Survival and change in physiological state of Bradyrhizobium japonicum in soybean (Glycine max L. Merril) liquid inoculants after long-term storage

Commercial liquid inoculants for soybean, stored at 20 °C for 1–8 years in 400 ml bottles or in 5000 ml containers, were assessed for their efficacy and changes in the physiological activity of Bradyrhizobium japonicum. A decrease in viable counts and in bacterial survival on seeds was observed in inoculants stored for several years. The number of nodules produced per plant in a growth chamber decreased and was correlated to the number of bacteria surviving on the seeds. Changes in physiological properties were assessed using biochemical, physiological and microscopic methods. The cell total sugars content decreased with increased storage of the inoculants. High calculated ratios of suspended solid dry matter/carbon/nitrogen/proteins weight per c.f.u. strongly suggested the presence of dead or viable but non-culturable (VBNC) cells in the inoculants. This was confirmed in a study of bacterial respiratory activity, using p-iodonitrotetrazolium reduction. The time of colony appearance on plates increased in the old inoculants stored for a long time, especially on yeast-free culture medium. The heterogeneity in colony size also increased with storage length. Inoculants stored for more than 2 years could be differentiated from the others by using nalidixic acid against cellular division. Nucleic acid staining of cells showed that the percentage of membrane-compromised bacteria in all the inoculants increased with increased storage length, whatever the type of packaging used for the inoculants. These results demonstrated that the physiological activity of B. japonicum cells in commercial liquid inoculants changes after storage. To complete c.f.u. determination, three methods were proposed to assess the fitness of stored bradyrhizobia, but they remain to be checked for reliability on a variety of commercial inoculants.     

Effect of soybean oil and glucose on sophorose lipid fermentation by Torulopsis bombicola in continuous culture

The effect of soybean oil and glucose on the growth of Torulopsis bombicola and sophorose lipid production in continuous culture was investigated. As the dilution rate in 100 g/l glucose and 100 g/l soybean oil medium was increased, the dry cell weight and sophorose lipid concentration decreased. Sophorose lipid productivity, however, was maximum at a dilution rate of 0.03 h⁻¹. The cell yield from glucose and the sophorose lipid production from soybean oil were approximately constant regardless of the dilution rate. The specific consumption rate of soybean oil was closely related to the specific production rate of sophorose lipid. These results suggest that soybean oil was used only for sophorose lipid production whereas glucose was used only for cell mass and maintenance. When the soybean oil concentration was varied at fixed dilution rate in 100 g/l glucose medium, a high concentration of soybean oil was found to inhibit sophorose lipid production. Received: 9 January 1997 / Received revision: 5 March 1997 / Accepted: 13 April 1997     

3-Carbamoyl-α-picolinic acid production by imidase-catalyzed regioselective hydrolysis of 2,3-pyridinedicarboximide in a water-organic solvent, two-phase system

3-Carbamoyl-α-picolinic acid, a versatile building block for the synthesis of agrochemicals and pharmaceuticals, was prepared by imidase-catalyzed regiospecific hydrolysis of 2,3-pyridinedicarboximide with intact Arthrobacter ureafaciens O-86 cells. Reactions were carried out in a water-organic solvent, two-phase system containing cyclohexanone at low pH to avoid spontaneous random hydrolysis. Under the optimized conditions, with the periodic addition of 2,3-pyridinedicarboximide (in total, 40 mM), the 3-carbamoyl-α-picolinic acid yield reached 36.6 mM in the water phase, with a molar conversion yield of 91.5% and a regioisomeric purity of 94.5%, in 2 h at pH 5.5. Received: 7 February 2000 / Received revision: 13 March 2000 / Accepted: 3 April 2000     

Thermophilic acidification of dairy wastewater

Acidification of simulated dairy wastewater was conducted in an upflow reactor at 55 °C. Results showed that the degree of acidification decreased with the increase in chemical oxygen demand (COD) loading rate, from 60.8% at 4 g l⁻¹ day⁻¹ to 27.1% at 24 g l⁻¹ day⁻¹. Carbohydrate was readily degraded at all loading rates, but degradation of protein and lipid decreased with the increase in loading rate. Most carbohydrate degradation occurred at the reactor bottom, whereas protein was degraded mainly after the carbohydrate became depleted. The predominant acidification products were acetate, propionate, butyrate and ethanol, whereas formate, i-butyrate, valerate, i-valerate, caproate, lactate, methanol, propanol and butanol were present in lesser quantities. The increase in loading rate resulted in the increase of propionate and the decrease of acetate, but had little effect on ethanol and butyrate productions. Only 2.5–8.8% of influent COD was converted to hydrogen and methane. The biomass yield was 0.30–0.43 mg VSS mg⁻¹ COD. Received: 8 December 1999 / Received revision: 14 February 2000 / Accepted: 25 February 2000     

