Screening of diazotrophic bacteria Azopirillum spp. for nitrogen fixation and auxin production in multiple field sites in southern Brazil

Isolation of microorganisms, screening for desirable characters and selection of efficient strains are important steps to optimize high crop yields and improve the sustainability of the ecosystem. The objective of this study was isolate and identify Azopirillum spp. with enhanced potential to promote plant growth among the natural bacterial population associated with rhizosphere soil, roots and stem of maize collected from five maize-growing regions within the southern state of Rio Grande do Sul in Brazil. Diazotrophic microorganisms were isolated using semi-solid N-free and solid selective media NFb. In order to select the most efficient isolates as candidates for plant growth promotion, the purified bacterial strains were studied for cell morphology, and Gram staining, streptomycin resistance, as well as screened for their potential for nitrogen fixation and auxin production under sterile conditions. Among 224 isolates obtained 121 were able to fix nitrogen and produce auxin. The 30 most promising isolates produced indole-3-acetic acid (IAA) ranging in concentration from 3.51 μg to 246.69 μg IAA mg⁻¹. Nitrogen fixation ranged from 15.43 μg to 95.21 μg of N mg protein⁻¹ day⁻¹ From the 30 most productive isolates, chromosomal DNA was extracted and a portion of the nifH gene was amplified and sequenced. Twenty-nine isolates were found to be similar to the Azospirillum genus and one isolate was found to be similar to Herbaspirillum seropedicae. These bacterial isolates revealed potential to increase crop productivity, however field crop experiments in Rio Grande do Sul climatic conditions should be done in order to formulate recommendations for their use as inoculants.     

Mating type of isolates derived from the spermogonial state ofPuccinia coronata var.coronata

Hyphal confrontation between two haploid cultures originating from single basidiospores was used to determine the mating type ofPuccinia coronata var.coronata. Pairs of 15 single-basidiospore cultures were placed approximately 1 mm apart on the medium in all possible combinations. Hyphae of the pairs of colonies came into contact with each other in all combinations approximately two weeks after confrontations. When the nuclear number of hyphal cells in a contact zone was investigated one month after confrontation, monokaryotic hyphae were observed in selfing combination. On the other hand, dikaryotic hyphae were observed in 90.5% of crossing combinations between different cultures. Two isolates are considered compatible if dikaryotic hyphae are present in the contact zone but incompatible if they are absent. The mating type of the fungus was found to be characterized by multiple-allelomorphic tetrapolar incompatibility controlled by the “A” and “B” incompatible factors. Contribution No. 116, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

Optimization of production of caffeine demethylase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. in a bioreactor

The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight⁻¹ h⁻¹ was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h⁻¹, indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors.     

Improvement in lactic acid production from starch using α-amylase-secreting <Emphasis Type="Italic">Lactococcus lactis</Emphasis> cells adapted to maltose or starch

To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting α-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l⁻¹ h⁻¹ lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l⁻¹ h⁻¹ lactate). Maximum volumetric lactate productivity was further increased (1.57 g l⁻¹ h⁻¹ lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of l-lactate) was achieved. In this study, we propose a new approach to lactate production by α-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.     

Acetylene reduction and indoleacetic acid production by Azospirillum isolates from Cactaceous plants

Azospirillum isolates were obtained from rhizosphere soil and roots of three cactaceae species growing under arid conditions. All Azospirillum isolates from rhizosphere and roots ofStenocereus pruinosus andStenocereus stellatus were identified asA. brasilense; isolates of surface-sterilized roots fromOpuntia ficus-indica were bothA. brasilense andA. lipoferum. Azospirilla per g of fresh root in the three species ranged from 70×10³ to 11×10³. The most active strains in terms of C₂H₂ reduction (25–49.6 nmol/h·ml) and indoleacetic acid (IAA) production (36.5–77 μg/ml) were those identified asA. brasilense and isolated from Stenocereus roots.A. lipoferum isolated from Opuntia roots produced low amounts of IAA (6.5–17.5 μg/ml) and low C₂H₂-reduction activity (17.8–21.2 nmol/h·ml).     

