Genetic modification of acetogens and optimization of fermentation process in C1-gas bioconversion / 生物工程学报

The current linear economy model relies on fossil energy and increases CO2 emissions, which contributes to global warming and environmental pollution. Therefore, there is an urgent need to develop and deploy technologies for carbon capture and utilization to establish a circular economy. The use of acetogens for C1-gas (CO and CO2) conversion is a promising technology due to high metabolic flexibility, product selectivity, and diversity of the products including chemicals and fuels. This review focuses on the physiological and metabolic mechanisms, genetic and metabolic engineering modifications, fermentation process optimization, and carbon atom economy in the process of C1-gas conversion by acetogens, with the aim to facilitate the industrial scale-up and carbon negative production through acetogen gas fermentation.

Engineering the glycogen metabolism in cyanobacterial photosynthetic cell factories: a review / 生物工程学报

Cyanobacteria are important photosynthetic autotrophic microorganisms and are considered as one of the most promising microbial chassises for photosynthetic cell factories. Glycogen is the most important natural carbon sink of cyanobacteria, playing important roles in regulating its intracellular carbon distributions. In order to optimize the performances of cyanobacterial photosynthetic cell factories and drive more photosynthetic carbon flow toward the synthesis of desired metabolites, many strategies and approaches have been developed to manipulate the glycogen metabolism in cyanobacteria. However, the disturbances on glycogen metabolism usually cause complex effects on the physiology and metabolism of cyanobacterial cells. Moreover, the effects on synthesis efficiencies of different photosynthetic cell factories usually differ. In this manuscript, we summarized the recent progress on engineering cyanobacterial glycogen metabolism, analyzed and compared the physiological and metabolism effects caused by engineering glycogen metabolism in different cyanobacteria species, and prospected the future trends of this strategy on optimizing cyanobacterial photosynthetic cell factories.

Media development and process parameter optimization using statistical experimental designs for the production of nonribosomal peptides in Escherichia coli

BACKGROUND: Nonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited bioprocess engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach. RESULTS: We found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions. CONCLUSIONS: We developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L-1, representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development.

Enhanced pyruvic acid yield in an osmotic stress-resistant mutant of Yarrowia lipolytica

BACKGROUND: Pyruvic acid (PA), a vital α-oxocarboxylic acid, plays an important role in energy and carbon metabolism. The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) has considerable potential for the production of PA. An increased NaCl concentration reportedly increases the biomass and PA yield of Y. lipolytica. RESULTS: To increase the yield of PA, the NaCl-tolerant Y. lipolytica A4 mutant was produced using the atmospheric and room temperature plasma method of mutation. The A4 mutant showed growth on medium containing 160 g/L NaCl. The PA yield of the A4 mutant reached 97.2 g/L at 120 h (0.795 g/g glycerol) in a 20-L fermenter with glycerol as the sole carbon source, which was 28.9% higher than that of the parental strain. CONCLUSION: The PA yield from Y. lipolytica can be improved by increasing its NaCl tolerance.

Changes induced by co-inoculation in nitrogen-carbon metabolism in cowpea under salinity stress

ABSTRACT To mitigate the deleterious effects of abiotic stress, the use of plant growth-promoting bacteria along with diazotrophic bacteria has been increasing. The objectives of this study were to investigate the key enzymes related to nitrogen and carbon metabolism in the biological nitrogen fixation process and to elucidate the activities of these enzymes by the synergistic interaction between Bradyrhizobium and plant growth-promoting bacteria in the absence and presence of salt stress. Cowpea plants were cultivated under axenic conditions, inoculated with Bradyrhizobium and co-inoculated with Bradyrhizobium sp. and Actinomadura sp., Bradyrhizobium sp. and Bacillus sp., Bradyrhizobium sp. and Paenibacillus graminis, and Bradyrhizobium sp. and Streptomycessp.; the plants were also maintained in the absence (control) and presence of salt stress (50 mmolL-1 NaCl). Salinity reduced the amino acids, free ammonia, ureides, proteins and total nitrogen content in nodules and increased the levels of sucrose and soluble sugars. The co-inoculations responded differently to the activity of glutamine synthetase enzymes under salt stress, as well as glutamate synthase, glutamate dehydrogenase aminating, and acid invertase in the control and salt stress. Considering the development conditions of this experiment, co-inoculation with Bradyrhizobium sp. and Bacillus sp. in cowpea provided better symbiotic performance, mitigating the deleterious effects of salt stress.

