Alginate: Microbial production, functionalization, and biomedical applications.

Alginates are natural polysaccharides widely participating in food, pharmaceutical, and environmental applications due to their excellent gelling capacity. Their excellent biocompatibility and biodegradability further extend their application to biomedical fields. The low consistency in molecular weight and composition of algae-based alginates may limit their performance in advanced biomedical applications. It makes microbial alginate production more attractive due to its potential for customizing alginate molecules with stable characteristics. Production costs remain the primary factor limiting the commercialization of microbial alginates. However, carbon-rich wastes from sugar, dairy, and biodiesel industries may serve as potential substitutes for pure sugars for microbial alginate production to reduce substrate costs. Fermentation parameter control and genetic engineering strategies may further improve the production efficiency and customize the molecular composition of microbial alginates. To meet the specific needs of biomedical applications, alginates may need functionalization, such as functional group modifications and crosslinking treatments, to achieve enhanced mechanical properties and biochemical activities. The development of alginate-based composites incorporated with other polysaccharides, gelatin, and bioactive factors can integrate the advantages of each component to meet multiple requirements in wound healing, drug delivery, and tissue engineering applications. This review provided a comprehensive insight into the sustainable production of high-value microbial alginates. It also discussed recent advances in alginate modification strategies and alginate-based composites for representative biomedical applications.

Azotobacter inoculation can enhance the resistance of Bt cotton to cotton bollworm, Helicoverpa armigera.

The rising trend in the cultivation of Bacillus thuringiensis (Bt) transgenic crops may cause a destabilization of agroecosystems, thus increasing concerns about the sustainability of Bt crops as a valid pest management method. Azotobacter can be used as a biological regulator to increase environmental suitability and improve the soil nitrogen utilization efficiency of crops, especially Bt cotton. A laboratory test investigated effects on the development and food utilization of Helicoverpa armigera fed with different Cry1Ab/Cry1Ac proteins and nitrogen metabolism-related compounds from cotton (transgenic variety SCRC 37 vs non-Bt cotton cv. Yu 2067) inoculated with Azospirillum brasilense (Ab) and Azotobacter chroococcum (Ac). The findings indicate that inoculation with Azotobacter significantly decreased the partial development and food utilization indexes (pupal weight; pupation rate; adult longevity; fecundity; relative growth rate, RGR; efficiency of conversion of digested food, ECD; and efficiency of conversion of ingested food, ECI) of H. armigera fed on Bt cotton, but contrasting trends were found among these indexes in H. armigera fed on non-Bt cotton inoculated with Azotobacter, as a result of differences in Bt toxin production. Overall, the results showed that inoculation with Azotobacter had negative effects on the development and food utilization of H. armigera fed on Bt cotton, leading to enhanced target insect resistance. Presumably, Azotobacter inoculation can be used to stimulate plant soil nitrogen uptake to increase nitrogen metabolism-related compounds and promote plant growth for Bt and non-Bt cotton, simultaneously raising Bt protein expression and enhancing resistance efficacy against cotton bollworm in Bt cotton.

Symbiotic interplay of Piriformospora indica and Azotobacter chroococcum augments crop productivity and biofortification of Zinc and Iron.

In the present study Piriformospora indica (Pi) a phyto-promotional fungus and Azotobacter chroococcumWR5 (AzWR5) a rhizobacterium, were symbiotically evaluated for their role in improving the nutritional quality of wheat (Triticum aestivum L.). Co-inoculation of Pi+AzWR5 modified root system architecture of host and along with increasing the proportion of finer roots by 88% and 92% in C306 and Hd2967 respectively. Furthermore, the synergistic impact of Pi+AzWR5 interplayed for enhanced accumulation of Zn and Fe in different plant parts including grains (3.12 and 1.33 fold respectively). Pi+AzWR5 increased the transfer factor of Zn (62%, 94%, 91% and 213%) and Fe (31%, 54%, 68% and 32%) in root, stem, leaves and grains, respectively, and translocation factor of Zn (20%, 18% and 63%) and Fe (18%, 29% and 29%) for root-stem, root-leaves and root-grains, respectively. In addition to these co-inoculation of endophytes led to several fold increase in expression of four ZIP transporter genes in roots and shoot. In addition to these symbiotic association of endophytes with host led to 3 fold increase in grain yield. We thereby conclude that co-inoculation of Pi+AzWR5 substantially improves mobilization of Zn and Fe from soil and increase its concentration in grains as well as improves crop yield.

