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Dowex® HCR-S ion-exchange resin was used to adsorb ectoine in a batch system under varying operation conditions in terms of contact time, temperature, pH value, initial concentration of ectoine, and type of salt. Six adsorption isotherm models (Langmuir, Freundlich, Temkin, Dubinin–Radushkevich, Sips, and Redlich–Peterson) and three kinetic models (pseudo-first-order, pseudo-second-order, and intraparticle diffusion) were used to investigate the ectoine adsorption mechanism of ion-exchange resin. According to the experimental results, the mechanism of ectoine adsorption using an ion exchanger includes the ion-exchange reaction and physisorption. Both the Langmuir and Freundlich models were found to have a high fitting. For the kinetic analysis, the pseudo-second-order and intraparticle diffusion models were suitable to describe the ectoine adsorption. Dowex® HCR-S resin has an average saturated adsorption capacity of 0.57 g/g and 93.6% of ectoine adsorption at 25~65 °C, with an initial concentration of 125 g/L. By changing the pH of the environment using NaOH solution, the adsorbed ectoine on the ion-exchange resin can be desorbed to 87.7%.

1,3-Propanediol (1,3-PDO) has numerous industrial applications in the synthesis of the monomer of the widely used fiber polytrimethylene terephthalate. In this work, the production of 1,3-PDO by Klebsiella pneumoniae is increased by dual-substrate cultivation and fed-batch fermentation. Experimental results indicate that the production of 1,3-PDO can be elevated to 16.09 g/L using a dual substrate ratio (of glucose to crude glycerol) of 1/30 and to 20.73 g/L using an optimized dual-substrate ratio of 1/20. Ultimately, the optimal dual-substrate feeding for a 5 L scale fed-batch fermenter that maximizes 1,3-PDO production (29.69 g/L) is determined. This production yield is better than that reported in most related studies. Eventually, the molecular weight and chemical structure of 1,3-PDO were obtained by FAB-MS, 1H-NMR, and 13C-NMR. Also, in demonstrating the effectiveness of the fermentation strategy in increasing the production and production yield of 1,3-PDO, experimental results indicate that the fermentation of 1,3-PDO is highly promising for commercialization.

Ectoine has fostered the development of products for skin care and cosmetics. In this study, we employed the marine bacterial strain Marinococcus sp. MAR2 to increase ectoine production by optimizi...

Hydroxyectoine, an ectoine derivative, is the most common compatible solute in halophilic microorganisms for resisting harsh environments. Compatible solutes can be utilized in fields such as cosmetics, medicine, and biochemistry. Moderately halophilic microorganisms produce much less hydroxyectoine as compared with ectoine. In this study, we first evaluate the effect of medium formulation (i.e., yeast extract (YE) medium and high yeast extract (HYE) medium) on hydroxyectoine production. In addition, an investigation of hydroxyectoine production by Halomonas salina under optimal conditions for vital factors (i.e., iron and α-ketoglutarate) and hydroxylase activity was also carried out. As a result, hydroxyectoine production was obviously elevated (0.9 g/L to 1.8 g/L) when the HYE medium was utilized. Furthermore, hydroxyectoine production further increased to 2.4 g/L when both the α-ketoglutarate and iron factors were added to the HYE medium in the early stationary phase. In addition, we found that ectoine hydroxylase activity increased more when a combination of iron and α-ketoglutarate was used than when either was used alone. The results showed that the alteration of iron and α-ketoglutarate clearly stimulated the expression of ectoine hydroxylase, which in turn affected hydroxyectoine synthesis. This study also showed that hydroxyectoine production was further raised from 2.4 g/L to 2.9 g/L when 50 mM of α-ketoglutarate and 1 mM of iron were added to the HYE medium. Ultimately, the experimental results showed using the optimal conditions further elevated the hydroxyectoine production yield to 2.90 g/L, which was over 3-fold higher than the best results obtained from the original medium.

Water lettuce (Pistia stratiotes) is one of the most well-known aquatic weeds as it causes problems in watercourses all over the world. This invasive species is fast-growing and thus has the potential for its use in preparing growth medium of microorganisms. Toward it, the pretreatment and enzymatic saccharification are positively the decisive processes. But there are other crucial processes, which are usually ignored by researchers. To the best of my knowledge, this presented work is the first time discovering that the juice obtained from water lettuce is valuable. Except for sterilization, no treatment is needed for the water lettuce’s juice and it can be directly served for cell growth. For Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Bacillus subtilis, Lactococcus Lactis, Lactobacillus rhamnosus, and Lactobacillus plantarum, the cell density in the broth of water lettuce’s juice as the only carbon source is 10–60% higher than that in LB, YPD, BHI, M17, MRS. We then examined the production of microbial lipid by YM prepared in the juice of water lettuce. In comparison to commercial medium YM broth, the YM with juice caused 84% increase in the production of microbial lipid. A simple process of collecting juice notably increased productivity.

