R. De Mot

Potentialities and limitations of direct alcoholic fermentation of starchy material with amylolytic yeasts

SummaryThe fermentation characteristics of a large number of starch-degrading yeasts were compared. None of the amylolytic yeasts currently recognized, appear to be entirely suitable for direct alcoholic fermentation of starchy biomass. The species capable of extensive starch hydrolysis produce only low amounts of ethanol from glucose and dextrin, one of the major limitations being their low ethanol tolerances. Some of the less-active yeasts have much better glucosefermentation characteristics, but dextrin conversion is limited probably due to the nature of their enzyme systems. Using an α-amylase dextrin (22.5% w/v), ethanol yields of about 70% were obtained with Saccharomyces diastaticus strains. Through associative fermentation of S. diastaticus and other selected amylolytic yeasts slightly better yields, however not exceeding 80%, were obtained.

Production of extracellular debranching activity by amylolytic yeasts

SummaryThe production of extracellular pullulan-degrading enzymes by several amylolytic yeast strains was studied. The highest activity was obtained with species of Endomycopsis, Lipomyces, Filobasidium, Leucosporidium, and Trichosporon, and also with some amylase-hyperproducing mutants. The most active strains are potentially valuable partners for intergeneric protoplast fusion.

Direct alcoholic fermentation of starchy biomass using amylolytic yeast strains in batch and immobilized cell systems

SummaryDirect alcoholic fermentation of dextrin or soluble starch with selected amylolytic yeasts was studied in both batch and immobilized cell systems. In batch fermentations, Saccharomyces diastaticus was capable of fermenting high dextrin concentrations much more efficiently than Schwanniomyces castellii. From 200 g·l−1 of dextrin S. diastaticus produced 77 g·l−1 of ethanol (75% conversion efficiency). The conversion efficiency decreased to 59% but a higher final ethanol concentration of 120 g·l−1 was obtained with a medium containing 400 g·l−1 of dextrin. With a mixed culture of S. diastaticus and Schw. castellii 136 g·l−1 of ethanol was produced from 400 g·l−1 of dextrin (67% conversion efficiency). S. diastaticus cells attached well to polyurethane foam cubes and a S. diastaticus immobilized cell reactor produced 69 g·l−1 of ethanol from 200 g·l−1 of dextrin, corresponding to an ethanol productivity of 7.6g·l−1·h−1. The effluent from a two-stage immobilized cell reactor with S. diastaticus and Endomycopsis fibuligera contained 70 g·l−1 and 80 g·l−1 of ethanol using initial dextrin concentrations of 200 and 250 g·l−1 respectively. The corresponding values for ethanol productivity were 12.7 and 9.6 g·l−1·h−1. The productivity of the immobilized cell systems was higher than for the batch systems, but much lower than for glucose fermentation.

Purification and characterization of an extracellular glucoamylase from the yeastCandida tsukubaensis CBS 6389

The starch-degrading yeastCandida tsukubaensis CBS 6389 secreted amylase at high activity when grown in a medium containing soluble starch. The extracellular α-amylase activity was very low. The major amylase component was purified by DEAE-Sephadex A-50 chromatography and Ultrogel AcA 44 gel filtration and characterized as a glucoamylase. The enzyme proved to be a glycoprotein with a molecular weight of 56000. The glucoamylase had a temperature optimum at 55°C and displayed highest activity in a pH range of 2.4–4.8. Acarbose strongly inhibited the purified glucoamylase. Debranching activity was present as demonstrated by the release of glucose from pullulan.

Identification and Characterization of the omaA Gene Encoding the Major Outer Membrane Protein of Azospirillum brasilense

The major outer membrane protein (MOMP) of Azospirillum brasilense was purified and degenerate oligo-nucleotides were constructed on the basis of partial internal amino acid sequences. PCR products were obtained using total DNA of A. brasilense as template. One of these, a 766-bp fragment, was DIG-labelled and used in Southern hybridization against A. brasilense DNA and a genomic library of A. brasilrnsc in Escherichia coli. A clone containing a 20-kb EcoRI insert in pLAFR3 was identified by PCR screening. From this insert, an EcoRI-SalI fragment of approximately 3.5-kb was subcloned in pUC19. The gene encoding the A. brasilense MOMP was sequenced and analyzed. The deduced amino acid sequence contains a putative signal peptide of 23 residues, followed by 367 amino acids of the mature protein with a molecular mass of 38,753 Da. The deduced amino acid sequence shows similarity to certain bacterial porins.