Stimulating effects of reduced graphene oxide on the growth and nitrogen fixation activity of nitrogen-fixing bacterium Azotobacter chroococcum.

Graphene has found important applications in various areas and hundred tons of graphene materials are annually produced. It is crucial to investigate both the negative and positive environmental effects of graphene materials to ensure the safe applications and develop environmental applications. In this study, we reported the stimulating effects of reduced graphene oxide (RGO) to nitrogen-fixing bacterium Azotobacter chroococcum. RGO stimulated the cell growth of A. chroococcum at 0.010-0.500 mg/mL according to the growth curves and the colony-forming unit (CFU) increases. RGO wrapped over the A. chroococcum cells without inducing ultrastructural changes. RGO decreased the leakage of cell membrane, but slight oxidative stress was observed in A. chroococcum. RGO promoted the nitrogen fixation activity of A. chroococcum at 0.5 mg/mL according to both isotope dilution method and acetylene reduction activity measurements. Consequently, the increases of soil nitrogen contents were evidenced, in particular about 30% increase of organic nitrogen occurred at 0.5 mg/mL of RGO. In addition, RGO might possibly benefit the plant growth through enhancing the indoleacetic acid production of A. chroococcum. These results highlighted the positive environmental effects of graphene materials to nitrogen-fixing bacteria in nitrogen cycle.

Mechanism of the small ATP-independent chaperone Spy is substrate specific.

ATP-independent chaperones are usually considered to be holdases that rapidly bind to non-native states of substrate proteins and prevent their aggregation. These chaperones are thought to release their substrate proteins prior to their folding. Spy is an ATP-independent chaperone that acts as an aggregation inhibiting holdase but does so by allowing its substrate proteins to fold while they remain continuously chaperone bound, thus acting as a foldase as well. The attributes that allow such dual chaperoning behavior are unclear. Here, we used the topologically complex protein apoflavodoxin to show that the outcome of Spy's action is substrate specific and depends on its relative affinity for different folding states. Tighter binding of Spy to partially unfolded states of apoflavodoxin limits the possibility of folding while bound, converting Spy to a holdase chaperone. Our results highlight the central role of the substrate in determining the mechanism of chaperone action.

Specific H+ level is crucial for accurate phosphate quantification using ascorbate as a reductant.

Owing to its essentiality for cellular metabolism, phosphate (PO43-) plays a pivotal role in ecosystem dynamics. Frequent testing of phosphate levels is necessary to monitor ecosystem health. Present investigations were aimed to identify the key factors that are essential for proper quantification of PO43-. Primarily, H+ levels played a critical role in the development of molybdenum blue complex by ammonium molybdate and PO43- with ascorbic acid as a reductant. Molybdenum blue complex formed in the presence of 8 to 12 mmol of H+ in 3 ml reaction mixture remained stable even after 72 h. Of different concentrations of ammonium molybdate and ascorbic acid tested, best molybdenum blue complex was formed when their concentrations were 24.3 and 5.68 µmol, respectively. More or less similar intensity of molybdenum blue complex (due to reduction of phosphomolybdic acid and not molybdic acid) was formed in the presence of H+ at levels ranging from 8 to 10 mmol in 3 ml reaction mixture. Our findings unequivocally demonstrated that (i) the reaction mixture containing 3% ammonium molybdate, 0.1% ascorbic acid and 5 M H2SO4 in the ratio of 1:1:1 is ideal for PO43- quantification; (ii) antimony (Sb) significantly curbs the formation of molybdenum blue under these ideal conditions; (iii) this fine-tuned protocol for PO43- quantification could be extended without any problem for determining the level of PO43- both in plant as well as soil samples; and (iv) Azotobacter possesses potential to enhance levels of total PO43- in leaves and grains and soluble/active PO43- in rhizosphere soils of wheat.

