John D. Sheppard

Ionic channels induced by surfactin in planar lipid bilayer membranes.

Surfactin is a lipopeptide produced by certain strains of Bacillus subtilis and has potent surface activity. Here, we present the first results showing that ion-conducting pores can be formed by surfactin in artificial lipid membranes. With a low aqueous concentration of surfactin (1 microM) and a restricted membrane area (5.10(-5) cm2) we observed conductance jumps that indicate the formation of individual ionic channels in the presence of K+, Rb+, Cs+, Na+ or Li+ chlorides. Although for every salt concentration (Ci), the distribution in amplitude of the conductance steps (lambda i) may be rather broad, there is always a step amplitude which is more frequent than the others. In addition, the channels corresponding to this most frequent step amplitude are the longest in duration. For Ci = 1 M, the cationic selectivity sequence deduced from these most frequent events is K+ greater than Rb+ greater than Na+ greater than Cs+ = Li+ with respective values for lambda Mi: 130, 110, 80 and 30 pS. In KCl solutions lambda MKCl increases as a function of Ci for low Ci, and shows a plateau for Ci greater than 0.5 M. When measured on larger area membranes (10(-2)cm2) with 1 M solutions of the monovalent salts KCl, NaCl, RbCl and CsCl or the divalent salt CaCl2, the macroscopic low voltage conductance (G0) increases with a slope of 2 on a log-log plot as a function of surfactin concentration. These results demonstrate that surfactin produces selective cationic channels in lipid bilayer membranes and suggest that at higher salt concentration, a dimer is involved in this functional channel-forming process.

Application of the Plackett-Burman experimental design to evaluate nutritional requirements for the production of Colletotrichum coccodes spores

Colletotrichum coccodes is being examined as a biological weed control agent for velvetleaf (Abutilon theophrasti). A modified Richards solution containing V-8 juice has been used to produce C. coccodes spores for growth-chamber and field experiments. Although C. coccodes sporulates well in this medium, V-8 is not available as a bulk commodity and is too expensive for commercial production. Eight substrates were evaluated as replacements for V-8 juice in modified Richards solution. Soy protein and casamino acids were equal to V-8 juice for sporulation of C. coccodes. The Plackett-Burman experimental design was used to test the relative importance of various components of a complex medium based on soy protein on mycelium biomass production and sporulation of C. coccodes. A new medium composed of sucrose (20 g/l), soy protein (5 g/l), KNO3 (5 g/l), KH2PO4 (5 g/l), MgSO4 (2 g/l), CaCl2 (0.5 g/l), and CuSO4 (0.05 g/l) was selected as the base medium for further study in the development of a low-cost and effective medium for C. coccodes spore production.

Hydrolysis of potato processing waste for the production of poly-β-hydroxybutyrate

Abstract The enzymatic hydrolysis of potato processing wastes has been studied as a possible source of a fermentable substrate for the production of the biopolymer poly-β-hydroxybutyrate (PHB) from the bacterium Alcaligenes eutrophus . PHB has been proposed as a biodegradable substitute for conventional thermoplastics. The results indicated that potato starch waste could be converted with high yield to a concentrated glucose solution. The most economical process used barley malt as a source of amylase enzyme with an optimal ratio of 10:90 g/g of potato waste. A conversion efficiency of 96% of theoretical was obtained with a final glucose concentration of 208 mg/ml. After dilution and addition of mineral salts the hydrolysate was converted by a batch culture of A. eutrophus to 5·0 g/l of PHB, comprising 77% of the biomass dry weight.

THE RESPONSE OF BACILLUS SUBTILIS ATCC 21332 TO MANGANESE DURING CONTINUOUS-PHASED GROWTH

SummaryBacillus subtilis ATCC 21332 produces an extracellular lipopeptide, called surfactin, a potent surfactant. Using a new technique for cell culturing, continuous phasing with feedback control, the effects of manganese on the growth, and the associated production of surfactin, were investigated. An intimate relationship was revealed between the availability of iron and manganese and the utilization of nitrogen. A critical proportionality of 920/7.7/1.0 (molar basis) of nitrogen to iron to manganese was identified. Increasing the manganese reduced the requirement for nitrogen and resulted in growth under iron limitation. The production of surfactin could be sustained for at least 30 consecutive generations growing under either an iron or nitrogen limitation, but only if manganese was available. The dynamic response of B. subtilis to changes in manganese concentration revealed a non-linear relationship that required several generations to stabilize.

