Iordanis Chatzipavlidis

Cultivation of Arthrospira (Spirulina) platensis in olive-oil mill wastewater treated with sodium hypochlorite.

The subject of this paper is the cultivation of the cyanobacterium Arthrospira (Sprirulina) platensis in olive-oil mill wastewater (OMWW) treated with sodium hypochlorite (NaOCl). The main positive effect of NaOCl on the OMWW characteristics is the decrease of the phenol concentration and turbidity, rendering the OMWW suitable for A. platensis growth. Maximum biomass production (1696 mg/l) was obtained when the concentration of OMWW in the cultivation medium was 10% with the supplementation of 1g/l NaNO(3) and 5 g/l NaHCO(3). However, the addition of NaHCO(3) has no significant effect, indicating that the only limited nutrient in this wastewater is nitrogen, while carbon is provided by the organic compounds of the wastewater. The maximum of the removals of chemical oxygen demand (COD) and carbohydrates was 73.18% and 91.19%, respectively, while phenols, phosphorus and nitrates in some runs was completely removed.

The Genetic Diversity of Culturable Nitrogen-Fixing Bacteria in the Rhizosphere of Wheat

A total of 17 culturable nitrogen-fixing bacterial strains associated with the roots of wheat growing in different regions of Greece were isolated and characterized for plant-growth-promoting traits such as auxin production and phosphate solubilization. The phylogenetic position of the isolates was first assessed by the analysis of the PCR-amplified 16S rRNA gene. The comparative sequence analysis and phylogenetic analysis based on 16S rRNA gene sequences show that the isolates recovered in this study are grouped with Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. The diazotrophic nature of all isolates was confirmed by amplification of partial nifH gene sequences. The phylogenetic tree based on nifH gene sequences is consistent with 16S rRNA gene phylogeny. The isolates belonging to Azospirillum species were further characterized by examining the partial dnaK gene phylogenetic tree. Furthermore, it was demonstrated that the ipdC gene was present in all Azospirillum isolates, suggesting that auxin is mainly synthesized via the indole-3-pyruvate pathway. Although members of P. stutzeri and A. zeae are known diazotrophic bacteria, to the best of our knowledge, this is the first report of isolation and characterization of strains belonging to these bacterial genera associated with wheat.

Modelling the effect of temperature and water activity on the growth rate and growth/no growth interface of Byssochlamys fulva and Byssochlamys nivea.

The purpose of the present study was to apply a modelling approach to define the growth rate and growth/no growth interface of Byssochlamys fulva and Byssochlamys nivea on a synthetic medium as a function of temperature and water activity. Both fungal species were grown on malt extract agar at different temperatures (10, 15, 20, 25, 30, 35, 40 and 45 degrees C) and a(w) levels (0.88, 0.90, 0.92, 0.94, 0.96 and 0.99) for a period of 30 days. Growth responses were evaluated over time in terms of colony diameter changes. Growth data were fitted to the primary model of Baranyi and the resulting growth rates were further modeled as a function of temperature and water activity using the cardinal model with inflection (CMI) (Rosso et al., 1993). A logistic regression quadratic polynomial model was also employed to predict the probability of growth over storage time. Estimated parameters for minimum, maximum and optimum temperatures for growth were 9.1 degrees C, 46.4 degrees C and 32.1 degrees C for B. fulva and 10.5 degrees C, 43.2 degrees C and 32.1 degrees C for B. nivea. The respective values for a(w) were 0.893, 0.993 and 0.985 for B. fulva and 0.892, 0.992 and 0.984 for B. nivea. No growth was observed at 0.88 a(w) regardless of temperature for both species, whereas B. nivea ascospores could not grow at 10 and 45 degrees C irrespective of a(w). Regarding growth boundaries, the degree of agreement between predictions and observations was >98% concordant for both species. The erroneously predicted growth cases were 1.4-4.2% false positive and 2.1-3.5% false negative for B. nivea and B. fulva, respectively. The developed logistic model was validated with two literature data sets as well as with data from independent experiments carried out on fruit juices. Validation results showed that agreement with literature data for growth was 25 out of 36 (69.4%) cases, whereas validation on fruit juice data failed in only 6 cases (5 false positives and 1 false negative) out of 128 cases.

Bioconversion of lignocellulosic residues by Agrocybe cylindracea and Pleurotus ostreatus mushroom fungi - Assessment of their effect on the final product and spent substrate properties

Nine agro-industrial and forestry by-products were subjected to solid-state fermentation by Agrocybe cylindracea and Pleurotus ostreatus, and the process and end-products were comparatively evaluated. Grape marc waste plus cotton gin trash was the best performing medium for both fungi, while substrate composition had a marked effect on most cultivation parameters. Biological efficiency was positively correlated with nitrogen, lignin and ash, and negatively with hemicelluloses and carbohydrate content of substrates. Spent substrates demonstrated high reductions in hemicelluloses and cellulose in contrast to lignin; fibre fractions were correlated with nitrogen, fat and ash content of initial materials, while residual mycelial biomass was affected by mushroom productivity. Mushroom proximate analysis revealed significant variations of constituents depending on the substrate. Crude protein and fat were correlated with substrates nitrogen for both species. Alternative cultivation substrates of high potential are proposed, while spent material could be exploited as animal feed due to its upgraded properties.