In this study, Saccharomyces cerevisiae was engineered to degrade and utilize xylan, one of the major polysaccharide chains present in hemicellulose. Different hemicellulases from Trichoderma reesei, namely: endoxylanase, β-xylosidase, acetylxylan esterase, α- d -glucuronidase and α- l -arabinofuranosidase, were heterologously secreted by S. cerevisiae. A mixture experimental design was adapted to statistically describe the synergistic interactions between the hemicellulases and to determine the optimum formulations for the hydrolysis of xylan substrates. The hydrolytic activities of the hemicellulase mixtures were then improved by displaying the hemicellulases on the yeast surface as whole-cell biocatalysts. The engineered yeast strains displaying hemicellulases were further engineered to express xylose-utilization genes xylose isomerase (XI) and xylulokinase (XK) which enable its utilization of xylose as a sole carbon source. The resulting consortium was then able to grow and produce ethanol from different xylan substrates.

The aim of this study was to utilize a new and highly effective bioreactor system, i.e., simultaneous saccharification and fermentation (SSCF), for bioethanol production by the cocultivation of Trichoderma reesei, Aspergillus niger, and Zymomonas mobilis by using a direct conversion process of pretreated-wood dust medium. Wood dust has been effectively used to obtain reducing sugars (glucose, xylose, and other byproducts) through the use of either a supercritical fluid extraction (SFE) or steam explosion (SE) pretreatment step. In addition, experimental results showed that polyurethane as a porous carrier could enhance total saccharification enzyme activity at an inoculum proportion of 1/1 of T. reesei, and A. niger and at a total inoculum concentration of 6.5 × 106 spores/ml. Furthermore, the concentration of alginate beads (3%) and immobilized proportion of Z. mobilis to alginate beads (1:4) were also examined. In accordance with previous reports, bioethanol production was carried out in a SSCF bioreactor by the cocultivation of T. reesei and A. niger in the polyurethane carrier and Z. mobilis immobilized in alginate beads using pretreated-wood dust medium. Experimental results revealed that, after 24 h of cultivation, the yield of bioethanol produced using pretreated-wood dust medium (1%) were 0.069 g/g and 0.049 g/g, for SFE and SE, respectively. Meanwhile, the sugar conversion rate reached 20.72% and 24.39% for SFE and SE, respectively. Thus, the results of this study show that pretreated-wood dust medium has significant potential for use in bioethanol production.

Summary Peanut sprout has associated with enhance the content of a nonflavonoids known as resveratrol, which influences a variety of functions including against cancer, cardiovascular and delaying ageing. Peanut from Liaoning province of China was used as a material to accumulate resveratrol during germination. The individual effects of soaking temperature and time as well as germination temperature, time and humidity on resveratrol accumulation in 5 days germinated peanut were investigated. Then, based on Box–Behnken design, interactive effects of the germination parameters were evaluated by response surface methodology. The optimum conditions were as follows: soaking temperature at 35 °C, soaking time at 8 h, germination temperature at 29 °C, germination humidity at 90%, and germination time at 5 day. Under the optimum condition, resveratrol content reached 32.87 μg g−1, which was almost 9.78 times higher than that in the peanut which did not germinate.

5-hydroxymethylfurfural (HMF) is a versatile biomass-derived chemical for synthesizing useful compounds. In this work an attempt is made to produce HMF by developing a low-cost process for doing that using cellulosic waste and deep eutectic solvents (DESs). The most effective DES was choline chloride/citric acid (ChCl/citric acid, 2/1), which provided a greater glucose conversion percentage (around 100 mol%), HMF yield (8 mol%), and HMF selectivity (8 %) than other DESs with 0.1 g of pure glucose. To increase the HMF yield further, the species of catalyst, reaction time and reaction temperature were varied. The highest glucose conversion percentage (91.76 mol%), HMF yield (15.02 g/g) and HMF selectivity (8%) were obtained with a reaction time of five minutes, a reaction temperature of 130 ℃, sulfuric acid as the catalyst, and a ChCl/citric acid ratio of the DES of 2/1. These optimal conditions were used with the enzymatic hydrolysate of pretreated wood dusts as a substrate. The experimental results thus obtained revealed a glucose conversion percentage, HMF yield and HMF selectivity of 34.86 mol%, 16.46 g/g and 67.43 %, respectively, under these conditions. These results reveal that using enzymatic hydrolysate from pretreated wood in the production of HMF can yield approximately as much (16.46 g/g) as the use of pure glucose, indicating that this low-cost process may be commercially feasible.