HylA, an Alternative Hydrolase for Initiation of Catabolism of the Phenylurea Herbicide Linuron in Variovorax sp. Strains

ABSTRACT Variovorax sp. strain WDL1, which mineralizes the phenylurea herbicide linuron, expresses a novel linuron-hydrolyzing enzyme, HylA, that converts linuron to 3,4-dichloroaniline (DCA). The enzyme is distinct from the linuron hydrolase LibA enzyme recently identified in other linuron-mineralizing Variovorax strains and from phenylurea-hydrolyzing enzymes (PuhA, PuhB) found in Gram-positive bacteria. The dimeric enzyme belongs to a separate family of hydrolases and differs in Km , temperature optimum, and phenylurea herbicide substrate range. Within the metal-dependent amidohydrolase superfamily, HylA and PuhA/PuhB belong to two distinct protein families, while LibA is a member of the unrelated amidase signature family. The hylA gene was identified in a draft genome sequence of strain WDL1. The involvement of hylA in linuron degradation by strain WDL1 is inferred from its absence in spontaneous WDL1 mutants defective in linuron hydrolysis and its presence in linuron-degrading Variovorax strains that lack libA. In strain WDL1, the hylA gene is combined with catabolic gene modules encoding the downstream pathways for DCA degradation, which are very similar to those present in Variovorax sp. SRS16, which contains libA. Our results show that the expansion of a DCA catabolic pathway toward linuron degradation in Variovorax can involve different but isofunctional linuron hydrolysis genes encoding proteins that belong to evolutionary unrelated hydrolase families. This may be explained by divergent evolution and the independent acquisition of the corresponding genetic modules.

Agar composition affects in vitro screening of biocontrol activity of antagonistic microorganisms.

Agar-based screening assays are the method of choice when evaluating antagonistic potential of bacterial biocontrol-candidates against pathogens. We showed that when using the same medium, but different agar compositions, the activity of a bacterial antagonist against Agrobacterium was strongly affected. Consequently, results from in vitro screenings should be interpreted cautiously.

Efficacy of hydrogen peroxide treatment for control of hairy root disease caused by rhizogenic agrobacteria.

Rhizogenic Agrobacterium strains are the causative agent of hairy root disease (HRD), an increasing problem in the hydroponic cultivation of tomato and cucumber in Europe. A previous study has demonstrated that different lineages of rhizogenic agrobacteria are able to form biofilms. Although hydrogen peroxide (H2O2) is a frequently used biocide in hydroponic systems, until now its effectiveness to remove rhizogenic agrobacteria has not been unequivocally demonstrated. Therefore, the main objective of this study was to assess the efficacy of H2O2 in controlling Agrobacterium‐containing biofilms.

Rhizogenic agrobacteria in hydroponic crops: epidemics, diagnostics and control

&NA; Rhizogenic Agrobacterium biovar 1, harbouring an Ri‐plasmid (root‐inducing plasmid), is the causative agent of hairy root disease (HRD) in the hydroponic cultivation of tomato, cucumber and aubergine. The disease is characterized by extensive root proliferation leading to strong vegetative growth and, in severe cases, substantial losses in marketable yield. Agrobacterium biovar 1 is a heterogeneous group of agrobacteria and includes at least 10 genomospecies, among which at least four (G1, G3, G8 and G9) have been associated with HRD in hydroponically grown vegetables. This review has synthesized the current knowledge on rhizogenic Agrobacterium biovar 1, including infection process, current taxonomic status, genetic and phenotypic diversity, detection methods and strategies for disease control. With regard to the latter, symptom reduction and prevention of infection through cultivation methods and chemical disinfection (e.g. by the use of chlorine‐based disinfectants and hydrogen peroxide) are discussed and biocontrol strategies are elaborated on. Recent research has led to the identification of a phylogenetically related clade of Paenibacillus strains that have antagonistic activity against rhizogenic Agrobacterium biovar 1 strains, holding great potential for HRD control. Finally, possible directions for future research are proposed.