Artificial biofilm model – a useful tool for biofilm research

For biofilm studies, artificial models can be very helpful in studying processes in hydrogels of defined composition and structure. Two different types of artificial biofilm models were developed. Homogeneous agarose beads (50–500 μm diameter) and porous beads (260 μm mean diameter) containing pores with diameters from 10 to 80 μm (28 μm on average) allowed the embedding of cells, particles and typical biofilm matrix components such as proteins and polysaccharides. The characterisation of the matrix structures and of the distribution of microorganisms was performed by confocal laser scanning microscopy. The physiological condition of the embedded bacteria was examined by redox activity (CTC-assay) and membrane integrity (Molecular Probes LIVE/DEAD-Kit). Approximately 35% of the immobilised cells (Pseudomonas aeruginosa SG81) were damaged due to the elevated temperature required for the embedding process. It was shown that the surviving cells were able to multiply when provided with nutrients. In the case of homogeneous agarose beads, cell growth only occurred near the bead surface, while substrate limitation prevented growth of more deeply embedded cells. In the porous hydrogel, cell division was observed across the entire matrix due to better mass transport. It could be shown that embedding in the artificial gel matrix provided protection of immobilized cells against toxic substances such as sodium hypochlorite (0.5 mg/l, 30 min) in comparison to suspended cells, as observed in other immobilized systems. Thus, the model is suited to simulate important biofilm matrix properties. Received: 21 December 1999 / Received revision: 7 March 2000 / Accepted: 10 March 2000     

Ice crystallization by Pseudomonas syringae

Several bacterial species can serve as biological ice nuclei. The best characterized of these is Pseudomonas syringae, a widely distributed bacterial epiphyte of plants. These biological ice nuclei find various applications in different fields, but an optimized production method was required in order to obtain the highly active cells which may be exploited as ice nucleators. The results presented here show that P. syringae cells reduce supercooling of liquid or solid media and enhance ice crystal formation at sub-zero temperatures, thus leading to a remarkable control of the crystallization phenomenon and a potential for energy savings. Our discussion focuses on recent and future applications of these ice nucleators in freezing operations, spray-ice technology and biotechnological processes. Received: 21 December 1999 / Received revision: 29 February 2000 / Accepted: 6 March 2000     

Metabolic characterization of high- and low-yielding strains of Penicillium chrysogenum

A recently developed method for analyzing metabolic networks using ¹³C-labels was employed for investigating the metabolism of a high- and a low-yielding strain of Penicillium chrysogenum. Under penicillin-producing conditions, the flux through the pentose phosphate (PP) pathway in the high- and the low-yielding strains was estimated to 70 and 66, respectively. When the high-yielding strain was cultivated in a medium without the penicillin side chain precursor, phenoxyacetic acid, the PP pathway flux was estimated as 71. Thus, in all three experiments, the flux through the PP pathway was almost constant with an average value of 69 ± 3, and the method therefore allows for a very reproducible estimation of the PP pathway flux. Phenoxyacetic acid was found to be a source of cytosolic acetyl-CoA and thereby a source of precursors for the biosynthesis of 2-aminoadipic acid, which is a central amino acid in penicillin biosynthesis. However, the labeling patterns also indicated the presence of an unrecognized pathway to cytosolic acetyl-CoA. Received: 20 December 1999 / Received revision: 7 March 2000 / Accepted: 10 March 2000     

A genetically modified solvent-tolerant bacterium for optimized production of a toxic fine chemical

The aim of the study was to investigate whether toxic fine chemical production can be improved using the solvent-tolerant Pseudomonas putida S12 in a two-liquid-phase system consisting of aqueous media and a water-immiscible octanol phase with production of 3-methylcatechol from toluene as the model conversion. For this purpose the genes involved in this conversion, todC1C2BAD from P. putida F1, were introduced into P. putida S12 with high stable expression. Production of 3-methylcatechol was monitored in batch incubations with different media using a single medium and a two-liquid medium–octanol system. The maximum concentration of 3-methylcatechol increased two-fold using the two-liquid medium–octanol system, irrespective of the selected medium. Received: 29 December 1999 / Received revision: 29 February 2000 / Accepted: 6 March 2000     