Cupriavidus taiwanensis 187 is reportedly efficient in achieving the degradation of phenol and accumulation of polyhydroxybutyrate (PHB). This study attempted to optimize the cultivation conditions and fermentation strategies for phenol degradation and PHA accumulation by C. taiwanensis 187. After the cultivation conditions were optimized, the conditions required for achieving phenol degradation (100%) and PHB accumulation (51 mg/L) by C. taiwanensis 187 were identified as 30 °C and 200 rpm, when the cultivation time was around 7 h. The accumulation of PHB was further increased from 72 to 213 mg/L by feeding phenol in three rounds into the fermenter along with the exhaustion of dissolved oxygen, which could totally degrade the phenol at around 1500 mg/L. Production of PHB by C. taiwanensis 187 was confirmed by GC, 1H-NMR, and 13C-NMR analyses. Each analytical result proved that C. taiwanensis 187 was able to use phenol as the sole carbon source for producing PHB. Finally, these results revealed that the phenol degraded by C. taiwanensis 187 mainly contributed to cell growth rather than PHB accumulation. These results indicated that the strain C. taiwanensis 187 could be used to degrade phenol to obtain usable biological polyesters.

The biosynthesis of prodigiosin (PG) from Serratia marcescens involves the coupling of a bipyrrole, 4-methoxy-2,2′-bipyrrole-5-carboxaldehyde (MBC), with a monopyrrole, 2-methyl-3-n-amyl-pyrrole (MAP), and formation of a linear tripyrrole (PG). We constructed mutant strains in which either the MBC biosynthesis by S. marcescens BMJ816 or the MAP biosynthesis by S. marcescens AMJ817. S. marcescens BMJ816 and AMJ817 confirmed that they lose the ability to synthesize PG when they are cultivated alone. An experiment was also conducted in which cultures of the two mutant strains were grown to the early exponential phase in a semi-defined medium, and one suspension culture was inoculated with the other. This approach yielded 103 mg/L PG. The findings suggest that the addition of precursors may enhance PG production by microorganisms.

Surfactin, one of the most powerful biosurfactants, is a lipopeptide-type biosurfactant that is generated by the gram-positive, endospore-producing, microorganism, Bacillus subtilis . The chemical structure of surfactin is composed of seven amino acids that are bonded to the carboxyl and hydroxy groups on long chain fatty acids (C 13 –C 15 ). The potential applications of surfactin are therapeutic applications, and environmental applications. However, the high cost of surfactin production and its low yields limit its range of commercial applications. This work therefore develops the natural production a surface active agent by microorganisms and evaluates the microbial production system, the purification process, the identification process and the potential applications of surfactin.

Thermosensitive hydrogels are attractive alternative scaffolding materials for minimally invasive surgery through a simple injection and in situ gelling. In this study, a novel poly(ester-amide) polymer, methoxy poly(ethylene glycol)-poly(pyrrolidone-co-lactide) (mPDLA, P3L7) diblock copolymer, was synthesized and characterized for cartilage tissue engineering. A series of amphiphilic diblock copolymers was synthesized by ring-opening polymerization of mPEG 550, D,L-lactide, and 2-pyrrolidone. By dynamic light scattering analysis and tube-flipped-upside-down method, viscoelastic properties of the mPDLA diblock copolymer solution exhibited sol-gel transition behavior as a function of temperature. An in vitro degradation assay showed that degradation acidity was effectively reduced by introducing the 2-pyrrolidone monomer into the polyester hydrogel. Besides, mPDLA exhibited great biocompatibility in vitro for cell encapsulation due to a high swelling ratio. Moreover, cell viability and biochemical analysis proved that the mPDLA hydrogel presented a great chondrogenic response. Taken together, these results demonstrate that mPDLA hydrogels are promising injectable scaffolds potentially applicable to cartilage tissue engineering.

