Evaluation of Growth and Production of High-Value-Added Metabolites in Scenedesmus quadricauda and Chlorella vulgaris Grown on Crude Glycerol under Heterotrophic and Mixotrophic Conditions Using Monochromatic Light-Emitting Diodes (LEDs).

This study aimed to examine the impact of crude glycerol as the main carbon source on the growth, cell morphology, and production of high-value-added metabolites of two microalgal species, namely Chlorella vulgaris and Scenedesmus quadricauda, under heterotrophic and mixotrophic conditions, using monochromatic illumination from light-emitting diodes (LEDs) emitting blue, red, yellow, and white (control) light. The findings indicated that both microalgae strains exhibited higher biomass yield on the mixotrophic growth system when compared to the heterotrophic one, while S. quadricauda generally performed better than C. vulgaris. In mixotrophic mode, the use of different monochromatic illumination affected biomass production differently on both strains. In S. quadricauda, growth rate was higher under red light (µmax = 0.89 d-1), while the highest biomass concentration and yield per gram of consumed glycerol were achieved under yellow light, reaching 1.86 g/L and Yx/s = 0.18, respectively. On the other hand, C. vulgaris demonstrated a higher growth rate on blue light (µmax = 0.45 d-1) and a higher biomass production on white (control) lighting (1.34 g/L). Regarding the production of metabolites, higher yields were achieved during mixotrophic mode in both strains. In C. vulgaris, the highest lipid (26.5% of dry cell weight), protein (63%), and carbohydrate (20.3%) contents were obtained under blue, red, and yellow light, respectively, thus indicating that different light wavelengths probably activate different metabolic pathways. Similar results were obtained for S. quadricauda with red light leading to higher lipid content, while white lighting caused higher production of proteins and carbohydrates. Overall, the study demonstrated the potential of utilizing crude glycerol as a carbon source for the growth and metabolite production of microalgae and, furthermore, revealed that the strains' behavior varied depending on lighting conditions.

Continuous Culture of Auxenochlorella protothecoides on Biodiesel Derived Glycerol under Mixotrophic and Heterotrophic Conditions: Growth Parameters and Biochemical Composition.

As crude glycerol comprises a potential substrate for microalga fermentation and value added products' biosynthesis, Auxenochlorella protothecoides was grown on it under heterotrophic and mixotrophic conditions and its growth kinetics were evaluated in a continuous system under steady state conditions. Increasing initial glycerol concentration (from 30 to 50 g/L) in the heterotrophic culture led to reduced biomass yield (Yx/S) and productivity (Px), but favored lipid accumulation. Under heterotrophic conditions, the microalga was found to grow better (biomass up to 7.888 g/L) and faster (higher growth rates), the system functioned more effectively (higher Px) and crude glycerol was exploited more efficiently. Heterotrophy also favored proteins synthesis (up to 53%), lipids (up to 9.8%), and carbohydrates (up to 44.6%) accumulation. However, different trophic modes had no significant impact on the consistency of proteins and lipids. Oleic acid was the most abundant fatty acid detected (55-61.2% of the total lipids). The algal biomass contained many essential and non-essential amino acids, especially arginine, glutamic acid, lysine, aspartic acid, leucine, and alanine. In all the experimental trials, the protein contents in the microalgal biomass increased with the increasing dilution rate (D), with a concomitant decrease in the lipids and carbohydrates fractions.

Enhancing the nutritional and functional properties of Pleurotus citrinopileatus mushrooms through the exploitation of winery and olive mill wastes.

Treatment and disposal of wineries and olive-oil mills waste is usually associated with complex processes, which are often of limited wide-scale applicability. Olive-leaves plus two-phase olive mill waste (OLW) or grape marc plus wheat straw (GMW) were assessed as substrates for the cultivation of the choice edible mushroom Pleurotus citrinopileatus. GMW led to increased mushroom biological efficiency and shorter production cycles. Antioxidant activities, triterpenic acids, free amino acids, lovastatin and ergosterol were significantly higher in fruitbodies from GMW; the latter compound was positively correlated with squalene concentrations in substrates. Glucans, resveratrol and fatty acids content showed minor differences among mushrooms from the three substrates examined, whereas ergothioneine was significantly higher in fruitbodies grown on OLW. High correlations were noted for oleanolic, ursolic and amino acid content in mushrooms and their respective substrates. Moreover, FTIR spectra revealed variations in fruitbodies content in bioactive compounds which were associated with the substrates used.

Co-composting of cotton residues with olive mill wastewater: process monitoring and evaluation of the diversity of culturable microbial populations.

