Strain screening and sodium lactate effect on spiramycin production in <Emphasis Type="Italic">Streptomyces spiramyceticus</Emphasis>

Strain improvement and addition of sodium lactate to fermentation medium to enhance the productivity of spiramycin were performed. Of the sodium lactate tolerant mutants that were screened, one mutant, Streptomyces spiramyceticus 16-10-12, produced 23 % more spiramycin than the original strain, Streptomyces spiramyceticus 5-1. The effect of sodium lactate on spiramycin production with S. spiramyceticus 16-10-12 was studied. The titer was improved by 16.9 % with the addition of 15 g L^?1 sodium lactate in the fermentation medium at the beginning. The results from using the new process in a 15 L bioreactor showed that there were more precursors in fermentation broth with a sodium lactate tolerant mutant, and that these precursors were used more than with the original strain. After adding sodium lactate, the titer was increased by 23.4 %, because the flux to TCA circulation was increased, more precursors had been produced and the activities of Acyl-CoA synthetases, Acylphosphotransferases and Acylkinases in synthesis phase were also increased.     

Research on the Solid State Fermentation of Jerusalem Artichoke Pomace for Producing <Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">R</Emphasis>-2,3-Butanediol by <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> ZJ-9

R,R-2,3-butanediol (R,R-2,3-BD) was produced by Paenibacillus polymyxa ZJ-9, which was capable of utilizing inulin without previous hydrolysis. The Jerusalem artichoke pomace (JAP) derived from the conversion of Jerusalem artichoke powder into inulin extract, which was usually used for biorefinery by submerged fermentation (SMF), was utilized in solid state fermentation (SSF) to produce R,R-2,3-BD. In this study, the fermentation parameters of SSF were optimized and determined in flasks. A novel bioreactor was designed and assembled for the laboratory scale-up of SSF, with a maximum yield of R,R-2,3-BD (67.90 g/kg (JAP)). This result is a 36.3% improvement compared with the flasks. Based on the same bath of Jerusalem artichoke powder, the total output of R,R-2,3-BD increased by 38.8% for the SSF of JAP combined with the SMF of inulin extraction. Overall, the utilization of JAP for R,R-2,3-BD production was beneficial to the comprehensive utilization of Jerusalem artichoke tuber.     

Establishment of Bioprocess for Synthesis of Nicotinamide by Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> Expressing High-Molecular-Mass Nitrile Hydratase

Application of engineered bacteria expressing nitrile hydratase for the production of amide is getting tremendous attention due to the rapid development of recombinant DNA technique. This study evaluated the effect of 3-cyanopyridine concentrations on nicotinamide production using recombinant Escherichia coli strain (BAG) expressing high-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1, and established proper process of whole-cell catalysis of 3-cyanopyridine and high cell-density cultivation. The process of substrate fed-batch was applied in the production of nicotinamide, and the concentration of product reached 390 g/L under the condition of low cell-density. After the high cell-density cultivation of BAG in 5 L bioreactor, the OD₆₀₀ of cell attained 200 and the total activity reached 2813 U/mL. Different high density of BAG after fermentation in the tank was used to catalyze 3-cyanopyridine, and the concentration of nicotinamide reached to 508 g/L in just 60 min. The productivity of BAG was 212% higher than that of R. rhodochrous J1, and it is possible that BAG is able to achieve industrial production of nicotinamide.     

Engineering <Emphasis Type="Italic">Pichia pastoris</Emphasis> for Efficient Production of a Novel Bifunctional <Emphasis Type="Italic">Strongylocentrotus purpuratus</Emphasis> Invertebrate-Type Lysozyme