Bioflocculant production from Streptomyces platensis and its potential for river and waste water treatment

ABSTRACT A bacterium isolated from Sterkfontein dam was confirmed to produce bioflocculant with excellent flocculation activity. The 16S rDNA nucleotide sequence analyses revealed the bacteria to have 99% similarity to Streptomyces platensis strain HBUM174787 and the sequence was deposited in the Genbank as Streptomyces platensis with accession number FJ 486385.1. Culture conditions for optimal production of the bioflocculant included glucose as a sole carbon source, resulting in flocculating activity of 90%. Other optimal conditions included: peptone as nitrogen source; presence of Mg2+ as cations and inoculum size of 1.0% (v/v) at neutral pH of 7. Optimum dose of the purified bioflocculant for the clarification of 4 g/L kaolin clay suspension at neutral pH was 0.2 mg/mL. Energy Dispersive X-ray analysis confirmed elemental composition of the purified bioflocculant in mass proportion (%w/w): carbon (21.41), oxygen (35.59), sulphur (26.16), nitrogen (0.62) and potassium (7.48). Fourier Transform Infrared Spectroscopy (FTIR) indicated the presence of hydroxyl, carboxyl, methoxyl and amino group in the bioflocculant. The bioflocculant produced by S. platensis removed chemical oxygen demand (COD) in river water and meat processing wastewater at efficiencies of 63.1 and 46.6% respectively and reduced their turbidity by 84.3 and 75.6% respectively. The high flocculating rate and removal efficiencies displayed by S. platensis suggests its industrial application in wastewater treatment.

Anaerobic biodegradation of benzo(a)pyrene by a novel Cellulosimicrobium cellulans CWS2 isolated from polycyclic aromatic hydrocarbon-contaminated soil

Abstract Cellulosimicrobium cellulans CWS2, a novel strain capable of utilizing benzo(a)pyrene (BaP) as the sole carbon and energy source under nitrate-reducing conditions, was isolated from PAH-contaminated soil. Temperature and pH significantly affected BaP biodegradation, and the strain exhibited enhanced biodegradation ability at temperatures above 30 °C and between pH 7 and 10. The highest BaP removal rate (78.8%) was observed in 13 days when the initial BaP concentration was 10 mg/L, and the strain degraded BaP at constant rate even at a higher concentration (50 mg/L). Metal exposure experimental results illustrated that Cd(II) was the only metal ion that significantly inhibited biodegradation of BaP. The addition of 0.5 and 1.0 g/L glucose enhanced BaP biodegradation, while the addition of low-molecular-weight organic acids with stronger acidity reduced BaP removal rates during co-metabolic biodegradation. The addition of phenanthrene and pyrene, which were degraded to some extent by the strain, showed no distinct effect on BaP biodegradation. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the five rings of BaP opened, producing compounds with one to four rings which were more bioavailable. Thus, the strain exhibited strong BaP degradation capability and has great potential in the remediation of BaP-/PAH-contaminated environments.

Fermentation optimization and enzyme characterization of a new ι-Carrageenase from Pseudoalteromonas carrageenovora ASY5

Background: A new ι-carrageenase-producing strain was screened from mangroves and authenticated as Pseudoalteromonas carrageenovora ASY5 in our laboratory. The potential application of this new strain was evaluated. Results: Medium compositions and culturing conditions in shaking flask fermentation were firstly optimized by single-factor experiment. ι-Carrageenase activity increased from 0.34 U/mL to 1.08 U/mL after test optimization. Optimal fermentation conditions were 20°C, pH 7.0, incubation time of 40 h, 15 g/L NaCl, 1.5% (w/v) yeast extract as nitrogen source, and 0.9% (w/v) ι-carrageenan as carbon source. Then, the crude ι-carrageenase was characterized. The optimum temperature and pH of the ι-carrageenase were 40°C and 8.0, respectively. The enzymatic activity at 35­40°C for 45 min retained more than 40% of the maximum activity. Meanwhile, The ι-carrageenase was inhibited by the addition of 1 mmol/L Cd2+ and Fe3+ but increased by the addition of 1 mmol/L Ag+, Ba2+, Ca2+, Co2+, Mn2+, Zn2+, Fe2+, and Al3+. The structure of oligosaccharides derived from ι-carrageenan was detected using electrospray ionization mass spectrometry (ESI-MS). The ι-carrageenase degraded ι-carrageenan, yielding disaccharides and tetrasaccharides as main products. Conclusions: The discovery and study of new ι-carrageenases are beneficial not only for the production of ι-carrageenan oligosaccharides but also for the further utilization in industrial production.