Effects of physical parameters of a gravel bed on the activity of sulphate‐reducing bacteria in the presence of acid mine drainage

The use of sulphate-reducing bacteria (SRB) in the passive treatment of acid mine drainage (AMD) requires the presence of a sediment, such as gravel bed, in which the bacteria can establish microenvironments conducive to their survival in the presence of oxygen and acidity. The characterization of relationships between the physical environment and SRB activity would be useful in the sizing of passive treatment systems using SRB. The effects of physical parameters of a gravel bed (i.e. void volume, total surface area of gravel, type of gravel) on SRB activity while in the presence of varying loads of fresh AMD was investigated. SRB activity was expressed as the proportion of sulphate removed after 7 days. The study was accomplished in a series of column reactors containing beds comprising different types and size classes of gravel. SRB were established in the beds and metal and sulphate removal were monitored. Quantifiable relationships were found to exist between SRB activity and the physical parameters of the bed. ©1997 SCI

Effects of carbon concentration and carbon-to-nitrogen ratio on growth, conidiation, spore germination and efficacy of the potential bioherbicide Colletotrichum coccodes

The effect of carbon concentration and carbon-to-nitrogen ratio (C:N) as well as their interaction on Colletotrichum coccodes growth and sporulation in submerged flask culture were evaluated. When C:N ratios were held constant, both mycelial dry biomass and spore yield increased with increasing carbon concentration. The specific spore yields (spore yield g−1 carbon), however, were not significantly different for the same C:N ratio in most cases. The highest spore yields (1.3 × 108 spores per ml) were obtained from media containing 20 g per liter carbon with C:N ratios ranging from 5:1 to 10:1. When the C:N ratio was greater than 15:1, spore yields were significantly decreased with increasing C:N ratios. High carbon concentration (20 g L−1) combined with high C:N ratios (above 15:1) reduced both mycelial growth and sporulation, and increased spore matrix production. Spores produced in medium containing 10 g L−1 carbon with C:N ratios from 10:1 to 15:1 had 90% germination on potato dextrose agar after 12 h and caused extensive shoot dry weight reduction on the target weed, velvetleaf. These results suggest that C:N ratios from 10:1 to 15:1 are optimal for C. coccodes spore production.

Application of self-cycling fermentation technique to the production of poly-β-hydroxybutyrate

The production of poly-beta-hydroxybutyrate (PHB) by Alcaligenes eutrophus DSM 545 in a cyclone bioreactor was compared using various culture methods: batch, fed-batch, and self-cycling fermentation (SCF) with and without extended periods of nutrient deprivation. SCF is a semi-continuous method that results in a nutrient limitation for every successive generation of cells and, therefore, may have advantages for products whose formation follow secondary metabolite kinetics. Use of the SCF technique without extended nutrient deprivation produced a PHB concentration of 1.2 g L(-1) as 40% of the biomass dry weight. With nitrogen deprivation for 4 or 6 h, the concentration of PHB decreased when compared to the standard SCF technique. However, nitrogen deprivation periods of 8 h resulted in an increase in PHB concentration to 2.7 g L(-1) or 59% of the biomass dry weight. The nutrient cycling may act to repress PHB accumulation during periods of nitrogen deprivation, unless a time threshold has been reached, after which PHB accumulation occurs as in normal batch culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 815-820, 1997.

Comparison of PCR‐DGGE and Selective Plating Methods for Monitoring the Dynamics of a Mixed Culture Population in Synthetic Brewery Wastewater

Enrichment of an activated sludge inoculum in synthetic brewery wastewater, which included glucose, maltose, and ethanol, was conducted in batch experiments to identify the dominant microbes present, to determine methodologies capable of monitoring the mixed culture population dynamics, and to determine the consortiumapos;s substrate degradation behavior. These results and methodologies were subsequently used in the determination of the population dynamics of suspended and attached microorganisms in a sequencing batch system in the second part of this research work. The three‐membered microbial community comprised two bacterial and one fungal species that were identified as Acinetobacter sp., Enterobacter sp., and Candida sp. PCR‐DGGE and plating on selective media were used to track the population dynamics of the consortium during the degradation of different substrates in synthetic wastewater containing glucose, maltose, and ethanol. Enterobacter sp. could degrade glucose and maltose but not ethanol, whereas Acinetobacter and Candida could degrade all three carbon sources. In buffered batch mixed culture experiments, Enterobacter was the predominant bacterium until the sugar concentrations decreased to levels that enabled Acinetobacter and Candida to degrade ethanol. PCR‐DGGE was effective for detecting the dominant species, but culture‐based methods were more accurate for monitoring the population dynamics of these microorganisms during growth in the wastewater medium.

A Kinetic Model for Suspended and Attached Growth of a Defined Mixed Culture

Kinetic experiments were carried out in a semicontinuous wastewater treatment process called self‐cycling fermentation (SCF) using a defined mixed culture and various concentrations of synthetic brewery wastewater. The same consortium, which had been previously identified as Acinetobacter sp., Enterobacter sp., and Candida sp., were used in these experiments. The overall rate of substrate removal was attributable to both suspended microbes and the biofilm that formed during the treatment process. A rate expression was developed for the SCF system for a range of synthetic wastewaters containing glucose and various initial concentrations of ethanol and maltose. The data indicated that substrate removal by the suspended cells was directly related to the biomass concentration. However, substrate removal by the biofilm was apparently not affected by the biofilm thickness and was a function of substrate concentration only.