Susan E. Withers

Energy sources of non-starter lactic acid bacteria isolated from Cheddar cheese

Abstract The range of carbon sources available in cheese curd during maturation that could be used as energy and growth substrates by 60 cultures of non-starter lactic acid bacteria isolated from Cheddar cheese was determined by the detection of tetrazolium salt reduction in Biolog MT1 microplates ™ . There were marked inter-species and strain differences in the range of carbon substrates catabolized by the 11 Lactobacillus spp. and 2 Weissella spp. examined. Sugars were used widely among the NSLAB with 90, 100 and 85% of the isolates metabolizing lactose, glucose and galactose, respectively. In addition, ribose, N -acetyl-galactosamine and sialic acid, potentially derived from nucleic acid and casein deglycosylation, were catabolized by 58, 48 and 22% of the isolates, respectively. Lactic acid was also a potential substrate for 15% of the isolates but Tween 80 was not an effective substrate. Although 50% of the NSLAB removed citric acid from the growth medium it was not an independent energy source. Peptides and amino acids were also catabolized by up to 27% of the NSLAB provided that an exogenous source of α -ketoglutaric acid was present to facilitate the aminotransferase-mediated transamination degradative pathway. The MT1 microplate method facilitates the rapid screening for isolates able to establish in the cheese curd and for the detection of specific metabolic activities in isolates undergoing evaluation for use as adjunct cultures in cheesemaking trials.

Glycoside hydrolases of rumen bacteria and protozoa

Sixteen strains of rumen bacteria and 21 protozoal preparations were screened for glycoside hydrolase and phosphatase activity, using 22 nitrophenyl glycoside substrates. The range and level of bacterial enzyme activities were species dependent, although, the glycosidases associated with plant cell wall breakdown were most active in the cellulolytic and hemicellulolytic species. Alkaline phosphatase occurred widely in the organisms examined, but was most active in the twoBacteroides ruminicola strains.A wide range of enzyme activities was also detected in the holotrich and Entodiniomorphid ciliates isolated from the rumen or cultured in vitro. The glycosidases involved in cellulose and hemicellulose breakdown were detected in all of the protozoa examined, and, with the exception ofEntodinium spp., were most active in the Entodiniomorphid protozoa; α-l-arabinofuranosidase, an essential hemicellulolytic glycoside hydrolase, was particularly active in this latter group of ciliates.

The production of hemicellulose-degrading enzymes byBacillus macerans in anaerobic culture

SummaryThe cell-associated and exocellular hemicellulolytic polysaccharide depolymerase and glycoside hydrolase activity ofBacillus macerans NCDO 1764 was monitored over a range of anaerobic growth conditions in batch and continuous culture. The enzymes were detectable throughout the complete growth cycle in batch culture reaching and maintaining maximum levels in the stationary phase. In continuous culture enzyme activity was largely independent of growth rate (D=0.025–0.1 h-1) although the activity was reduced at higher dilution rates (0.125–0.15 h-1). Although activity was detectable over a wide pH range (pH 5.5–7.5) it was pH dependent, and maximum activities of both the cell-associated and exocellular enzymes were measured in cultures maintained at pH 6.5–7.0±0.1.The principal metabolites formed anaerobically from xylose byB. macerans in batch and continuous culture were acetic acid, formic acid and ethanol which represented 95–99% of the products formed. Smaller amounts of acetone,d,l-lactic acid and succinic acid were formed together with traces of butyric acid (<5 nmol/ml) and isovaleric acid (<25 nmol/ml). The proportions of the metabolites produced varied with growth conditions and were influenced by the pH of the culture and the rate and stage of growth of the microorganism.