Identification and molecular characterization of Azotobacter chroococcum and Azotobacter salinestris using ARDRA, REP, ERIC, and BOX.

Azotobacter chroococcum and A. salinestris do not possess significant and distinct morphological and physiological differences and are often mistaken with each other in microbiological research. In this study, 12 isolates of Azotobacter isolated by standard protocol from soils were identified morphologically and physiologically as A. chroococcum. The isolates were more closely investigated for the molecular differentiation and diversity of A. chroococcum and A. salinestris. For this purpose, the ARDRA technique including HpaII, RsaI, and AluI restriction enzymes, and REP, ERIC, and BOX markers were used. The nifD and nifH genes were also utilized to evaluate the molecular identification of these two species. The 16S rDNA evaluation showed that only four out of the 12 isolates were identified as A. chroococcum and the rest were A. salinestris. The results revealed that HpaII was able to differentiate A. chroococcum from A. salinestris whereas RsaI and AluI were not able to separate them. Moreover, BOX and REP markers were able to differentiate between A. chroococcum and A. salinestris. However, ERIC marker and nifD and nifH genes were unable to separate these species. According to the results, HpaII restriction enzyme is suggested to save time and cost. BOX and REP markers are recommended for differentiation and clear discrimination not only between A. chroococcum and A. salinestris but also among their strains.

Azotobacters as biofertilizer.

Azotobacters have been used as biofertilizer since more than a century. Azotobacters fix nitrogen aerobically, elaborate plant hormones, solubilize phosphates and also suppress phytopathogens or reduce their deleterious effect. Application of wild type Azotobacters results in better yield of cereals like corn, wheat, oat, barley, rice, pearl millet and sorghum, of oil seeds like mustard and sunflower, of vegetable crops like tomato, eggplant, carrot, chillies, onion, potato, beans and sugar beet, of fruits like mango and sugar cane, of fiber crops like jute and cotton and of tree like oak. In addition to the structural genes of the enzyme nitrogenase and of other accessory proteins, A. vinelandii chromosomes contain the regulatory genes nifL and nifA. NifA must bind upstream of the promoters of all nif operons for enabling their expression. NifL on activation by oxygen or ammonium, interacts with NifA and neutralizes it. Nitrogen fixation has been enhanced by deletion of nifL and by bringing nifA under the control of a constitutive promoter, resulting in a strain that continues to fix nitrogen in presence of urea fertilizer. Additional copies of nifH (the gene for the Fe-protein of nitrogenase) have been introduced into A. vinelandii, thereby augmenting nitrogen fixation. The urease gene complex ureABC has been deleted, the ammonia transport gene amtB has been disrupted and the expression of the glutamine synthase gene has been regulated to enhance urea and ammonia excretion. Gluconic acid has been produced by introducing the glucose dehydrogenase gene, resulting in enhanced solubilization of phosphate.

Stable Isotope-Labeled Single-Cell Raman Spectroscopy Revealing Function and Activity of Environmental Microbes.

Microorganisms play a key role in driving the global element (C, N, H, P, and S) cycling. However, the function and activity of environmental microbes remain largely elusive because the vast majority of them are yet uncultured. Recent achievements in single cell stable isotope-labeled Raman spectroscopy enable direct investigation of function and activity of individual microbes in complex environmental communities. Here, this protocol describes a workflow to investigate environmental microbes in soil and water by combining 15N, 2D, and 13C stable isotope labeling with different single-cell Raman techniques, including normal Raman, resonance Raman (RR), and surface-enhanced Raman spectroscopy (SERS). Their applications in investigating functional bacteria driving the N and C cycles, and metabolically active cells are described.

Integrated Effects of Co-Inoculation with Phosphate-Solubilizing Bacteria and N2-Fixing Bacteria on Microbial Population and Soil Amendment Under C Deficiency.