Axenic aerobic biofilms inhibit corrosion of SAE 1018 steel through oxygen depletion

Corrosion inhibition of SAE 1018 steel by pure-culture biofilms of Pseudomonas fragi and Escheri-chia coli DH5α has been evaluated in complex Luria-Bertani medium, seawater-mimicking medium, and modified Baar's medium at 30 °C. In batch cultures, both bacteria inhibited corrosion three to six fold compared to sterile controls, and the corrosion was comparable to that observed in anaerobic sterile media. To corroborate this result, a continuous reactor and electrochemical impedance spectroscopy were used to show that both P. fragi K and E. coli DH5α decreased the corrosion rate by 4- to 40-fold as compared to sterile controls; this matched the decrease in corrosion found with sterile medium in the absence of oxygen and with E. coli DH5α grown anaerobically. In addition, the requirement for live respiring cells was demonstrated by the increase in the corrosion rate that was observed upon killing the P. fragi K biofilm in continuous cultures, and it was shown that fermentation products do not cause an increase in corrosion. Hence, pure-culture biofilms inhibit corrosion of SAE 1018 steel by depleting oxygen at the metal surface. Received: 16 December 1996 / Received revision: 18 March 1997 / Accepted: 27 March 1997     

Effect of dissolved oxygen and carbon–nitrogen loads on denitrification by an aerobic consortium

Four samples of natural ecosystems and one sample from an activated sludge treatment plant were mixed together and progressively adapted to alternating aerobic/anoxic phases in the presence of nitrate in order to enrich the microflora in aerobic denitrifiers. Aerobic denitrifying performances of this mixed ecosystem at various dissolved oxygen concentrations and various carbon–nitrogen loads were evaluated and compared to those obtained with the aerobic denitrifier Microvirgula aerodenitrificans. The consortium and the pure strain exhibited an aerobic denitrifying activity at air saturation conditions (7 mg dissolved oxygen l^–1), i.e. there was co-respiration of the two electron acceptors with significant specific nitrate reduction rates. Dissolved oxygen concentrations had no influence on denitrifying performances above a defined threshold: 0.35 mg l^–1 for the consortium and 4.5 mg l^–1 for M. aerodenitrificans respectively. Under these thresholds, decreasing the dissolved oxygen concentrations enhanced the denitrifying activity of each culture. The higher the carbon and nitrogen loads, the higher the performance of the aerobic denitrifying ecosystem. However, for M. aerodenitrificans, the nitrate reduction percentage was affected more by variations in nitrogen load than in carbon load. Received: 6 December 1999 / Received revision: 8 March 2000 / Accepted: 10 March 2000     

Purification, characterization, and primary structure of a chitinase from Pseudomonas sp. YHS-A2

A chitinase gene (chiA) from Pseudomonas sp. YHS-A2 was cloned into Escherichia coli using pUC19. The nucleotide sequence determination revealed a single open reading frame of chiA comprised of 1902 nucleotide base pairs and 633 deduced amino acids with a molecular weight of 67,452 Da. Amino acid sequence alignment showed that ChiA contains two putative chitin-binding domains and a single catalytic domain. Two proline-threonine repeat regions, which are linkers between catalytic and substrate-binding domains in some cellulases and xylanases, were also found. From E. coli, ChiA was purified 12.8-fold relative to the periplasmic fraction. The Michaelis constant and maximum initial velocity for p-nitrophenyl-N,N′-diacetylchitobiose were 1.06 mM and 44.4 μmol/h per mg protein, respectively. The purified ChiA binds not only to colloidal chitin but also to other substrates (avicel, chitosan, and xylan), but the binding affinity of avicel, chitosan, and xylan is around 10 times lower than that of colloidal chitin. The reaction of ChiA with colloidal chitin and chitooligosaccharides (trimer-hexamer) produced an end product of N,N′-diacetylchitobiose, indicating that ChiA is a chitobiosidase. Received: 29 October 1999 / Received revision: 16 March 2000 / Accepted: 24 March 2000     

Female-biased sex allocation in relation to growth rate of fruit in Erythronium japonicum

Using four populations of the liliaceous perennial Erythronium japonicum, I examined the hypothesis that sex allocation will be female-biased if the duration of sink-limited growth of fruits, during which fruits grow exponentially, is long. I found that all marked fruits in each population had a period of sink-limited growth. Among the four populations, the mean length of sink-limited growth increased, and the mean dry mass ratio of the sum of the corolla and androecium/fruit decreased, in a consistent order. Thus, plants in populations where the duration of sink-limited growth was long allocated relatively more of their resources to their female functions. This result was consistent with the above hypothesis. Received: 21 March 1998 / Accepted: 27 August 1998     