This study attempted to utilize Halomonas salina BCRC17875 to produce ectoine by optimizing the agitation speed and medium composition. In addition, the chemical structure of ectoine produced by H. salina BCRC17875 was determined. The results indicate that ectoine production reached 3.65 g/L at 38 h of cultivation when the agitation rate and NaCl concentration were fixed at 200 rpm and 2.0 M, respectively. It reached 9.20 g/L at 44 h of cultivation when the major medium components were yeast extract (56 g/L), glutamate (74.40 g/L), and ammonium sulfate (14 g/L). After the nitrogen concentration had been evaluated, evaluation of the nitrogen concentration revealed that the ectoine production reached 11.80 g/L at 44 h of cultivation when 56 g/L of yeast extract and 28 g/L of ammonium sulfate were used. Ectoine production reached 13.96 g/L at 44 h of cultivation when the carbon/nitrogen ratio was fixed at 3/1 using 84 g/L of yeast extract and 28 g/L of ammonium sulfate. Furthermore, the identification of ectoine were identified and characterized by fast atom bombardment mass spectrometry (FAB-MS) and 1H NMR. The results demonstrated a fermentation strategy was successful in increasing ectoine production, and that the fermentation medium of ectoine had commercialization potential.

The aim of this work was to study the feasibility of surface displaying synthetic phytochelatin (EC) on Saccharomyces cerevisiae to overcome the inhibitory effect of heavy metals on ethanol production. Via the fusion of a gene encoding EC to an α-agglutinin gene, the engineered S. cerevisiae was able to successfully display EC on its surface. This surface engineered yeast strain exhibited an efficient cadmium adsorption capability and a remarkably enhanced cadmium tolerance. Moreover, its ethanol production efficiency was significantly improved as compared to a control strain in the presence of cadmium. Similar results could also be observed in the presence of other metals, such as nickel, lead and copper. Overall, this method allows simultaneous biorefinery and heavy metal removal when using heavy metal-contaminated biomass as raw materials.

This work studies a series of strategies in the production of lutein by Scenedesmus obliquus CWL-1 under mixotrophic cultivation. Our experimental results revealed that the optimal conditions associated with light-related strategies were 12 h light period followed by a 12 h dark period and blue to red light under mixotrophic cultivation. Under such conditions, the biomass, lutein content and lutein productivity were maximized to 9.88 (g/L), 1.78 (mg/g) and 1.43 (mg/L/day), respectively. Moreover, the assimilation of 4.5 g/L of calcium nitrate into S. obliquus CWL-1 increased the maximal biomass (12.73 g/L) and the highest maximal lutein productivity (3.06 mg/L/day), while the assimilation of 1.5 g/L of calcium nitrate yielded the highest maximal lutein content of 2.45 mg/g. The highest maximal lutein productivity of 4.96 (mg/L/day) was obtained when fed-batch fermentation was conducted, and this value was approximately 11-folds that obtained using the batch system.

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (PHBV) are biopolymers produced by microorganisms and have commercial potential as bioplastics. The compositions of polymers influence their physical and mechanical properties. PHBV polymers with high composition of 3-hydroxybutyrate (3HB) are brittle. Increasing the fraction of 3-hydroxyvalerate (3HV) monomer in the PHBV can reduce the crystallinity, resulting in more flexibility. To increase the fraction of 3HV in PHBV and reduce the antibiotic supplementation to maintain the recombinant plasmid, we cloned the prpE gene encoding propionyl-CoA synthase, the vgb gene encoding bacterial hemoglobin (VHb), and the polyhydroxyalkanoates (PHAs) synthesis operon (phaCAB) in one recombinant plasmid transformed into Escherichia coli XL1-blue, termed CT-2E. The prpP gene encoding propionate permease and the bktB gene encoding beta-ketothiolase were also cloned to evaluate their effects on PHBV accumulated in E. coli containing phaCAB-vgb, termed CT-2-BP. The results showed that the 3HV fraction in PHBV and the molecular weight of PHBV increased when prpE was expressed in CT-2E, compared with E. coli containing phaCAB-vgb (termed CT-2). The polydispersity index (PDI) of the PHBV derived from CT-2-BP was 1.33, indicating that polymer uniformity was greater than that from CT-2, which had a PDI of 1.68. Compared with CT-2, the enthalpies and melting temperature of PHBV from CT-2E and CT-2-BP decreased, indicating the advantages of processing.