Lysozymes are known as ubiquitously distributed immune effectors with hydrolytic activity against peptidoglycan, the major bacterial cell wall polymer, to trigger cell lysis. In the present study, the full-length cDNA sequence of a novel sea urchin Strongylocentrotus purpuratus invertebrate-type lysozyme (sp-iLys) was synthesized according to the codon usage bias of Pichia pastoris and was cloned into a constitutive expression plasmid pPIC9K. The resulting plasmid, pPIC9K-sp-iLys, was integrated into the genome of P. pastoris strain GS115. The bioactive recombinant sp-iLys was successfully secreted into the culture broth by positive transformants. The highest lytic activity of 960 U/mL of culture supernatant was reached in fed-batch fermentation. Using chitin affinity chromatography and gel-filtration chromatography, recombinant sp-iLys was produced with a yield of 94.5 mg/L and purity of >?99%. Recombinant sp-iLys reached its peak lytic activity of 8560 U/mg at pH 6.0 and 30 °C and showed antimicrobial activities against Gram-negative bacteria (Vibrio vulnificus, Vibrio parahemolyticus, and Aeromonas hydrophila) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In addition, recombinant sp-iLys displayed isopeptidase activity which reached the peak at pH 7.5 and 37 °C with the presence of 0.05 M Na⁺. In conclusion, this report describes the heterologous expression of recombinant sp-iLys in P. pastoris on a preparative-scale, which possesses lytic activity and isopeptidase activity. This suggests that sp-iLys might play an important role in the innate immunity of S. purpuratus.     

Fumaric Acid Production from Alkali-Pretreated Corncob by Fed-Batch Simultaneous Saccharification and Fermentation Combined with Separated Hydrolysis and Fermentation at High Solids Loading

Production of fumaric acid from alkali-pretreated corncob (APC) at high solids loading was investigated using a combination of separated hydrolysis and fermentation (SHF) and fed-batch simultaneous saccharification and fermentation (SSF) by Rhizopus oryzae. Four different fermentation modes were tested to maximize fumaric acid concentration at high solids loading. The highest concentration of 41.32 g/L fumaric acid was obtained from 20 % (w/v) APC at 38 °C in the combined SHF and fed-batch SSF process, compared with 19.13 g/L fumaric acid in batch SSF alone. The results indicated that a combination of SHF and fed-batch SSF significantly improved production of fumaric acid from lignocellulose by R. oryzae than that achieved with batch SSF at high solids loading.     

High Cell Density Process for Constitutive Production of a Recombinant Phytase in Thermotolerant Methylotrophic Yeast <Emphasis Type="Italic">Ogataea thermomethanolica</Emphasis> Using Table Sugar as Carbon Source

The yeast Ogataea thermomethanolica has recently emerged as a potential host for heterologous protein expression at elevated temperature. To evaluate the feasibility of O. thermomethanolica as heterologous host in large-scale fermentation, constitutive production of fungal phytase was investigated in fed-batch fermentation. The effect of different temperatures, substrate feeding strategies, and carbon sources on phytase production was investigated. It was found that O. thermomethanolica can grow in the temperature up to 40 °C and optimal at 34 °C. However, the maximum phytase production was observed at 30 °C and slightly decreased at 34 °C. The DOT stat control was the most efficient feeding strategy to obtain high cell density and avoid by-product formation. The table sugar can be used as an alternative substrate for phytase production in O. thermomethanolica. The highest phytase activity (134 U/mL) was obtained from table sugar at 34 °C which was 20-fold higher than batch culture (5.7 U/mL). At a higher cultivation temperature of 38 °C, table sugar can be used as a low-cost substrate for the production of phytase which was expressed with an acceptable yield (85 U/mL). Lastly, the results from this study reveal the industrial favorable benefits of employing O. thermomethanolica as a host for heterologous protein production.     

Simultaneous Production of Amyloglucosidase and Exo-Polygalacturonase by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a Rotating Drum Reactor

Simultaneous production of amyloglucosidase (AMG) and exo-polygalacturonase (exo-PG) was carried out by Aspergillus niger in substrate of defatted rice bran in a rotating drum bioreactor (RDB) and studied by a 3¹ × 2² factorial experimental design. Variables under study were A. niger strains (A. niger NRRL 3122 and A. niger t0005/007-2), types of inoculum (spore suspension and fermented bran), and types of inducer (starch, pectin, and a mix of both). Solid-state fermentation process (SSF) was conducted at 30 °C under 60-vvm aeration for 96 h in a pilot scale. Production of AMG and exo-PG was significantly affected by the fungal strain and the type of inoculum, but inducers did not trigger any significant effect, an evidence of the fact that these enzymes are constitutive. The maximum activity of exo-PG was 84 U g_dm ^?1 whereas the maximum yield of AMG was 886.25 U g_dm ^?1.     