The inoculation of beneficial microorganisms to improve plant growth and soil properties is a promising strategy in the soil amendment. However, the effects of co-inoculation with phosphate-solubilizing bacteria (PSB) and N2-fixing bacteria (NFB) on the soil properties of typical C-deficient soil remain unclear. Based on a controlled experiment and a pot experiment, we examined the effects of PSB (M: Bacillus megaterium and F: Pseudomonas fluorescens), NFB (C: Azotobacter chroococcum and B: Azospirillum brasilence), and combined PSB and NFB treatments on C, N, P availability, and enzyme activities in sterilized soil, as well as the growth of Cyclocarya Paliurus seedlings grow in unsterilized soil. During a 60-day culture, prominent increases in soil inorganic N and available P contents were detected after bacteria additions. Three patterns were observed for different additions according to the dynamic bacterial growth. Synergistic effects between NFB and PSB were obvious, co-inoculations with NFB enhanced the accumulation of available P. However, decreases in soil available P and N were observed on the 60th day, which was induced by the decreases in bacterial quantities under C deficiency. Besides, co-inoculations with PSB and NFB resulted in greater performance in plant growth promotion. Aimed at amending soil with a C supply shortage, combined PSB and NFB treatments are more appropriate for practical fertilization at intervals of 30-45 days. The results demonstrate that co-inoculations could have synergistic interactions during culture and application, which may help with understanding the possible mechanism of soil amendment driven by microorganisms under C deficiency, thereby providing an alternative option for amending such soil.

Response surface optimization of conditions for culturing Azotobacter chroococcum in Agaricus bisporus industrial wastewater.

In the present study, the conditions for Azotobacter chroococcum fermentation using Agaricus bisporus wastewater as the culture medium were optimized. We analyzed the total number of living A. chroococcum in the fermentation broth, using multispectral imaging flow cytometry. Single-factor experiments were carried out, where a Plackett-Burman design was used to screen out three factors from the original six processing factors wastewater solubility, initial pH, inoculum size, liquid volume, culture temperature, and rotation speed that affected the total number of viable A. chroococcum. The Box-Behnken response surface method was used to optimize the interactions between the three main factors and to predict the optimal fermentation conditions. Factors significantly affecting the total number of viable A. chroococcum, including rotation speed, wastewater solubility, and culture temperature, were investigated. The optimum conditions for A. chroococcum fermentation in A. bisporus wastewater were a rotation speed of 200 rpm, a solubility of 0.25%, a culture temperature of 26°C, an initial pH of 6.8, a 5% inoculation volume, a culture time of 48 h, and a liquid volume of 120 mL in a 250 mL flask. Under these conditions, the concentration of total viable bacteria reached 4.29 ± 0.02 ✕ 107 Obj/mL A. bisporus wastewater can be used for the cultivation of A. chroococcum.

Application of rice (Oryza sativa L.) root endophytic diazotrophic Azotobacter sp. strain Avi2 (MCC 3432) can increase rice yield under green house and field condition.

Use of plant-associated beneficial microbes, especially endophytes are getting popular day by day as they occupy a relatively privileged niche inside different plant tissues with lesser competition for food and shelter than rhizosphere. The effects of different physical factors like temperature, rainfall, and seasonal variation and UV radiation on plant growth promoting endophytic communities are less pronounced than those on the rhizospheric and phylloplane microbes. This present work has been compromised with further utilization of an indigenous rice (Oryza sativa L.) root endophytic Azotobacter sp. strain Avi2 (MCC 3432) (AzA) as a bio-formulation for sustainable rice production based on several physiological parameters (plant height, root length/weight, leaf area, yield, chlorophyll contain), in-vitro comparative plant growth promoting assays, greenhouse and field experiments (dry and wet season). Treatments with AzA exhibited higher yield as well as maximal chlorophyll fluorescence (Fm) of flag leaves in flowering and grain filling stages indicating higher photosynthetic rates. Scanning electron microscopic image of rice roots demonstrated accumulation of bacterial biofilm at the junction of primary and lateral root confirming the root-colonizing ability of the bacterium. The results of the study were quite encouraging as AzA exhibited better vegetative and reproductive growth of rice in pot and field experiment compared to formulated rhizospheric Azotobacter sp. (commercial product). Apart from that plants treated with AzA (supplemented 50% nitrogenous fertilizer of recommended dose) exhibited similar yield parameters when it was compared with the recommended dose of fertilizer (RDF; 120:60:60 mg N:P:K kg-1 soil/ without any bacterial). Therefore, it can be concluded that application of this plant growth promoting endophyte can reduce a substantial amount of N-fertilizer for field application.