Serratia marcescens C3 produces a natural red-pigment, prodigiosin, which exhibits immunosuppressive properties, in vitro apoptotic effects, and in vivo anti-tumor activities. This work seeks to improve the production of prodigiosin by S. marcescens C3 using various strategies. Starch and peptone were identified as the optimized carbon and nitrogen sources for the production of prodigiosin, yielding a prodigiosin concentration of 2.3 g/L. This value was significantly increased to 6.7 g/L using a carbon/nitrogen ratio of 6/4 (starch/peptone = 16 g/L/10.67 g/L). To enhance prodigiosin production even further, a statistical experimental design methodology was utilized to optimize the composition of the culture medium that is utilized in the production of prodigiosin. Prodigiosin production of 7.07 g/L was achieved when the concentrations of two trace compounds, FeSO 4 ·4H 2 O and MnSO 4 ·4H 2 O, were optimized using the statistical experimental design methodology. Their optimal concentrations were 0.56 mM and 3.25 mM, respectively. Ultimately, the production of prodigiosin was increased from 2.3 g/L to 15.6 g/L, or by a factor of nearly seven by immobilizing microorganisms in 3% calcium alginate beads.

The aim of this study was to use a modified bioreactor system for simultaneous saccharification of cellulose and bioethanol production. We tested Aspergillus niger and Trichoderma reesei for cellulose saccharification and Zymomonas mobilis for bioethanol production simultaneously in this modified bioreactor. The results showed that various carboxymethylcellulose (CMC) concentrations (10, 15, or 20 g/L) as a substrate for A. niger and T. reesei yielded bioethanol production of 0.51, 0.78, and 0.89 g/L and a CMC conversion rate of 10.2%, 10.7%, and 8.89%, respectively. These data suggested that at 10 g/L CMC as a substrate, the CMC conversion rate was higher than that at the other concentrations. When CMC concentration exceeded 15 g/L, bioethanol production was prolonged to 40 h. These results were attributed to the viscosity of CMC. This study also tested Napier grass (an agricultural byproduct) for bioethanol production. The results revealed bioethanol production and the theoretical Napier grass conversion rate were 0.38 g/L and 12.6%, respectively, for 13-h cultivation when the feeding concentration of Napier grass was 10 g/L. When Napier grass concentration was increased to 15 g/L, bioethanol production and the Napier grass conversion rate reached 0.51 g/L and 23%, respectively, after 14-h cultivation. Eventually, the experimental results indicated using agricultural waste for bioethanol production has been become a potential strategy.

Lignocellulose was converted into reducing sugars by using saccharification enzymes from cocultivated Trichoderma reesei and Aspergillus niger and reducing sugars as nutrients for Zymomonas mobilis to produce bioethanol in an immobilization system. After 96 h of cultivation, cocultivated T. reesei and A. niger had enzymatical synergistic effects that enabled a reducing sugar production of 1.29 g/L and a cellulose conversion rate of 23.27%. An 18% total inoculum concentration and a 1/1 inoculation ratio of T. reesei to A. niger obtained a reducing sugar production rate and a cellulose conversion rate of 2.57 g/L and 46.27%, respectively. The co-immobilization cultivation results showed that using polyurethane as a carrier optimized total saccharification enzyme activity at an inoculum ratio of 1/1 and a total inoculum concentration of 6.5 × 106 spores/mL. Based on the experimental results, the bioreactor design was further modified to enhance bioethanol production. The three strains (A. niger, T. reesei and Z. mobilis) were cocultivated with a co-immobilization cultivation system. The experimental results showed that, after 24 h cultivation, bioethanol production reached 0.56 g/L, and reducing sugar conversion rate reached 11.2% when using carboxymethylcellulose (CMC) substrates. The experimental results confirmed that the modified bioreactor enhances bioethanol production. However, further experiments are needed to determine how to prevent multi-stage failure of reducing medium volume.