The potential of bacteria isolated from ruminal contents of seaweed‐eating North Ronaldsay sheep to hydrolyse seaweed components and produce methane by anaerobic digestion in vitro

The production of methane biofuel from seaweeds is limited by the hydrolysis of polysaccharides. The rumen microbiota of seaweed‐eating North Ronaldsay sheep was studied for polysaccharidic bacterial isolates degrading brown‐seaweed polysaccharides. Only nine isolates out of 65 utilized > 90% of the polysaccharide they were isolated on. The nine isolates (eight Prevotella spp. and one Clostridium butyricum) utilized whole Laminaria hyperborea extract and a range of seaweed polysaccharides, including alginate (seven out of nine isolates), laminarin and carboxymethylcellulose (eight out of nine isolates); while two out of nine isolates additionally hydrolysed fucoidan to some extent. Crude enzyme extracts from three of the isolates studied further had diverse glycosidases and polysaccharidase activities; particularly against laminarin and alginate (two isolates were shown to have alginate lyase activity) and notably fucoidan and carageenan (one isolate). In serial culture rumen microbiota hydrolysed a range of seaweed polysaccharides (fucoidan to a notably lesser degree) and homogenates of L. hyperborea, mixed Fucus spp. and Ascophyllum nodosum to produce methane and acetate. The rumen microbiota and isolates represent potential adjunct organisms or enzymes which may improve hydrolysis of seaweed components and thus improve the efficiency of seaweed anaerobic digestion for methane biofuel production.

Formation of polysaccharide depolymerase and glycoside hydrolase enzymes byBacteroides ruminicola subsp.ruminicola grown in batch and continuous culture

The activity of ten polysaccharide depolymerase and glycoside hydrolase enzymes was monitored inBacteroides ruminicola subsp.ruminicola throughout the growth cycle and over a range of dilution rates in carbon-limited continuous (chemostat) culture. The enzymes were principally cell associated, and the specific activities increased throughout the growth cycle to reach maximum levels in the late exponential and stationary growth phases. In chemostat-grown cells the activities were growth rate dependent and were highest at the lowest dilution rates examined (0.025/h), but remained constant over a wide range of growth rates (D=0.05–0.15/h). The specific activities were lower in cells with a generation time of 3 h (D=0.225/h). The major metabolites formed from xylose, in batch and continuous culture, were lactic, acetic, and succinic acids, with traces (∼1%–2% of total acid production) of branched and straight-chain C3−C5 volatile fatty acids. The proportions of the metabolites produced varied with the stage and rate of growth.

Induction of xylan-degrading enzymes inButyrivibrio fibrisolvens

The formation of xylanolytic enzymes byButyrivibrio fibrisolvens NCFB 2249 was induced by xylan, xylo-oligosaccharides, and xylobiose. Inhibition of RNA or protein synthesis prevented inducetion, and enzyme formation occured only when anaerobiosis was maintained. The rate of enzyme inducetion by xylan was affected by pH and inducer concentration, and highest levels of activity occurred when the initial pH and xylan concentration were pH 6.5–7 and ≥2 mg/ml respectively. The ability of the cells to respond to the inducer was reduced in slowly growing cells, although cells that were grown at dilution rates that appertain in the rumen ecosystem responded rapidly to the inducer.Butyrivibrio fibrisolvens also exhibited diauxic characteristics of carbohydrate utilization, and in consequence enzyme induction and xylanolysis were delayed until readily metabolized sugars (e. g., glucose, arabinose) had been consumed.

A modified method for the quantitative enzymic determination of d-xylose with commercially available reagents

Abstract A two stage procedure for the quantitative determination of d -xylose is described. The monosaccharide is first chemically reduced with sodium borohydride to the sugar alditol; the xylitol formed is subsequently quantified enzymically with sheep liver d -glucitol-dehydrogenase. The reduced nicotinamide adenine dinucleotide formed is either determined directly or through formazan production in a linked assay with pig heart diaphorase and iodonitrotetrazolium chloride. The assay is an end point determination and uses reagents that are all available commercially, either singly or in kit form. The potential application of the assay in the determination of other monosaccharides of biological importance (e.g., ribose) is discussed. Data on the estimation of xylose during the breakdown of oat spelt xylan by the rumen fungus Piromonas communis and the formation of extracellular bioppolymer from xyclose by Pseudomonas NCIB 11264 are presented to exemplify applications of the assay procedure.