Genetic, structural, and functional diversity of low and high-affinity siderophores in strains of nitrogen fixing Azotobacter chroococcum.

To increase iron (Fe) bioavailability in surface soils, microbes secrete siderophores, chelators with widely varying Fe affinities. Strains of the soil bacterium Azotobacter chroococcum (AC), plant-growth promoting rhizobacteria used as agricultural inoculants, require high Fe concentrations for aerobic respiration and nitrogen fixation. Recently, A. chroococcum str. NCIMB 8003 was shown to synthesize three siderophore classes: (1) vibrioferrin, a low-affinity α-hydroxy carboxylate (pFe = 18.4), (2) amphibactins, high-affinity tris-hydroxamates, and (3) crochelin A, a high-affinity siderophore with mixed Fe-chelating groups (pFe = 23.9). The relevance and specific functions of these siderophores in AC strains remain unclear. We analyzed the genome and siderophores of a second AC strain, A. chroococcum str. B3, and found that it also produces vibrioferrin and amphibactins, but not crochelin A. Genome comparisons indicate that vibrioferrin production is a vertically inherited, conserved strategy for Fe uptake in A. chroococcum and other species of Azotobacter. Amphibactin and crochelin biosynthesis reflects a more complex evolutionary history, shaped by vertical gene transfer, gene gain and loss through recombination at a genomic hotspot. We found conserved patterns of low vs. high-affinity siderophore production across strains: the low-affinity vibrioferrin was produced by mildly Fe limited cultures. As cells became more severely Fe starved, vibrioferrin production decreased in favor of high-affinity amphibactins (str. B3, NCIMB 8003) and crochelin A (str. NCIMB 8003). Our results show the evolution of low and high-affinity siderophore families and conserved patterns for their production in response to Fe bioavailability in a common soil diazotroph.

Heavy metal induced oxidative damage and root morphology alterations of maize (Zea mays L.) plants and stress mitigation by metal tolerant nitrogen fixing Azotobacter chroococcum.

Heavy metals are one of the major abiotic stresses that adversely affect the quantity and nutritive value of maize. Microbial management involving the use of plant growth promoting rhizobacteria (PGPR) is a promising inexpensive strategy for metal clean up from polluted soils. Considering these, metal tolerant plant growth promoting nitrogen fixing rhizobacterial strain CAZ3 identified by 16SrRNA gene sequence analysis as Azotobacter chroococcum was recovered from metal polluted chilli rhizosphere. When exposed to varying levels of metals, A. chroococcum survived up to 1400 and 2000 µg mL-1 of Cu and Pb, respectively and expressed numerous plant growth promoting activities even under metal stress. Strain CAZ3 secreted 65.5 and 60.8 µg mL-1 IAA at 400 µg mL-1 each of Cu and Pb, respectively and produced siderophores, ammonia and ACC deaminase under metal pressure. The melanin extracted from A. chroococcum revealed metal chelating ability under EDX. Following application, strain CAZ3 enhanced growth and yield of maize grown both in the presence of Cu and Pb. The dry biomass of roots of inoculated plants grown with 2007 mg Cu kg-1 and 585 mg Pb kg-1 was increased by 28% and 20%, respectively. At 585 mg Pb kg-1, the bioinoculant also increased the kernel attributes. At 2007 mg Cu kg-1 strain CAZ3 enhanced the number, yield and protein of kernels by 10%, 45% and 6%, respectively. Interestingly, strain CAZ3 significantly reduced the levels of proline, malondialdehyde and antioxidant enzymes in foliage. The roots of inoculated plants accumulated greatest amounts of metals compared to other organs. In kernels, the concentration of Pb was more as compared to Cu. The metal concentrations in roots, shoots and kernels, however, declined following CAZ3 inoculation. Copper and lead had substantial distortive impact on root and leaf morphology while cell death were visible under CLSM and SEM. Conclusively, A. chroococcum CAZ3 could be a most suitable and promising option to increase maize production in metal polluted soils despite the soils being contaminated with heavy metals.