Use of single-protoplast isolates in the study of the mating phenomena of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> (<Emphasis Type="Italic">Thanatephorus cucumeris</Emphasis>) AG-1 IC and IA

This study evaluates the effectiveness of using single-protoplast isolates (SPIs) to study the mating phenomena of Rhizoctonia solani AG-1 IC and IA. SPIs obtained from three field isolates (F-1, Rh28, and RO2) of AG-1 IC were paired with representative single-basidiospore isolate (SBI)-M1/-M2 testers, each from their own field isolates, or paired in all possible combinations. Tufts were formed between SPIs and SBI-M1/-M2 testers and between SPIs-M1 and -M2. The separation ratios of SPIs-M1 and -M2 were approximately 1:1, which were similar to the results obtained with SBIs. SPIs obtained from three isolates (GNSD, R59, and Tr8) of AG-1 IA, which failed to form basidiospores, were paired in all possible combinations. Although no tufts formed among SPIs from Tr8 and R59, tufts did form between SPIs from GNSD. SPIs from GNSD were separated into homokaryotic (-M1 or -M2) and heterokaryotic isolates, and the separation ratio of -M1 and -M2 was also around 1:1. Amplified fragment length polymorphism (AFLP) phenotypes of the tuft isolates formed between GNSD SPIs-M1 and -M2 suggested that these tuft isolates were all heterokaryotic. These results indicate that all three isolates of AG-1 IC and one isolate GNSD of AG-1 IA are heterokaryotic, and that the other two isolates of Tr8 and R59 of AG-1 IA are homokaryotic. Single-protoplast isolates are effective for studies of the mating phenomena of isolates belonging to different AGs of R. solani that could not form a perfect stage.     

Occurrence of extended-spectrum β-lactamases in <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from piggery farms in Imo State,Nigeria

A total of 600 Escherichia coli isolates recovered from pig wastes from three Senatorial Zones of Imo State, Nigeria, were tested for production of extended-spectrum β-lactamases (ESBLs) by the double-disk (DD) potentiation method. Of the numbers of isolates made, 190 (32%) were positive for the ESBLs production. Results of testing 190 positive isolates for ESBL production by several recommended methods were as follows (percentage detection in parentheses): DD method with aztreonam (91), ceftazidime (82), ceftriaxone (84), or cefpodoxime (94); broth microdilution method with ceftazidime (77) or cefotaxime (96) alone or in combination with clavulanate; and the standard disk diffusion method with new breakpoints and standard concentrations of aztreonam (69), ceftazidime (81), ceftriaxone (77), or cefpodoxime (98) or a novel concentration (5 μg) of ceftazidime (84). These data indicate that ESBLs occur at a relatively high incidence in our piggery farms and that the standard disks diffusion method with cefpodoxime and the DD method with several β-lactams are practical and cost-effective methods for detecting ESBL-producing isolates of E. coli.      

Studies of four thelytokous puerto rican isolates ofMuscidifurax uniraptor [Hymenoptera: Pteromalidae]

Field collections in Puerto Rico revealed 4 behaviorally distinct isolates ofMuscidifurax uniraptor Kogan & Legner that differed initially in diapause and nondiapause emergence, and the age when ♀ progeny were produced. Subsequent F₁ and F₂ progeny differed in sex ratio and total progeny production. Mating F₂ ♀ ♀ from nondiapause isolates to naturally emerging ♂ ♂ from thelytokous populations significantly reduced total progeny and the proportion of ♀ ♀ toca. 20 %. These mated ♀ ♀ at first resembled in behavior those which originated from diapausing parents. Random mating within all isolates beginning in the F₃, resulted in a general lower survival and progeny production but was accompanied by a rise in sex ratio toca. 50% ♀ ♀ by the F₆ generation. The interinvolvement of extranuclear and genic factors is considered.      