Bacterial degradation of dissolved organic matter released by Planktothrix agardhii (Cyanobacteria) / Degradação bacteriana da matéria orgânica dissolvida liberada por Planktothrix agardhii (Cyanobacteria)

Abstract Although Planktothrix agardhii often produces toxic blooms in eutrophic water bodies around the world, little is known about the fate of the organic matter released by these abundant Cyanobacteria. Thus, this study focused in estimating the bacterial consumption of the DOC and DON (dissolved organic carbon and dissolved organic nitrogen, respectively) produced by axenic P. agardhii cultures and identifying some of the bacterial OTUs (operational taxonomic units) involved in the process. Both P. agardhii and bacterial inocula were sampled from the eutrophic Barra Bonita Reservoir (SP, Brazil). Two distinct carbon degradation phases were observed: during the first three days, higher degradation coefficients were calculated, which were followed by a slower degradation phase. The maximum value observed for particulate bacterial carbon (POC) was 11.9 mg L-1, which consisted of 62.5% of the total available DOC, and its mineralization coefficient was 0.477 day-1 (t½ = 1.45 days). A similar pattern of degradation was observed for DON, although the coefficients were slightly different. Changes in the OTUs patterns were observed during the different steps of the degradation. The main OTUs were related to the classes Alphaproteobacteria (8 OTUs), Betaproteobacteria (2 OTUs) and Gammaproteobacteria (3 OTUs). The genus Acinetobacter was the only identified organism that occurred during the whole process. Bacterial richness was higher at the slower degradation phase, which could be related to the small amounts of DOM (dissolved organic matter) available, particularly carbon. The kinetics of the bacterial degradation of P. agardhii-originated DOM suggests minimal loss of DOM from the Barra Bonita reservoir.

Resumo Embora Planktothrix agardhii frequentemente forme florações tóxicas em corpos d'água pelo mundo, pouco ainda se sabe sobre o destino da matéria orgânica liberada por essa abundante Cyanobacteria. Assim, este estudo foi focado na estimativa do consumo bacteriano do carbono orgânico dissolvido (DOC) e nitrogênio orgânico dissolvido (DON) produzido por culturas axênicas de P. agardhii e identificação de algumas das unidades taxonômicas operacionais (OTUs) bacterianas envolvidas no processo. Ambos a linhagem de P. agardhii e o inóculo bacteriano foram amostrados do reservatório eutrófico de Barra Bonita (SP, Brasil). Foram observadas duas fases distintas da degradação do DOC: durante os três primeiros dias, coeficientes mais altos de degradação foram calculados, que foram então seguidos por uma fase mais lenta da degradação do carbono. O valor máximo calculado para o carbono bacteriano particulado (POC) foi de 11,9 mgL-1, o que equivale a aproximadamente 62,5% do DOC disponível para consumo, e o seu coeficiente de mineralização foi de 0,477 dia-1 (t1/2 = 1,45 dias). Um padrão similar de degradação foi observado para DON, embora os coeficientes sejam ligeiramente diferentes. Foram observadas mudanças nos padrões de OTUs durante os diferentes passos da degradação. As principais OTUs foram relacionadas às classes Alphaproteobacteria (8 OTUs), Betaproteobacteria (2 OTUs) e Gammaproteobacteria (3 OTUs). O gênero Acinetobacter foi o único organismo identificado que ocorreu durante todo o processo. A maior riqueza bacteriana foi observada durante a fase lenta de degradação, o que pode estar relacionado às pequenas quantidades de matéria orgânica dissovida (DOM) disponíveis, particularmente o carbono. A cinética da degradação bacteriana da MOD de P. agardhii, quando comparada ao tempo de retenção do reservatório, sugere que existe uma perda mínima após sua liberação em Barra Bonita.

Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast

Abstract Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable. Therefore, this review focuses on the selection of the best promoter in the recombination process, considering different inductors, and the temperature as a culture medium variable in methylotrophic Pichia pastoris yeast. The goal is to understand the effects associated with different factors that influence its cell metabolism and to reach the construction of an expression system that fulfills the requirements of the yeast, presenting an optimal growth and development in batch, fed-batch or continuous cultures, and at the same time improve its yield in heterologous protein production.

Comparative proteomic analyses of Hyphozyma roseonigra ATCC 20624 in response to sclareol

Abstract Sclareol is an important intermediate for ambroxide synthesis industries. Hyphozyma roseonigra ATCC 20624 was the only reported strain capable of degrading sclareol to the main product of sclareol glycol, which is the precursor of ambroxide. To date, knowledge is lacking about the effects of sclareol on cells and the proteins involved in sclareol metabolism. Comparative proteomic analyses were conducted on the strain H. roseonigra ATCC 20624 by using sclareol or glucose as the sole carbon source. A total of 79 up-regulated protein spots with a >2.0-fold difference in abundance on 2-D gels under sclareol stress conditions were collected for further identification. Seventy spots were successfully identified and finally integrated into 30 proteins. The up-regulated proteins under sclareol stress are involved in carbon metabolism; and nitrogen metabolism; and replication, transcription, and translation processes. Eighteen up-regulated spots were identified as aldehyde dehydrogenases, which indicating that aldehyde dehydrogenases might play an important role in sclareol metabolism. Overall, this study may lay the fundamentals for further cell engineering to improve sclareol glycol production.

Effects of fermentation conditions on valuable products of ethanolic fungus Mucor indicus

Background: Mucor indicus is a dimorphic fungus used in the production of ethanol, oil, protein, and glucosamine. It can ferment different pentoses and hexoses; however, the yields of products highly depend on the nutrients and cultivation conditions. In this study, the effects of different morphologic forms, cultivation time and temperature, presence or absence of oxygen, carbon sources, and concentration of nitrogen source on the products of M. indicus were investigated. Results: The fungus with all morphologies produced high yields of ethanol, in the range of 0.32­0.43 g/g, on glucose. However, the fungus with filamentous morphology produced higher amounts of oil, protein, phosphate, and glucosamine together with ethanol, compared with other morphologies. A higher amount of oil (0.145 g/g biomass) was produced at 28°C, while the best temperature for protein and glucosamine production was 32 and 37°C, respectively. Although ethanol was produced at a higher yield (0.44 g/g) under anaerobic conditions compared with aerobic conditions (yield of 0.41 g/g), aerobic cultivation resulted in higher yields of protein (0.51 g/g biomass), glucosamine (0.16 g/g alkali insoluble material, AIM), and phosphate (0.11 g/g AIM). Conclusions: It is not possible to have the maximum amounts of the products simultaneously. The fermentation conditions and composition of culture media determine the product yields. Carbon source type and the addition of nitrogen source are among the most influencing factors on the product yields. Moreover, all measured products were made with higher yields in cultivation on glucose, except glucosamine, which was produced with higher yields on xylose.

Effect of synthetic and natural media on lipid production from Fusarium oxysporum

Background: Dependence on fossil resources, for the production of fuels and energy, has resulted in environmental and financial problems, which require our immediate action in order to reverse the situation. Use of renewable sources for the production of fuels and energy is an important alternative with biodiesel remains as one of the promising options. Aim of this work is to evaluate the fungus Fusarium oxysporum for its potentials to accumulate microbial lipids when grown on synthetic media and saccharified sweet sorghum stalks. Results: The effect of different carbon sources, nitrogen sources and C/N ratio on the lipid production was initially examined, which resulted in a lipid concentration of 4.4 g/L, with lipid content of 42.6% w/w. Sweet sorghum stalks were able to support growth and lipid production of the fungus, both as carbon source and as nitrogen source. It was also shown that saccharification of the dried stalks is an important step to increase lipid production. Removal of the remaining stalk solids enabled the lipid production during cultivation in increased initial solids of up to 16 w/w. This resulted in a lipid production of 3.81 g/L. Conclusions: It was demonstrated that F. oxysporum can be used as an efficient oleaginous microorganism, with sweet sorghum serving as an excellent raw material for the cultivation of the fungus. The lipids obtained during this work were also found to have a fatty acid profile with good potentials to be used for biodiesel production.