Optimization of Levan Production by Cold-Active <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> ANT 179 and Fructooligosaccharide Synthesis by Its Levansucrase

Fructooligosaccharides (FOS) and levan attract much attention due to a wide range of applications in food technology and pharmaceutical and cosmetic industry. Bacillus licheniformis ANT 179, isolated from Antarctica soil, produced levansucrase and levan in a medium containing sucrose as carbon substrate. In this study, characterization of levansucrase and production of short-chain FOS and levan were investigated. Temperature and pH optimum of the enzyme were found to be 60 °C and pH 6.0, respectively. The optimization of fermentation conditions for levan production using sugarcane juice by response surface methodology (RSM) was carried out. Central composite rotatable design was used to study the main and the interactive effects of medium components: sugarcane juice and casein peptone concentration on levan production by the bacterium. The optimized medium with sugarcane juice at 20 % (v/v) and casein peptone at 2 % (w/v) was found to be optimal at an initial pH of 7.0 and incubation temperature of 35 °C for 48 h. Under these conditions, the maximum levan concentration was 50.25 g/L on wet weight basis and 16.35 g/L on dry weight basis. The produced inulin type FOS (kestose and neokestose) and levan were characterized by Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) analysis. The study revealed that the levansucrase could form FOS from sucrose. The locally available low-cost substrate such as sugarcane juice in the form of a renewable substrate is proposed to be suitable even for scale-up production of enzyme and FOS for industrial applications. The levan and FOS synthesized by the bacterium are suitable for food applications and biomedical uses as the bacterium has GRAS status and devoid of endotoxin as compared to other Gram-negative bacteria.     

Harnessing the Effect of pH on Lipid Production in Batch Cultures of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> SKY7

The objective of this research was to investigate the kinetics of lipid production by Yarrowia lipolytica SKY7 in the crude glycerol-supplemented media with and without the control of pH. Lipid and citric acid production were improved with the pH control condition. There was no significant difference observed in the biomass concentration with or without the pH control. In the pH-controlled experiments, the biomass and lipid concentration reached 18 and 7.78 g/L, (45.5% w/w), respectively, with lipid yield (Yp/s) of 0.179 g/g at 60 h of fermentation. The lipid production was directly correlated with growth and the process was defined as growth associated. After 60 h of fermentation, the lipid degradation was noticed in the pH-controlled reactor whereas it occurred after 84 h in the pH-uncontrolled reactor. Apart from lipid, citric acid was produced as the major extracellular product in both fermentations but the much lower concentration in uncontrolled pH. Based on the experimental results, it is evident that controlling the pH will enhance the lipid production by 15% compared to pH-uncontrolled fermentation.     

Combined De-Algination Process as a Fractionation Strategy for Valorization of Brown Macroalga <Emphasis Type="Italic">Saccharina japonica</Emphasis>

A combined process, de-algination followed by enzymatic saccharification, was designed to produce alginate and glucose from Saccharina japonica consecutively. The process conditions of de-algination were optimized separately for each stage of acidification and alkaline extraction. Collectively, the de-algination yield was 70.1% under the following optimized conditions: 2.4 wt% of Na₂CO₃, 70 °C, and 100 min with the acidified S. japonica immersed in a 0.5 wt% H₂SO₄ solution for 2 h at room temperature. The glucan content in the de-alginated S. japonica increased to 38.0%, which was approximately fivefold higher than that of the raw S. japonica. The enzymatic hydrolysis of the de-alginated S. japonica almost completed in 9 h, affording 5.2 g (96.8% of glucan digestibility) of glucose at a de-alginated S. japonica loading of 14.2 g.     