RAD51 is essential for homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs) in mammalian cells. RAD51 is an attractive target for anticancer drugs, given high RAD51 levels are frequently observed in many human tumors and associated with increased resistance to DSBs-inducing chemotherapeutics. Prodigiosin is a bacterial tripyrrole pigment with potent anticancer activity and also provokes DSBs. We hereby aimed to elucidate the role of RAD51 in prodigiosin-induced cytotoxicity. Prodigiosin was found to down-regulate RAD51 in multiple human breast carcinoma cell lines irrespective of p53 status. Mechanistically, prodigiosin lowered RAD51 mRNA expression, whereas blockade of proteasome-mediated degradation failed to restore RAD51 levels following prodigiosin treatment. In addition, prodigiosin triggered phosphorylation of JNK and p38 MAPK, while pharmacological inhibition of JNK or p38 MAPK attenuated prodigiosin-mediated inhibition of RAD51 mRNA expression. Lastly, cells with enforced RAD51 expression showed increased resistance to prodigiosin-induced cytotoxicity as well as inhibition of colony formation. Collectively, we conclude that RAD51 down-regulation represents one of the modes of prodigiosin's cytotoxic action, ostensibly by augmenting the genotoxic effect of prodigiosin through suppression of RAD51-mediated HR repair. Our findings further implicate the use of prodigiosin to potentiate the cytotoxicity of DSB-inducing chemotherapeutics through RAD51 down-regulation.

For eco-friendly recycling and reuses of biomaterials with sustainability, this feasibility study tended to use indigenous pollutant degrading bacteria for the production of biodegradable polymers – polyhydroxyalkanotes (PHAs) during wastewater treatment. First, feasible PHA-producing strains were qualitatively screened among pollutant-degrading microbes via Sudan black B staining (SB staining). Next, according to batch cultures using lauric acid as sole carbon source, the promising PHA-generating strains were obtained via comparative analysis upon the characteristics of cell growth and poly 3-hydroxybutyrate (PHB) production. Aeromonas hydrophila NIU01, YTl1, KB23 and A. salmonicida 741 were found to generate intracellular PHA content at higher levels of 19.35, 24.48, 22.52%, respectively. Plus, Klebsiella pneumoniae ZMd31, Pseudomonas plecoglossicida NIU-Y3 and Chromobacterium violaceum P1 produced lower PHA contents at 18.25, 5.37 and 4.17%, respectively.

This study tended to use indigenous PHA-generating and dye-decolorizing microorganisms Aeromonas hydrophila NIU01, YTl1, KB23 and Aeromonas salmonicida 741, Klebsiella pneumoniae ZMd31 for comparing PHA production performance at various dye or amine-bearing conditions. Batch cultures using lauric acid (C12) at various concentrations as sole carbon source were carried out to determine optimal concentration for microbial growth and the production of poly 3-hydroxybutyrate (PHB). At optimal concentration of C12, cultures laden with dyes at different concentrations were conducted to explore whether dye-bearing cultures could stimulate or inhibit capabilities of PHA production. In addition, as aromatic amines were generated during reductive decolorization, effects of supplemented amines on capabilities of PHA-production were also conducted to uncover whether PHA generation during dye decolorization was attenuated. According to biostatistics, the finding indicated that strains NIU01, YTl1, KB23 could significantly tolerate dye pollutant(s) and aromatic amines for cell growth and PHA-production, suggesting promising feasibility to be used for PHA production during wastewater decolorization.

Cupriavidus necator is well known for its ability to accumulate polyhydroxybutyrate (PHB). When supplemented with propionic acid (or sodium propionate) in the growth medium, the bacterium is also able to synthesize polyhydroxybutyrate-co-hydroxyvalerate (PHBV). In order to increase the fraction of 3-hydroxyvalerate (3HV) in PHBV, we cloned the propionate permease gene prpP from C. necator and the propionyl-CoA synthase gene prpE from Cupriavidus taiwanensis and transformed into an Escherichia coli containing phaCAB operon of C. necator. The effects on PHBV accumulation in cells co-expressed with phaCAB and prpE or prpP in the media contained mixed carbon sources (glucose and sodium propionate) were evaluated. The HV fraction in PHBV increased when prpE or prpP was overexpressed in the cells. Concentrations of yeast extracts could also affect the fraction of HV. In addition, when glucose was replaced by sodium pyruvate, sodium succinate, or sodium gluconate, only PHB were detected in the recombinant strains.