An overview of biotechnological production of propionic acid: from upstream to downstream processes

The increasing demand for propionic acid (PA) production and its wide applications in several industries, especially the food industry (as a preservative and satiety inducer), have led to studies on the low-cost biosynthesis of this acid. This paper gives an overview of the biotechnological aspects of PA production and introduces Propionibacterium as the most popular organism for PA production. Moreover, all process variables influencing the production yield, different simple and complex carbon sources, the metabolic pathway of production, engineered mutants with increased productivity, and modified tolerance against high concentrations of acid have been described. Furthermore, possible methods of extraction and analysis of this organic acid, several applied bioreactors, and different culture systems and substrates are introduced. It can be concluded that maximum biomass and PA production may be achieved using metabolically engineered microorganisms and analyzing the most significant factors influencing yield. To date, the maximum reported yield for PA production is 0.973 g·g-1, obtained from Propionibacterium acidipropionici in a three-electrode amperometric culture system in medium containing 0.4 mM cobalt sepulchrate. In addition, the best promising substrate for PA bioproduction may be achieved using glycerol as a carbon source in an extractive continuous fermentation. Simultaneous production of PA and vitamin B12 is suggested, and finally, the limitations of and strategies for competitive microbial production with respect to chemical process from an economical point of view are proposed and presented. Finally, some future trends for bioproduction of PA are suggested.

Mannheimia haemolytica growth and leukotoxin production for vaccine manufacturing: a bioprocess review

Mannheimia haemolytica leukotoxin (LKT) is a known cause of bovine respiratory disease (BRD) which results in severe economic losses in the cattle industry (up to USD 1 billion per year in the USA). Vaccines based on LKT offer the most promising measure to contain BRD outbreaks and are already commercially available. However, insufficient LKT yields, predominantly reflecting a lack of knowledge about the LKT expression process, remain a significant engineering problem and further bioprocess optimization is required to increase process efficiency. Most previous investigations have focused on LKT activity and cell growth, but neither of these parameters defines reliable criteria for the improvement of LKT yields. In this article, we review the most important process conditions and operational parameters (temperature, pH, substrate concentration, dissolved oxygen level, medium composition and the presence of metabolites) from a bioprocess engineering perspective, in order to maximize LKT yields.

Cultivation of Pichia pastoris carrying the scFv anti LDL (-) antibody fragment. Effect of preculture carbon source

Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.

Production of docosahexaenoic acid by Aurantiochytrium sp. ATCC PRA-276

Abstract The high costs and environmental concerns associated with using marine resources as sources of oils rich in polyunsaturated fatty acids have prompted searches for alternative sources of such oils. Some microorganisms, among them members of the genus Aurantiochytrium, can synthesize large amounts of these biocompounds. However, various parameters that affect the polyunsaturated fatty acids production of these organisms, such as the carbon and nitrogen sources supplied during their cultivation, require further elucidation. The objective of this investigation was to study the effect of different concentrations of carbon and total nitrogen on the production of polyunsaturated fatty acids, particularly docosahexaenoic acid, by Aurantiochytrium sp. ATCC PRA-276. We performed batch system experiments using an initial glucose concentration of 30 g/L and three different concentrations of total nitrogen, including 3.0, 0.44, and 0.22 g/L, and fed-batch system experiments in which 0.14 g/L of glucose and 0.0014 g/L of total nitrogen were supplied hourly. To assess the effects of these different treatments, we determined the biomass, glucose, total nitrogen and polyunsaturated fatty acids concentration. The maximum cell concentration (23.9 g/L) was obtained after 96 h of cultivation in the batch system using initial concentrations of 0.22 g/L total nitrogen and 30 g/L glucose. Under these conditions, we observed the highest level of polyunsaturated fatty acids production (3.6 g/L), with docosahexaenoic acid and docosapentaenoic acid ω6 concentrations reaching 2.54 and 0.80 g/L, respectively.