Crochelins: Siderophores with an Unprecedented Iron-Chelating Moiety from the Nitrogen-Fixing Bacterium Azotobacter chroococcum.

Microbes use siderophores to access essential iron resources in the environment. Over 500 siderophores are known, but they utilize a small set of common moieties to bind iron. Azotobacter chroococcum expresses iron-rich nitrogenases, with which it reduces N2 . Though an important agricultural inoculant, the structures of its iron-binding molecules remain unknown. Here, the "chelome" of A. chroococcum is examined using small molecule discovery and bioinformatics. The bacterium produces vibrioferrin and amphibactins as well as a novel family of siderophores, the crochelins. Detailed characterization shows that the most abundant member, crochelin A, binds iron in a hexadentate fashion using a new iron-chelating γ-amino acid. Insights into the biosynthesis of crochelins and the mechanism by which iron may be removed upon import of the holo-siderophore are presented. This work expands the repertoire of iron-chelating moieties in microbial siderophores.

Sequence of inoculation influences the nature of extracellular polymeric substances and biofilm formation in Azotobacter chroococcum and Trichoderma viride.

Extracellular polymeric substances (EPS) are important structural components of biofilms. In the present study, the EPS in biofilms developed using two agriculturally beneficial organisms-Azotobacter chroococcum (Az) and Trichoderma viride (Tv) were quantified and characterised. Time course experiments were undertaken to optimise the EPS yield of biofilm samples resulting from coculture and staggered inoculation. The EPS produced during biofilm formation was found to differ quantitatively and qualitatively in individual cultures (Az alone, Tv alone), and in treatments differing in the sequence of inoculation of bacterium and fungus (Az + Tv coculture, staggered inoculation of Az followed by Tv i.e. Az - Tv, or Tv followed by Az i.e. Tv - Az). Significant enhancement in terms of growth and biofilm formation, as compared to individual inoculation was recorded, with Tv - Az exhibiting higher values of these attributes. The EPS from biofilms showed significantly higher concentrations of protein, acetyl, and uronic acids, while planktonic EPS recorded higher total carbohydrates. Fourier transform infrared spectroscopy analyses illustrated the significant influence on chemical and structural aspects of EPS (planktonic and biofilm). This represents a first report correlating EPS production, cell aggregation and biofilm formation during bacterial-fungal biofilm development, which can have implications in the colonisation of soil and plants.

Co-cultivation of Chlamydomonas reinhardtii with Azotobacter chroococcum improved H2 production.

OBJECTIVES: To improve H2 production, the green algae Chlamydomonas reinhardtii cc849 was co-cultured with Azotobacter chroococcum. RESULTS: The maximum H2 production of the co-culture was 350% greater than that of the pure algal cultures under optimal H2 production conditions. The maximum growth and the respiratory rate of the co-cultures were about 320 and 300% of the controls, and the dissolved O2 of co-cultures was decreased 74%. Furthermore, the in vitro maximum hydrogenase activity of the co-culture was 250% greater than that of the control, and the in vivo maximum hydrogenase activity of the co-culture was 1.4-fold greater than that of the control. In addition, the maximum starch content of co-culture was 1400% that of the control. CONCLUSIONS: Azotobacter chroococcum improved the H2 production of the co-cultures by decreasing the O2 content and increasing the growth and starch content of the algae and the hydrogenase activity of the co-cultures relative to those of pure algal cultures.

Role of microbial inoculation and industrial by-product phosphogypsum in growth and nutrient uptake of maize (Zea mays L.) grown in calcareous soil.