Use of sulfur to control pH in composts derived from olive processing by-products

Two composts were prepared from olive press cake (OPC) repeatedly turned and moistened with either olive mill wastewater (OPC+OMW) or water (OPC+W). When phytotoxicity was drastically reduced and the pH of the composts had reached 8.6 and 7.55 respectively, elemental sulfur was added at 0.9% of dry weight to the OPC+OMW compost and at five different doses (0.1 – 1.0% of dry wt) to the OPC+W compost. During the following six months, an exponential pH decline was observed in both compost materials. The pH reached a final value of 5.8 in the OPC+OMW compost whereas a pH decline related to the amount of added sulfur was observed in the OPC+W compost (final values from 6.8 to 4.3). Over 80% of the pH decline occurred during the first two months following the sulfur addition. Sulfur was applied following the stabilization of the material in the case of OPC+W. No phytotoxic effects of the final products were observed at sulfur application doses up to 0.5% of dry compost weight, but a significant germination index reduction was observed at the 1% dose, probably related to the increased conductivity of the compost leachate. Sulfur was applied before stabilization of the compost material, in the case of OPC+OMW, to also investigate the effects of sulfur addition on the composting process. A thermophilic phase similar to that observed after the last OMW application exceeding 50°C followed, and no effects on microbial activity profiles of the compost were observed. The results indicate that small amounts of elemental sulfur may efficiently control high pH values in the final compost products and could be safely applied at late composting stages or after composting. This may broaden the utilization of these composts in potting media and alkaline soils.

Biochemical and molecular characterization of an Azotobacter vinelandii strain with respect to its ability to grow and fix nitrogen in olive mill wastewater

Abstract The bacterial strain A belongs to a collection of nitrogen fixing bacteria isolated from soil treated with olive mill wastewater (OMW). This strain can grow in OMW showing significant nitrogen fixing capacity. The study of growth and nitrogenase activity of the above strain during its growth into the waste showed that the maximum value of total acetylene reduction activity (expressed in nmol Ethyl/24 h/ml of culture) was obtained after 24 h of incubation as well as the maximum value of bacterial population. When the above nitrogen fixing capacity was expressed in reference to the bacterial population (nmol Ethyl/24 h/μg bacterial protein) its maximum value was observed earlier, since the first 7 h of incubation. Western blot analysis of total bacterial proteins, extracted at specific time intervals showed that nitrogenase activity was induced 30 mins after the inoculation of the waste with the strain A . The respective time of the enzymes induction in chemical media (N-free) was 1 h. Southern blot analysis of total genomic DNA of strain A using as probes the three structural genes (nifH, nifD, nifK) encoding nitrogenase-1 in Azotobacter vinelandii gave hybridization patterns which are conserved between the above two bacteria. These results strongly support parallel biochemical taxonomy data indicating that strain A may belong to Azotobacter vinelandii species.

Characterization of nitrogen-fixing bacteria isolated from field-grown barley, oat, and wheat

Diazotrophic bacteria were isolated from the rhizosphere of field-grown Triticum aestivum, Hordeum vulgare, and Avena sativa grown in various regions of Greece. One isolate, with the highest nitrogen-fixation ability from each of the eleven rhizospheres, was selected for further characterisation. Diazotrophic strains were assessed for plant-growth-promoting traits such as indoleacetic acid production and phosphate solubilisation. The phylogenies of 16S rRNA gene of the selected isolates were compared with those based on dnaK and nifH genes. The constructed trees indicated that the isolates were members of the species Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. Furthermore, the ipdC gene was detected in all A. brasilence and one A. zeae isolates. The work presented here provides the first molecular genetic evidence for the presence of culturable nitrogen-fixing P. stutzeri and A. zeae associated with field-grown A. sativa and H. vulgare in Greece.

Characterization of a Mesorhizobium loti α-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation

Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO(2) to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an alpha-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active alpha-CA. The biochemical and physiological roles of the CAA1 gene in free-living and symbiotic rhizobia were examined by using an M. loti R7A disruption mutant strain. Our analysis revealed that CAA1 is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH. L. japonicus plants inoculated with the CAA1 mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and delta(13)C abundance. Based on these results, we propose that although CAA1 is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH(3) protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule delta(13)C abundance suggest the recycling of at least part of the HCO(3)(-) produced by CAA1.

The Nitrogen-Fixation Island Insertion Site Is Conserved in Diazotrophic Pseudomonas stutzeri and Pseudomonas sp. Isolated from Distal and Close Geographical Regions

The presence of nitrogen fixers within the genus Pseudomonas has been established and so far most isolated strains are phylogenetically affiliated to Pseudomonas stutzeri. A gene ortholog neighborhood analysis of the nitrogen fixation island (NFI) in four diazotrophic P. stutzeri strains and Pseudomonas azotifigens revealed that all are flanked by genes coding for cobalamin synthase (cobS) and glutathione peroxidise (gshP). The putative NFIs lack all the features characterizing a mobilizable genomic island. Nevertheless, bioinformatic analysis P. stutzeri DSM 4166 NFI demonstrated the presence of short inverted and/or direct repeats within both flanking regions. The other P. stutzeri strains carry only one set of repeats. The genetic diversity of eleven diazotrophic Pseudomonas isolates was also investigated. Multilocus sequence typing grouped nine isolates along with P. stutzeri and two isolates are grouped in a separate clade. A Rep-PCR fingerprinting analysis grouped the eleven isolates into four distinct genotypes. We also provided evidence that the putative NFI in our diazotrophic Pseudomonas isolates is flanked by cobS and gshP genes. Furthermore, we demonstrated that the putative NFI of Pseudomonas sp. Gr65 is flanked by inverted repeats identical to those found in P. stutzeri DSM 4166 and while the other P. stutzeri isolates harbor the repeats located in the intergenic region between cobS and glutaredoxin genes as in the case of P. stutzeri A1501. Taken together these data suggest that all putative NFIs of diazotrophic Pseudomonas isolates are anchored in an intergenic region between cobS and gshP genes and their flanking regions are designated by distinct repeats patterns. Moreover, the presence of almost identical NFIs in diazotrophic Pseudomonas strains isolated from distal geographical locations around the world suggested that this horizontal gene transfer event may have taken place early in the evolution.