Bioremediation of polychlorinated biphenyl-contaminated soil using carvone and surfactant-grown bacteria

Partial bioremediation of polychlorinated biphenyl (PCB)-contaminated soil was achieved by repeated applications of PCB-degrading bacteria and a surfactant applied 34 times over an 18-week period. Two bacterial species, Arthrobacter sp. strain B1B and Ralstonia eutrophus H850, were induced for PCB degradation by carvone and salicylic acid, respectively, and were complementary for the removal of different PCB congeners. A variety of application strategies was examined utilizing a surfactant, sorbitan trioleate, which served both as a carbon substrate for the inoculum and as a detergent for the mobilization of PCBs. In soil containing 100 μg Aroclor 1242 g⁻¹ soil, bioaugmentation resulted in 55–59% PCB removal after 34 applications. However, most PCB removal occurred within the first 9 weeks. In contrast, repeated addition of surfactant and carvone to non-inoculated soil resulted in 30–36% PCB removal by the indigenous soil bacteria. The results suggest that bioaugmentation with surfactant-grown, carvone-induced, PCB-degrading bacteria may provide an effective treatment for partial decontamination of PCB-contaminated soils. Received: 9 March 2000 / Received revision: 27 June 2000 / Accepted: 16 July 2000     

Saccharide production from methanol by transposon 5 mutants derived from the extracellular polysaccharide-producing bacterium Methylobacillus sp. strain 12S

A CH₃OH-utilizing bacterium that has the ability to produce extracellular polysaccharide (EPS) was isolated from a soil sample, and was identified as the obligate methylotroph Methylobacillus sp. strain 12S on the basis of its 16S rDNA sequence and growth-substrate specificity. The EPS produced by strain 12S was purified and the sugar composition was analysed by GC-MS and HPLC to reveal that the EPS was a heteropolymer composed of glucosyl, galactosyl, and mannosyl residues in the molar ratio 3:1:1. In order to produce mono- and/or oligosaccharides by single-step fermentation from CH₃OH, stain 12S was mutagenized by transposon 5. Among eleven EPS-deficient mutants, three strains were found to accumulate significant amounts of reducing sugars in the media. The amounts of the reducing sugars produced by the mutants (>ca. 700 mg glucose equivalent/l) were >11–22 times higher than those produced by the wild-type strain (<ca. 60 mg glucose equivalent/l). The GC-MS analysis showed that all the mutants accumulated glucose, erythrose, threose and a disaccharide-like compound in the media. Received: 25 August 1999 / Received revision: 15 March 2000 / Accepted: 24 March 2000     

Enzymology of Phanerochaete chrysosporium with respect to the degradation of recalcitrant compounds and xenobiotics

The archetypal white-rot fungus Phanerochaete chrysosporium has been shown to degrade a variety of persistent environmental pollutants. Many of the enzymes responsible for pollutant degradation, which are normally involved in the degradation of wood, are extracellular. Thus, P. chrysosporium is able to degrade toxic or insoluble chemicals more efficiently than other microorganisms. P. chrysosporium has a range of oxidative and reductive mechanisms and uses highly reactive, nonspecific redox mediators which increase the number of chemicals that can be effectively degraded. This review gives an overview of the enzymes that are believed to be important for bioremediation and briefly discusses the degradation of some individual chemicals. Received: 25 April 2000 / Received revision: 05 June 2000 / Accepted: 04 July 2000     

Composition of the cell walls of several yeast species

Cell walls, representing 26%–32% of the cell dry weight, were prepared from several strains of the yeasts Kloeckera apiculata, Debaryomyces hansenii, Zygosaccharomyces bailii,Kluyveromyces marxianus and Saccharomyces cerevisiae. Extraction of the walls with potassium hydroxide at 4 °C, followed by saturation of the alkali-soluble extract with ammonium sulphate gave fractions of mannoprotein, alkali-soluble glucan and alkali-insoluble glucan. Chitin was associated with the alkali-insoluble glucan. The proportions of the different fractions within the walls varied with the species and strain. Mannoprotein comprised between 25% and 34% of the walls, the content of alkali-insoluble glucan ranged from 15% to 48%, and the content of alkali-soluble glucan ranged from 10% to 48%. There was significant variation in the physical appearance of the alkali-soluble glucans and the relative viscosity of suspensions of these glucans. The yeasts could represent novel sources of polysaccharides with industrial and medical applications. Received: 30 December 1997 / Received revision: 24 March 1998 / Accepted: 27 March 1998     