Co-production of Fructooligosaccharides and Levan by Levansucrase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> natto with Potential Application in the Food Industry

Fructooligosaccharides and levan have a wide range of applications in the food industry due to their physiological and functional properties. The enzymatic synthesis of these molecules exhibits great advantages when compared with microbial fermentation. In this study, the production of levansucrase from Bacillus subtilis natto and its utilization in fructooligosaccharides and levan syntheses using different reaction conditions were described. The best condition for levansucrase production was 420.7 g L^?1 of sucrose at pH 7.0, which reached 23.9 U ml^?1 of transfructosylation activity. In a bioreactor, the highest production of fructooligosaccharides was 41.3 g L^?1 using a medium containing 350 g L^?1 sucrose at 35 °C for 36 h. The enzymatic synthesis of levan resulted in 86.9 g L^?1 when conditions similar to those used for fructooligosaccharides synthesis were applied. These results indicate that the levansucrase from B. subtilis natto could be applied for the co-production of fructooligosaccharides and levan, which are biomolecules that have health benefits and are used successfully in the food industry.     

The Feasibility of Thermophilic <Emphasis Type="Italic">Caldimonas manganoxidans</Emphasis> as a Platform for Efficient PHB Production

Recently, poly(3-hydroxybutyrate) (PHB) has been found in a few thermophilic strains where several advantages can be gained from running fermentation at high temperatures. Caldimonas manganoxidans, a thermophilic gram-negative bacterium, was investigated for the feasibility as a PHB-producing strain. It is suggested that the best fermentation strategy for achieving the highest PHB concentration of 5.4?±?1.1 g/L (from 20 g/L glucose) in 24 h is to use the fermentation conditions that are favored for the bacterial growth, yet temperature and pH should be chosen at conditions that are favored for the PHB content. Besides, the above fermentation conditions produce PHB that has a high molecular weight of 1274 kDa with a low polydispersity index (PDI) of 1.45, where the highest Mw of PHB of 1399?kDa (PDI of 1.32) is obtained in this study. To the best knowledge of authors, C. manganoxidans has the best PHB productivity among the thermophiles and is comparable to those common PHB-producing mesophiles.     

Production of <Emphasis Type="Italic">R</Emphasis>-Mandelic Acid Using Nitrilase from Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Cells Immobilized with Tris(Hydroxymethyl)Phosphine

Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH 8.0 and 55 °C. The half-lives of THP-immobilized cells measured at 35, 40, and 50 °C were 1800, 965, and 163 h, respectively. The concentration of R-mandelic acid (R-MA) reached 358 mM after merely 1-h conversion by the immobilized cells with 500 mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307 g L^?1 day^?1 and the space-time productivity of 143.2 mmol L^?1 h^?1 g^?1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100 mM R,S-MN as substrate at 40 °C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA.     

CO<Subscript>2</Subscript> Biofixation of <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> Through Novel Amine-Functionalized Polystyrene Microsphere Materials

CO₂-derived succinate production was enhanced by Actinobacillus succinogenes through polystyrene (PSt) microsphere materials for CO₂ adsorption in bioreactor, and the adhesion forces between A. succinogenes bacteria and PSt materials were characterized. Synthesized uniformly sized and highly cross-linked PSt microspheres had high specific surface areas. After modification with amine functional groups, the novel amine-functionalized PSt microspheres exhibited a high adsorption capacity of 25.3 mg CO₂/g materials. After addition with the functionalized microspheres into the culture broth, CO₂ supply to the cells increased. Succinate production by A. succinogenes can be enhanced from 29.6 to 48.1 g L^?1. Moreover, the characterization of interaction forces between A. succinogenes cells and the microspheres indicated that the maximal adhesive force was about 250 pN. The amine-functionalized PSt microspheres can adsorb a large amount of CO₂ and be employed for A. succinogenes anaerobic cultivation in bioreactor for high-efficiency production of CO₂-derived succinate.     