Cerrena unicolor isolated from the mycangia of a horntail,Tremex longicollis,in Kochi Prefecture,Japan

A fungus was found to be stored in the mycangia of a horntail,Tremex longicollis, as hyphal fragments. All fungal isolates from the mycangia of 31 adult females of the horntail produced the same colonies on PDA. Basidiocarps ofCerrena unicolor occurred near the emergence hole of the horntail on a dead hackberry tree (Celtis sinensis). The cultures of this fungus were similar to those from the mycangia of the horntail in cultural characteristics. Mating between single-basidiospore mycelia ofC. unicolor and single-arthrospore mycelia from the mycangia of the horntail showed that they were compatible. These results revealed that the fungus isolated from the mycangia ofT. longicollis wasC. unicolor.     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.     

Expression of the Neisseria gonorrhoeae major outer membrane protein PI in Escherichia coli

Summary To actively express an outer membrane protein, protein I (PI), from different strains of Neisseria gonorrhoeae in E.␣coli, PI gene fragments from two reference strains and four clinical isolates of Neisseria gonorrhoeae were obtained with PCR amplification. They were cloned into the PCR cloning vector pBS-T to form pBS-T-PI and sequenced. Subsequently, they were cloned into an expression vector pET-30b (+) to generate pET-PI recombinants. After inducing with isopropyl-β-d-thiogalactopyranoside (IPTG), the expressed PI proteins were analysed by SDS-PAGE, Western blotting and ELISA. The results implied that we had successfully constructed the PI gene recombinants from both reference strains and clinical isolates and obtained the recombinant proteins expressed in E. coli at relatively high levels, and the expressed proteins had the immunological activity with the corresponding antibodies. This research will be very helpful for the further study of these proteins in generating preventive vaccines on Neisseria gonorrhoeae infection and clinical diagnosis.     

Selection and characterization of mannanase-producing bacteria useful for the formation of prebiotic manno-oligosaccharides from copra meal

In order to select bacterial strains effectively secreting mannanase activity for the production of prebiotic mannooligosaccharides, a two-step screening procedure was performed. Enriched cultures from isolation medium containing copra meal were primary screened on an isolation agar medium containing 1% locust bean gum (LBG), which resulted in 48 mannanase-producing bacterial isolates with significant clearing zones on the mannan-containing agar. However, only nine isolates showed appreciable mannanase activities against copra meal in their culture supernatants (0.054–0.185 U/mg of protein) as determined in a standard assay based on the detection of reducing sugars released from this substrate. The isolates CW2-3 and ST1-1 displayed the highest activity against LBG and copra meal, respectively. Copra mannan hydrolysates that were obtained by using crude mannanase from these nine isolates were further used for a secondary screening towards a growth-enhancing activity on Lactobacillus reuteri and inhibitory activity against Escherichia coli as well as Salmonella Enteritidis, resulting in 0.09–2.15 log CFU/ml enhancing activity and low inhibitory activity of 0.46–1.78 log CFU/ml as well as 0.37–1.72 log CFU/ml, respectively. The hydrolysate of CW2-3 mannanase showed the highest enhancing activity of 2.15 log CFU/ml while isolate ST1-1 was most effective with respect to growth inhibition against E. coli E010 and S. Enteritidis S003 with 0.76 and 1.61 log CFU/ml, respectively. Based on morphological, physical, biochemical and genetics properties, isolates CW2-3 and ST1-1 were identified as Klebsiella oxytoca and Acinetobacter sp., respectively. Crude mannanase activity from these two strains was characterized preliminarily. The pH optima of mannanase activity from Klebsiella oxytoca CW2-3 and Acinetobacter sp. ST1-1 were 7 and 6, respectively. The enzymes were stable at 4°C over a pH range of 3–6 and 3–10, respectively.     

