m) and its conversion to maltose is achieved by synthetic followed by hydrolytic events, which result in the very high levels of maltose observed and preclude glucose formation. This system is distinguished from other very high maltose-producing amylases by virtue of its high temperature maximum, very low affinity for maltotriose and the absence of glucose in the final saccharide mixture.

The isomaltulose synthesising enzyme ofSerratiaplymuthica

SummaryAn intracellular enzyme was located inSerratiaplymuthica which produced isomaltulose from sucrose. The enzyme was purified giving a preparation with a specific activity of 1,285. It has pH and temperature optima of 6.0 and 30°C, respectively. The enzyme was stable retaining 100% activity after 2 weeks at 30°C. It had an isoelectric point at pH 9.0, a Mr of 79,500 and the Km for sucrose was 65.3mM. The enzyme converted 40% (w/v) sucrose to isomaltulose with an efficiency of 87%.

Bacterial community dynamics during the ensilage of wilted grass

Aims:  Grass silage is the product formed by a natural lactic acid bacterial fermentation when grass is stored under anaerobic conditions, and represents an important ruminant feedstuff on farms during winter. Of the two commonly employed methods of ensiling forage, baled silage composition frequently differs from that of comparable precision‐chop silage reflecting a different ensiling environment. The aim of this study was to investigate the dynamics of the silage fermentation in wilted grass and between ensiling systems.