With the aim to recommend an integrated alternative for the combined treatment of olive mill wastewater (OMW) and cotton residues (CR), and the production of high value and environmentally friendly products, two compost piles were set up. The first pile (control, pile 1) consisted of ginned CR, whereas the second (pile 2) was made of CR with the addition of OMW. A series of physicochemical parameters and the culturable microbial diversity in both piles were assessed. Co-composting (pile 2) displayed higher temperatures during the whole process, a prolonged second thermophilic phase and temperature values higher than 40 °C even after the thermophilic stage. Comparing the physicochemical parameters of the pile 2 with those of the pile 1, it was deduced that pH in the former was more acidic during the onset of the process; the EC values were higher throughout the process, while the levels of ammonium and nitrate nitrogen, as well as the NH4+/NO3- ratios, were lower at most of the sampling dates. By evaluating the abovementioned results, it was estimated that the co-composting process headed sooner toward stability and maturity, Isolated microorganisms from both piles were identified as members of the genera Brevibacillus, Serratia, Klebsiella, and Aspergillus, whereas active thermotolerant diazotrophs were detected in both piles at the 2nd thermophilic phase emerging a promising prospect upon further evaluation for enhancing the end-product quality. Our findings indicate that co-composting is an interesting approach for the exploitation of large quantities of agro-industrial residues with a final product suitable for improving soil fertility and health.

Effects of Monochromatic Illumination with LEDs Lights on the Growth and Photosynthetic Performance of Auxenochlorella protothecoides in Photo- and Mixotrophic Conditions.

This study examined the effects of monochromatic illumination (blue, red, green and yellow) employing light-emitting diodes (LEDs), trophic conditions (photoautotrophic and mixotrophic), and nitrogen availability (high and low peptone concentration) on the growth and biochemical composition of Auxenochlorella protothecoides. The results revealed that mixotrophic conditions did not favor A. protothecoides, giving lower growth rates compared to heterotrophy (dark conditions). However, mixotrophy gave significantly higher growth rates compared to photoautotrophy. The best light wavelengths for mixotrophic cultivation were that of white and red. In all cases investigated in this study, high peptone concentration (4 g/L) resulted in decreased growth rates. Regarding the biochemical composition of A. protothecoides, the strongest effect, irrespective of trophic conditions, was caused by nitrogen availability (peptone concentration). Specifically, at nitrogen replete conditions (4 g/L peptone), biomass was rich in proteins (32-67%), whereas under deplete conditions (0.5 g/L peptone), A. protothecoides accumulated mainly carbohydrates (up to 56%). Mixotrophic conditions generally favored higher carbohydrate content, whereas photoautotrophic conditions favored higher protein content. The different illumination spectra did not have any clear effect on the biochemical composition (metabolites content), except that, in all trophic conditions, the use of the green spectrum resulted in higher chlorophyll b content. Chlorophyll a fluorescence studies revealed that the trophic conditions and the high peptone concentrations impacted the photosystem II (PSII) performance, and also affected plastoquinone re-oxidation kinetics and the heterogeneity of the PSII reaction centers.

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.

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.

Effects of phosphorus concentration and light intensity on the biomass composition of Arthrospira (Spirulina) platensis.

This paper presents the effects of various phosphorus concentrations (10, 50, 250 and 500 mg l(-1) K(2)HPO(4)) on the biomass production and composition of Arthrospira (Spirulina) platensis in relation to light intensity (24, 42 and 60 µE m(-2) s(-1)). The maximum biomass production was 3,592 ± 392 mg l(-1) and this was observed in 250 mg l(-1) K(2)HPO(4) at 60 µE m(-2) s(-1) light intensity after 32 days of cultivation. A maximum specific growth rate (µ(max)) of 0.55 d(-1) was obtained in 500 mg l(-1) K(2)HPO(4) at 60 µE m(-2) s(-1). The protein, lipid and chlorophyll contents of the biomass varied from 33.59 to 60.57 %, 5.34 to 13.33 % and 0.78 to 2.00 %, respectively. The most significant finding was that phosphorus limitation (10 mg l(-1) K(2)HPO(4)) caused a drastic increase of the carbohydrate content (59.64 %). The effect of phosphorus limitation on the carbohydrate content was independent of the light intensity. The accumulated carbohydrates are proposed to be used as substrate for biofuel generation via one of the appropriate biomass energy conversion technologies. Also, it was observed that phosphorus removal is a function of biomass density, phosphorus concentration and light intensity.

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.

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.

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.

Characterization of a Mesorhizobium loti alpha-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.