BACKGROUND: Alkaline soils with high calcium carbonate and low organic matter are deficient in plant nutrient availability. Use of organic and bio-fertilizers has been suggested to improve their properties. Therefore, a greenhouse experiment was conducted to evaluate the integrative role of phosphogypsum (PG; added at 0.0, 10, 30, and 50 g PG kg-1 ), cow manure (CM; added at 50 g kg-1 ) and mixed microbial inoculation (Incl.; Azotobacter chroococcum, and phosphate-solubilizing bacteria Bacillus megaterium var. phosphaticum and Pseudomonas fluorescens) on growth and nutrients (N, P, K, Fe, Mn, Zn and Cu) uptake of maize (Zea mays L.) in calcareous soil. Treatment effects on soil chemical and biological properties and the Cd and Pb availability to maize plants were also investigated. RESULTS: Applying PG decreased soil pH. The soil available P increased when soil was inoculated and/or treated with CM, especially with PG. The total microbial count and dehydrogenase activity were enhanced with PG+CM+Incl. TREATMENTS: Inoculated soils treated with PG showed significant increases in NPK uptake and maize plant growth. However, the most investigated treatments showed significant decreases in shoot micronutrients. Cd and Pb were not detected in maize shoots. CONCLUSIONS: Applying PG with microbial inoculation improved macronutrient uptake and plant growth. © 2017 Society of Chemical Industry.

Biosynthesis of poly(3-hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: A precursor feeding strategy.

A precursor feeding strategy for effective biopolymer producer strain Azotobacter chroococcum 7B was used to synthesize various poly(3-hydroxybutyrate) (PHB) copolymers. We performed experiments on biosynthesis of PHB copolymers by A. chroococcum 7B using various precursors: sucrose as the primary carbon source, various carboxylic acids and ethylene glycol (EG) derivatives [diethylene glycol (DEG), triethylene glycol (TEG), poly(ethylene glycol) (PEG) 300, PEG 400, PEG 1000] as additional carbon sources. We analyzed strain growth parameters including biomass and polymer yields as well as molecular weight and monomer composition of produced copolymers. We demonstrated that A. chroococcum 7B was able to synthesize copolymers using carboxylic acids with the length less than linear 6C, including poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB-4MHV) using Y-shaped 6C 3-methylvaleric acid as precursor as well as EG-containing copolymers: PHB-DEG, PHB-TEG, PHB-PEG, and PHB-HV-PEG copolymers using short-chain PEGs (with n ≤ 9) as precursors. It was shown that use of the additional carbon sources caused inhibition of cell growth, decrease in polymer yields, fall in polymer molecular weight, decrease in 3-hydroxyvalerate content in produced PHB-HV-PEG copolymer, and change in bacterial cells morphology that were depended on the nature of the precursors (carboxylic acids or EG derivatives) and the timing of its addition to the growth medium.

Rhizobacteria and plant symbiosis in heavy metal uptake and its implications for soil bioremediation.

Certain species of plants can benefit from synergistic effects with plant growth-promoting rhizobacteria (PGPR) that improve plant growth and metal accumulation, mitigating toxic effects on plants and increasing their tolerance to heavy metals. The application of PGPR as biofertilizers and atmospheric nitrogen fixators contributes considerably to the intensification of the phytoremediation process. In this paper, we have built a system consisting of rhizospheric Azotobacter microbial populations and Lepidium sativum plants, growing in solutions containing heavy metals in various concentrations. We examined the ability of the organisms to grow in symbiosis so as to stimulate the plant growth and enhance its tolerance to Cr(VI) and Cd(II), to ultimately provide a reliable phytoremediation system. The study was developed at the laboratory level and, at this stage, does not assess the inherent interactions under real conditions occurring in contaminated fields with autochthonous microflora and under different pedoclimatic conditions and environmental stresses. Azotobacter sp. bacteria could indeed stimulate the average germination efficiency of Lepidium sativum by almost 7%, average root length by 22%, average stem length by 34% and dry biomass by 53%. The growth of L. sativum has been affected to a greater extent in Cd(II) solutions due its higher toxicity compared to that of Cr(VI). The reduced tolerance index (TI, %) indicated that plant growth in symbiosis with PGPR was however affected by heavy metal toxicity, while the tolerance of the plant to heavy metals was enhanced in the bacteria-plant system. A methodology based on artificial neural networks (ANNs) and differential evolution (DE), specifically a neuro-evolutionary approach, was applied to model germination rates, dry biomass and root/stem length and proving the robustness of the experimental data. The errors associated with all four variables are small and the correlation coefficients higher than 0.98, which indicate that the selected models can efficiently predict the experimental data.