A Novel 2-Keto-<Emphasis Type="SmallCaps">d</Emphasis>-Gluconic Acid High-Producing Strain <Emphasis Type="Italic">Arthrobacter globiformis</Emphasis> JUIM02

2-Keto-d-gluconic acid (2KGA) is mainly used for industrial production of erythorbic acid, a food antioxidant. In this study, a 2KGA producing strain JUIM02 was firstly identified as Arthrobacter globiformis by morphological observation and 16S rDNA sequencing. The 2KGA synthetic capacity of A. globiformis JUIM02 was evaluated by both fermentation and bioconversion, with 180 g/L dextrose monohydrate as substrates, in shake flasks and 5 L fermenters. For fermentation, 2KGA titer, yield, molar yield, and productivity of JUIM02 reached 159.05 g/L, 0.97 g/g, 90.18%, and 6.63 g/L/h in 24 h. For non-sterile and buffer-free bioconversion by free resting cells (~?3.2 g/L dry cell weight) of JUIM02, these data were 172.96 g/L, 1.06 g/g, 98.07%, and 5.41 g/L/h in 32 h. Moreover, JUIM02 resting cells could be repeatedly used. Resting cells stored at 4 °C within 30 days showed stable bioconversion capacity, with 2KGA titers ≥?171.50 g/L, yields ≥?1.04 g/g, and molar yields ≥?97.24%. The 2KGA synthetic pathway in A. globiformis, which was rarely reported, was also speculated similar to Pseudomonas and verified preliminarily. In conclusion, A. globiformis JUIM02 is a promising 2KGA industrial-producing strain suitable for various production methods and a suitable object for 2KGA metabolism research of A. globiformis.     

Synthesis and characterization of a new monophosphate (C<Subscript>6</Subscript>H<Subscript>16</Subscript>N<Subscript>2</Subscript>)(H<Subscript>2</Subscript>PO<Subscript>4</Subscript>)<Subscript>2</Subscript>

Chemical preparation, crystal structure, and NMR spectroscopy of a new trans-2,5-dimethylpiperazinium monophosphate are given. This new compound crystallizes in the triclinic system, with the space group P-1 and the following parameters: a = 6.5033(3), b = 7.6942(4), c = 8.1473(5) Å, α = 114.997(3), β = 92.341(3), γ = 113.136(3), V = 329.14(3) ų, Z = 1, and D_x = 1.565 g cm^?3. The crystal structure has been determined and refined to R = 0.030 and R _w(F ²) = 0.032 using 1558 independent reflections. The structure can be described as infinite [H₂PO₄] _n ^n? chains with (C₆H₁₆N₂)²⁺ organic cations anchored between adjacent polyanions to form columns of anions and cations running along the b axis. This compound has also been investigated by IR, thermal, and solid-state, ¹³C and ³¹P MAS NMR spectroscopies and Ab initio calculations.     

Bioaccumulation of <Superscript>137</Superscript>Cs and <Superscript>60</Superscript>Co by bacteria isolated from spent nuclear fuel pools

This paper is focused on a characterization of bacterial contamination in pool water of the interim spent fuel storage (JAVYS Inc.) in Slovak Republic and on bioaccumulation of ¹³⁷Cs and ⁶⁰Co by isolated bacteria. Bacterial community in pool water is kept on very low level by extremely low concentration of solutes in deionized water and by the efficient water filtration system. Based on standard methods and sequencing of 16S rDNA four pure bacterial cultures were identified as Kocuria palustris, Micrococcus luteus, Ochrobactrum spp. and Pseudomonas aeruginosa. Isolated aerobic bacteria were able to bioaccumulate ¹³⁷Cs and ⁶⁰Co in laboratory experiments. The mechanism of Co and Cs binding involve rapid interactions with anionic groups of the components of cell surface and in the case of Cs⁺ ions is followed by transport processes across cytoplasm membranes and by intracellular distribution. The maximum specific uptake of Cs⁺ after 48 h cultivation in mineral medium (MM) reached 7.54 ± 0.48 μmol g^?1 dw (Ochrobactrum spp.), 19.6 ± 0.1 μmol g^?1 dw (M. luteus) and 20.1 ± 2.2 μmol g^?1 dw (K. palustris). The maximum specific uptake of Co²⁺ after 24 h cultivation in MM reached 31.1 ± 3.5 μmol g^?1 dw (Ochrobactrum spp.), 86.6 ± 12.2 μmol g^?1 dw (M. luteus) and 16.9 ± 1.2 μmol g^?1 dw (K. palustris). These results suggest that due to the long lasting uptake of ¹³⁷Cs, ⁶⁰Co and other radionuclides by biofilm in pool water high specific radioactivities (Bq m^?2) can be expected on stainless steel walls of pools.     