Discoveries of tumor-resistant pharmacological drugs have mainly resulted from screening of natural products and their analogs. Some are also discovered incidentally when studying organisms. The great biodiversity of microorganisms raises the possibility of producing secondary metabolites (e.g., mevastatin, lovastatin, epothilone, salinosporamide A) to cope with adverse environments. Recently, natural plant pigments with anti-tumor activities such as β-carotene, lycopene, curcumin and anthocyanins have been proposed. However, many plants have a long life cycle. Therefore, pigments from microorganisms represent another option for the development of novel anti-tumor drugs. Prodigiosin (PG) is a natural red pigment produced by microorganisms, i.e., Serratia marcescens and other gram-negative bacteria. The anti-tumor potential of PG has been widely demonstrated. The families of PG (PGs), which share a common pyrrolylpyrromethene (PPM) skeleton, are produced by various bacteria. PGs are bioactive pigments and are known to exert immunosuppressive properties, in vitro apoptotic effects, and in vivo anti-tumor activities. Currently the most common strain used for producing PGs is S. marcescens. However, few reports have discussed PGs production. This review therefore describes the development of an anti-tumor drug, PG, that can be naturally produced by microorganisms, and evaluates the microbial production system, fermentation strategies, purification and identification processes. The application potential of PGs is also discussed.

A halophilic bacterium isolated from a salt environment in southern Taiwan was identified as a Marinococcus sp. ECT1. This bacterium could synthesize and accumulate intracellular ectoine as a compatible solute capable of resisting osmotic stress in a hyper-osmotic environment. This study also developed a semi-synthesized medium (YAMS medium), capable of facilitating the growth of this Marinococcus sp. ECT1 with 600 mg/L crude ectoine production. Moreover, Marinococcus sp. ECT1 was grown on YAMS medium containing different initial yeast extract concentrations (C(YE)) (0 to 60 g/L) to demonstrate how C(YE) affects crude ectoine production. While the maximum cell concentration was increased by 23-fold when the C(YE) was 40 g/L, the maximum crude ectoine production reached 2.5 g/L when C(YE) was 40 g/L. In addition to demonstrating the success of the fermentation strategy of ectoine in increasing the production and production yield, experimental results further demonstrated that the fermentation medium of ectoine is highly promising for commercialization. Furthermore, the molecular weight and chemical structure of ectoine were identified and characterized by FAB-MS and (1)H-NMR.

Polyhydroxyalkanoates (PHAs) are biodegradable plastics. Currently most PHAs are produced using microbial fermentation. The separation of PHAs is a key factor in the design of a successful production scheme. This study chose a recombinant Escherichia coli to produce poly(3-hydroxybutyrate) (PHB), an important one among PHAs; and to use different separation methods to recover PHB. The diversity of PHB obtained by different separation methods was characterized. Among different methods used, sodium dodecyl sulfate (SDS)–NaOH method was selected as the best in separation from the results obtained from experiments using a combinatorial design. In the combinatorial design, only 16 runs are needed to generate 150 sets of experimental data. By using a two-stage design, PHB with a purity of 99% was obtained when the first-stage digestion used SDS digestion method (50 °C, 125 rpm, biomass/digestion solution (100 g/L) = 1/10 (w/v) and 10 min) and the second-stage used NaOH method (50 °C, 125 rpm, biomass/digestion solution (1 M) = 1/10 (w/v) and 10 min).

Polyhydroxyalkanoate (PHA) is a biodegradable material with many potential biomedical applications, including medical implants and drug delivery. This study developed a system for screening production strains in order to optimize PHA production in Cupriavidus taiwanensis 184, 185, 186, 187, 204, 208, 209 and Pseudomona oleovorans ATCC 29347. In this study, Sudan black B staining, Infrared (IR) and Gas Chromatography (GC) analysis indicated that the best strain for PHA synthesis is C. taiwanensis 184, which obtains polyhydroxybutyrate (PHB). Cultivation of C. taiwanensis 184 under a pH of 7.0, at 30 °C, and at an agitation rate of 200 rpm, obtained a PHB content of 10% and PHB production of 0.14 g/L. The carbon and nitrogen types selected for analysis of PHB production by C. taiwanensis 184 were gluconic acid and NH(4)Cl, respectively. Optimal carbon/nitrogen ratio for PHB production was also determined. This study demonstrated a PHB content of 58.81% and a PHB production of 2.44 g/L when the carbon/nitrogen ratio of 8/1 was selected for C. taiwanensis 184. A two-stage fermentation strategy significantly enhanced PHB content and PHB production. Under a two-stage fermentation strategy with nutrient-limited conditions, C. taiwanensis 184 obtained a PHB content of 72% and a PHB concentration of 7 g/L. Finally, experimental results confirmed that optimizing the growth medium and fermentation conditions for cultivating the indigenous C. taiwanensis 184 strain substantially elevated PHB content from 10% to 72% and PHB production from 0.14 g/L to 7 g/L, respectively.