Stimulatory effect of growth in the presence of alcohols on biotransformation of penicillin G into cephalosporin-type antibiotics by resting cells of Streptomyces clavuligerus NP1

Growth of Streptomyces clavuligerus NP1 in the presence of methanol or ethanol resulted in a marked increase in production of cephalosporin(s) from penicillin G by resting cells. The mycelium produced in alcohol-supplemented medium was fragmented and dispersed as compared with growth in control medium. HPLC analysis showed that at least two products were present in the biotransformation supernatant fluid after 1 h incubation. One of them has been identified as deacetoxycephalosporin G (DAOG). Received: 9 December 1998 / Received revision: 29 March 1999 / Accepted: 16 April 1999     

Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max)

A full-length cDNA, GmNRT2, encoding a putative high-affinity nitrate transporter was isolated from a Glycine max (L.) root cDNA library and sequenced. The deduced GmNRT2 protein is 530 amino acids in length and contains 12 putative membrane-spanning domains and a long, hydrophilic C-terminal domain. GmNRT2 is related to high-affinity nitrate transporters in the eukaryotes Chlamydomonas reinhardtii and Aspergillus nidulans, and to putative high-affinity nitrate transporters in barley and tobacco. Southern blot analysis indicated that GmNRT2 is part of a small, multigene family in soybean. Expression of GmNRT2 in roots was regulated by the type of nitrogen source provided to plants: GmNRT2 mRNA levels were barely detectable in ammonium-grown plants, higher in nitrogen-deprived plants, and highest in nitrate-grown plants. Induction of GmNRT2 mRNA levels in roots occurred within 1 h after exposure of plants to nitrate. Nitrate induction of GmNRT2 mRNA levels was accompanied by a fourfold increase in net nitrate uptake by soybean roots at 100 μM external nitrate. The molecular and physiological evidence indicates that GmNRT2 is probably a high-affinity nitrate transporter involved in nitrate uptake by soybean roots. Received: 22 November 1997 / Accepted: 26 January 1998     

Chemostat study of xylitol production by Candida guilliermondii

The mechanism of production of xylitol from xylose by Candida guilliermondii was studied using chemostat cultures and enzymatic assays. The maximum dilution rate in aerobic conditions was 0.34 1/h. No xylitol was produced. Under oxygen-limited conditions xylose uptake was impaired and glycerol accumulated but no xylitol was detected. Under transient oxygen limitation, caused by a gradual decrease in the agitation rate, onset of xylitol, acetate and residual xylose accumulation occurred simultaneously when q _O2 dropped below 25 mmol/C-mmol cell dry weight (CDW) per hour. Ethanol and glycerol started to accumulate when q _O2 dropped below 20 mmol/C-mmol CDW per hour. The highest in vitro enzyme activities were found at the lowest dilution rate studied (0.091/h) under aerobic conditions. The amount of active enzymes or cofactor availability did not limit the rate of xylose consumption. Our results confirm that a surplus of NADH during transient oxygen limitation inhibited the activity of xylitol dehydrogenase which resulted in xylitol accumulation. Phosphoglucoisomerase (E.C. 5.3.1.9.) and glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) activities suggest re-shuttling of the metabolites into the pentose phosphate pathway. Received: 7 March 2000 / Received revision: 9 June 2000 / Accepted: 18 June 2000     

The role of the alternative respiratory pathway in the stimulation of cephalosporin C formation by soybean oil in Acremonium chrysogenum

Addition of soybean oil to Acremonium chrysogenum cultures growing on sugars doubled the specific production of cephalosporin C during the idiophase of growth. While the addition of soybean oil had no effect on the total rate of respiration, the respiration that proceeded via the alternative, cyanide-insensitive pathway exhibited a more than twofold increase. Addition of soybean oil also stimulated the formation of isocitrate lyase activities. Inhibition of oxidative metabolism of one of the products of isocitrate lyase (succinate) by thenoyltrifluoroacetone completely inhibited the alternative respiratory pathway. The role of soybean-oil-stimulated alternative respiration in the stimulation of cephalosporin C production and the role of isocitrate lyase are discussed. Received: 13 October 1998 / Revised revision: 14 January 1999 / Accepted: 22 January 1999