Phenol degradation and genotypic analysis of dioxygenase genes in bacteria isolated from sediments

Abstract The aerobic degradation of aromatic compounds by bacteria is performed by dioxygenases. To show some characteristic patterns of the dioxygenase genotype and its degradation specificities, twenty-nine gram-negative bacterial cultures were obtained from sediment contaminated with phenolic compounds in Wuhan, China. The isolates were phylogenetically diverse and belonged to 10 genera. All 29 gram-negative bacteria were able to utilize phenol, m-dihydroxybenzene and 2-hydroxybenzoic acid as the sole carbon sources, and members of the three primary genera Pseudomonas, Acinetobacter and Alcaligenes were able to grow in the presence of multiple monoaromatic compounds. PCR and DNA sequence analysis were used to detect dioxygenase genes coding for catechol 1,2-dioxygenase, catechol 2,3-dioxygenase and protocatechuate 3,4-dioxygenase. The results showed that there are 4 genotypes; most strains are either PNP (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is positive) or PNN (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is negative). The strains with two dioxygenase genes can usually grow on many more aromatic compounds than strains with one dioxygenase gene. Degradation experiments using a mixed culture representing four bacterial genotypes resulted in the rapid degradation of phenol. Determinations of substrate utilization and phenol degradation revealed their affiliations through dioxygenase genotype data.

Efficient production of pullulan by Aureobasidium pullulans grown on mixtures of potato starch hydrolysate and sucrose

Abstract Pullulan is a natural exopolysaccharide with many useful characteristics. However, pullulan is more costly than other exopolysaccharides, which limits its effective application. The purpose of this study was to adopt a novel mixed-sugar strategy for maximizing pullulan production, mainly using potato starch hydrolysate as a low-cost substrate for liquid-state fermentation by Aureobasidium pullulans. Based on fermentation kinetics evaluation of pullulan production by A. pullulans 201253, the pullulan production rate of A. pullulans with mixtures of potato starch hydrolysate and sucrose (potato starch hydrolysate:sucrose = 80:20) was 0.212 h−1, which was significantly higher than those of potato starch hydrolysate alone (0.146 h−1) and mixtures of potato starch hydrolysate, glucose, and fructose (potato starch hydrolysate:glucose:fructose = 80:10:10, 0.166 h−1) with 100 g L−1 total carbon source. The results suggest that mixtures of potato starch hydrolysate and sucrose could promote pullulan synthesis and possibly that a small amount of sucrose stimulated the enzyme responsible for pullulan synthesis and promoted effective potato starch hydrolysate conversion effectively. Thus, mixed sugars in potato starch hydrolysate and sucrose fermentation might be a promising alternative for the economical production of pullulan.

Characterization of rhizobia isolates obtained from nodules of wild genotypes of common bean

Abstract This study aimed to evaluate the tolerance to salinity and temperature, the genetic diversity and the symbiotic efficiency of rhizobia isolates obtained from wild genotypes of common bean cultivated in soil samples from the States of Goiás, Minas Gerais and Paraná. The isolates were subjected to different NaCl concentrations (0%, 1%, 2%, 4% and 6%) at different temperatures (28 °C, 33 °C, 38 °C, 43 °C and 48 °C). Genotypic characterization was performed based on BOX-PCR, REP-PCR markers and 16S rRNA sequencing. An evaluation of symbiotic efficiency was carried out under greenhouse conditions in autoclaved Leonard jars. Among 98 isolates about 45% of them and Rhizobium freirei PRF81 showed a high tolerance to temperature, while 24 isolates and Rhizobium tropici CIAT899 were able to use all of the carbon sources studied. Clustering analysis based on the ability to use carbon sources and on the tolerance to salinity and temperature grouped 49 isolates, R. tropici CIAT899 and R. tropici H12 with a similarity level of 76%. Based on genotypic characterization, 65% of the isolates showed an approximately 66% similarity with R. tropici CIAT899 and R. tropici H12. About 20% of the isolates showed symbiotic efficiency similar to or better than the best Rhizobium reference strain (R. tropici CIAT899). Phylogenetic analysis of the 16S rRNA revealed that two efficient isolates (ALSG5A1 and JPrG6A8) belong to the group of strains used as commercial inoculant for common bean in Brazil and must be assayed in field experiments.