Cryptococcus neoformans and Cryptococcus gattii Isolated from the Excreta of Psittaciformes in a Southern Brazilian Zoological Garden

Cryptococcus neoformans, a major pathogen in immunocompromised patients, is a ubiquitous free-living fungus that can be isolated from soils, avian excreta and plant material. To further study potential saprophytic sources of this yeast in the Southern Brazilian State Rio Grande do Sul, we analyzed fecal samples from 59 species of captive birds kept in cages at a local Zoological Garden, belonging to 12 different orders. Thirty-eight environmental isolates of C. neoformans were obtained only from Psittaciformes (Psittacidae, Cacatuidae and Psittacula). Their variety and serotype were determined, and the genetic structure of the isolates was analyzed by use of the simple repetitive microsatellite specific primer M13 and the minisatellite specific primer (GACA)₄ as single primers in the PCR. The varieties were confirmed by pulsed-field gel electrophoresis (PFGE). Thirty-three isolates (87%) were from the var. grubii, serotype A, molecular type VNI and five (13%) were Cryptococcus gattii, serotype B, molecular type VGI. All the isolates were mating type α. Isolates were screened for some potential virulence factors. Quantitative urease production by the environmental isolates belonging to the C. gattii was similar to the values usually obtained for clinical ones.     

Effect of carbon and nitrogen sources, pH and temperature on in vitro culture of several isolates of Amanita caesarea (Scop.:Fr.) Pers.

Several isolates were obtained from sporocarps of Amanita caesarea (Scop.: Fr.) Pers. associated with Quercus suber and Castanea sativa coming from the southwest of Spain. Culture conditions were optimized for these isolates. The largest radial growth was obtained at pH 6–7, and optimal growth temperature was 24–28°C depending on the isolate. Albumin bovine and nitrate produced the largest patch size diameters, but the greatest mycelium dry weight yields were obtained with ammonium. Mannitol produced the largest radial growth, and mannitol and glucose yielded the biggest mycelium dry weights. Although variations in growth behaviour between isolates were observed, only one internal spacer sequence–restriction fragment length polymorphism type was obtained.     

Isolation and characterization of <Emphasis Type="Italic">Pseudoalteromonas ruthenica</Emphasis> (SBT033), an EPS-producing biofilm bacterium from the seawater intake point of a tropical power station

Biofilm formation in bacteria is closely linked with production of exopolysaccharides (EPS). This study examined the quantitative variations in EPS production and biofilm-forming ability among bacteria isolated from the seawater intake point of a power station located on the east coast of India. Of the 233 isolates obtained from the intake site, 71 bacterial isolates displayed different colony morphological characteristics. Thirteen isolates that produced wide and thick mucoid colonies were further tested for their ability to attach and form biofilms by microtitre plate assay and confocal microscopy. EPS production among the selected bacterial isolates ranged from 826 to 1838 μg ml⁻¹. Strain SBT033, which produced the maximum amount of EPS also displayed the maximum biofilm-forming ability among the 13 isolates. This strain was selected for further characterization using biochemical and molecular methods. The pale orange-pigmented isolate was a Gram negative, aerobic, short rod-shaped and grew well only in the presence of 2% NaCl. On the basis of phenotypic characteristics the isolate SBT033 is shown to belong to the genus Pseudoalteromonas. Analysis of 16S rRNA of the isolate revealed 99% homology with Pseudoalteromonas ruthenica.     

Very high ethanol productivity in an innovative continuous two-stage bioreactor with cell recycle

The performance of an innovative two-stage continuous bioreactor with cell recycle—potentially capable of giving very high ethanol productivity—was investigated. The first stage was dedicated to cell growth, whereas the second stage was dedicated to ethanol production. A high cell density was obtained by an ultrafiltration module coupled to the outlet of the second reactor. A recycle loop from the second stage to the first one was tested to improve cell viability and activity. Cultivations of Saccharomyces cerevisiae in mineral medium on glucose were performed at 30°C and pH 4. At steady state, total biomass concentrations of 59 and 157 gDCW l⁻¹ and ethanol concentrations of 31 and 65 g l⁻¹ were obtained in the first and second stage, respectively. The residual glucose concentration was 73 g l⁻¹ in the first stage and close to zero in the second stage. The present study shows that a very high ethanol productivity (up to 41 g l⁻¹ h⁻¹) can indeed be obtained with complete conversion of the glucose and with a high ethanol titre (8.3°GL) in the two-stage system.     