Recovery of Phenolic Acid and Enzyme Production from Corn Silage Biologically Treated by <Emphasis Type="Italic">Trametes versicolor</Emphasis>

Corn silage is used as high-energy forage for dairy cows and more recently for biogas production in a process of anaerobic co-digestion with cow manure. In this work, fresh corn silage after the harvest was used as a substrate in solid-state fermentations with T. versicolor with the aim of phenolic acid recovery and enzyme (laccase and manganese peroxidase) production. During 20 days of fermentation, 10.4-, 3.4-, 3.0-, and 1.8-fold increments in extraction yield of syringic acid, vanillic acid, p-hydroxybenzoic acid, and caffeic acid, respectively, were reached when compared to biologically untreated corn silage. Maximal laccase activity was gained on the 4th day of fermentation (V.A. = 180.2 U/dm³), and manganese peroxidase activity was obtained after the 3rd day of fermentation (V.A. = 30.1 U/dm³). The addition of copper(II) sulfate as inducer during solid state fermentation resulted in 8.5- and 7-fold enhancement of laccase and manganese peroxidase activities, respectively. Furthermore, the influence of pH and temperature on enzyme activities was investigated. Maximal activity of laccase was obtained at T = 50 °C and pH = 3.0, while manganese peroxidase is active at temperature range T = 45–70 °C with the maximal activity at pH = 4.5.     

Synthesis,crystal structure of a new tetranuclear Ni<Stack><Subscript>2</Subscript><Superscript>II</Superscript></Stack>Cu<Stack><Subscript>2</Subscript><Superscript>II</Superscript></Stack> complex containing a macrocyclic ligand

The first inorg/organic hybrid complex incorporating the macrocyclic oxamide, of formula [(NiL)₂Cu₂(μ-NSC)₂(NSC)₂] (1), (NiL, H₂L = 2, 3-dioxo-5,6,14,15-dibenzo-1,4,8,12-tetraazacyclo-pentadeca-7,13-dien), have been synthesized and structurally characterized. The crystals crystallize in the triclinic system, space group P-1, for (1) a = 8.319(3) Å, b = 10.434(4) Å, c = 14.166(5) Å, a = 107.030(5)°, β  =  91.257(5)°, γ = 107.623(5)°. The complex involved both bridging N, S-ligand, and oxamide ligand, C–H?S interactions and NCS → Ni weak coordination interactions making the complex superamolecular.     

Producing Acetic Acid of <Emphasis Type="Italic">Acetobacter pasteurianus</Emphasis> by Fermentation Characteristics and Metabolic Flux Analysis

The acetic acid bacterium Acetobacter pasteurianus plays an important role in acetic acid fermentation, which involves oxidation of ethanol to acetic acid through the ethanol respiratory chain under specific conditions. In order to obtain more suitable bacteria for the acetic acid industry, A. pasteurianus JST-S screened in this laboratory was compared with A. pasteurianus CICC 20001, a current industrial strain in China, to determine optimal fermentation parameters under different environmental stresses. The maximum total acid content of A. pasteurianus JST-S was 57.14?±?1.09 g/L, whereas that of A. pasteurianus CICC 20001 reached 48.24?±?1.15 g/L in a 15-L stir stank. Metabolic flux analysis was also performed to compare the reaction byproducts. Our findings revealed the potential value of the strain in improvement of industrial vinegar fermentation.