Continuous production of citric acid from dairy wastewater using immobilized<Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9142

The continuous production of citric acid from dairy wastewater was investigated using calcium-alginate immobilizedAspergillus niger ATCC 9142. The citric acid productivity and yield were strongly affected by the culture conditions. The optimal pH, temperature, and dilution rate were 3.0, 30°C, and 0.025 h⁻¹, respectively. Under optimal culture conditions, the maximum productivity, concentration, and yield of citric acid produced by the calcium-alginate immobilizedAspergillus niger were 160 mg L⁻¹ h⁻¹, 4.5 g/L, and 70.3% respectively. The culture was continuously perfored for 20 days without any apparent loss in citric acid productivity. Conversely, under the same conditions with a batch shake-flask culture, the maximum productivity, citric acid concentration, and yield were only 63.3 mg L⁻¹ h⁻¹, 4.7 g/L and 51.4%, respectively. Therefore, the results suggest that the bioreactor used in this study could be potentially used for continuous citric acid production from dairy wastewater by applying calcium-alginate immobilizedAspergillus niger.     

Colonisation of phase II compost by biotypes of Trichoderma harzianum and their effect on mushroom yield and quality

Colonisation assessments confirmed that Trichoderma harzianum biotypes Th1, Th2a, Th2b and Th3 inoculated into two distinct compost types at spawning became established by first flush assessment; the extension rate of two Th2 isolates was over 1000 times that of Th1 and Th3. Results subsequently confirmed that while Th1 and Th3 did not significantly affect yield, Th2 could reduce mushroom quality and productivity by as much as 80%. Analysis of compost type also indicated that the speed and magnitude of T. harzianum colonisation was influenced by key compost characteristics, most notably, moisture, ash content and degree of fermentation. This study has shown that compost parameters which have a positive influence on Agaricus growth and productivity also resulted in increased compost colonisation by T. harzianum. Commercially acceptable yields obtained from uninoculated compost confirmed that production of a high quality, productive substrate does not confer inherent immunity to colonisation by T. harzianum. Received: 25 September 1998 / Received revision: 30 November 1998 / Accepted: 5 December 1998     

Improvement of pristinamycin-producing Streptomyces pristinaespiralis by rational screening

Summary According to the biosynthetic pathway of pristinamycin, a rational selection procedure with u.v. mutation was performed to obtain a high pristinamycin-producing strain. Aminoacetic acid-resistant mutants (AA^r), valine hydroxamate-resistant mutants (VH^r), kitasamycin-resistant mutants (KTM^r) and 2-deoxy-D-glucose-resistant mutants (DOG^r) were selected, successively. A strain Streptomyces pristinaespiralis 12–55 with AA^r, Val^r, KTM^r, and DOG^r was obtained, and its production of pristinamycin reached 3000 u/ml which is 100 times higher than that of the parent strain S. pristinaespiralis ATCC 25486. It is inferred that S. pristinaespiralis 12–55 can alleviate catabolite repression caused by carbon sources, provide more acetic acid and valine for pristinamycin biosynthesis and increase its resistance to pristinamycin produced by itself, all of which are favorable for pristinamycin production. The subculture experiments indicated that the hereditary character of high productivity of S. pristinaespiralis 12–55 is stable. The pristinamycin production of S. pristinaespiralis 12–55 in a 15-l fermentor could reach 3010 u/ml after a 56 h batch fermentation.     

High cell density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> with surface anchored transglucosidase for use as whole-cell biocatalyst for α-arbutin synthesis

A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for α-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of α-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.