Phenol biodegradation in batch systems using Cupriavidus taiwanesis 187 has been experimentally studied. To determine the various parameters of a kinetic model, combinations of rearranged equations have been evaluated using inverse polynomial techniques for parameter estimation. The correlations between lag phase and phase concentration suggest that considering phenol inhibition in kinetic analysis is helpful for characterizing phenol degradation. This study proposes a novel method to determine multiplicity of steady states in continuous stirred tank reactors (CSTRs) in order to identify the most appropriate kinetics to characterize the dynamics of phenol biodegradation.

This work was undertaken to develop an improved gas chromatography (GC) analysis of poly (3-hydroxyalkanoate) (PHA) quantification method based on acidic methanolysis. This is achieved by investigating the kinetics of acidic hydrolysis of PHAs with sulfuric acid in the chloroform/aqueous solution to identify suitable hydrolytic pretreatment conditions for quantitative analysis of PHAs. The target parameters included sulfuric acid concentration, salt (NaCl) addition, kind of PHAs (commercial products of poly-3-hydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV-8%) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx-3.8% and-10.5%), as well as the type of PHA-producing microorganisms (Cupriavidus taiwanensis 184 and Burkholderia sp. PTU9). These results show that esters would preferentially accumulate in the organic phase when NaCl was added in the two-phase system, thereby enhancing the accuracy of GC analysis. Decomposition efficiency of different types of PHAs was found to be dependent on sulfuric acid concentration, such as 1% H2SO4 was favorable for PHB decomposition, while 5 and 7% H2SO4 should be used to decompose PHBV and PHBHHx. Copyright © 2009 Curtin University of Technology and John Wiley & Sons, Ltd.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)) was produced by Aeromonas sp. KC014 strain isolated from Taiwan in soil environment in the flask and fermentor cultures. The medium optimiza- tion, such as carbon source and nitrogen source, carbon-nitrogen ratio was conducted to obtain the optimum 3-hydroxy- hexanoate content. The defined medium with dodecanoic acid as the carbon source and (NH4)2SO4 as the nitrogen source was obtained as the main medium. When cells grown in medium containing 30 g/L dodecanoic acid, 15 g/L sodium gluconate and 1 g/L soytone (C/N was 30/1) as final PHA concentration, the cell dry weight and HB content of 5.16 g/ L, 14.0 g/L and 36.0%, respectively, were obtained. The maximum HHx/PHA content increased from 0.1% to 1.3% nearly 12-fold when the dissolved oxygen was decreased from 40% to 20%. P(3HB-co-3HHx) biosynthesis was trig- gered by the addition of limited nitrogen, phosphorus and magnesium to get a maximum HHx/PHA content of 14% in 95 hours.

Addition of a small quantity of solid porous carriers (e.g., activated carbon or expanded clay) into fermentation broth significantly increased surfactin production with Bacillus subtilis ATCC 21332. Culture medium containing 25 g L(-1) of activated carbon gave an optimal surfactin yield of 3600 mg L(-1), which was approximately 36-fold higher than that obtained from carrier-free liquid culture. The marked increase in surfactin production was primarily attributed to stimulation of cell growth due to the presence of activated carbon carriers. Concentration of limiting carbon substrate (glucose) is also an important factor affecting the production of surfactin, as an initial glucose concentration of 40 g L(-1) resulted in optimal surfactin production. An appropriate agitation rate also benefited surfactin production, as the best yield appeared at an agitation rate of 200 rpm. Surfactin was purified from fermentation broth via a series of acidic precipitation and solvent extraction. The resulting product was nearly 90% pure with a recovery efficiency of ca. 72%. The purified surfactin reduced the surface tension of water from 72 to 27 mN m(-1) with a critical micelle concentration of ca. 10 mg L(-1). The surfactin product also attained an emulsion index of 70% for kerosene and diesel at a low concentration of 100 and 600 mg L(-1), respectively.

An indigenous strain Pseudomonas aeruginosa S2 (P. aeruginosa S2), isolated from diesel-contaminated soil, produced extracellular surface-active material identified as rhamnolipid. Due to its excellent surface activity, rhamnolipid is known to be well-suited for stimulating the bioremediation efficiency of oil contaminated sites. To improve production yield of rhamnolipid with P. aeruginosa S2, various carbon and nitrogen sources were screened to select favorable ones leading to better biosurfactant production yield. It was found that using 4% glucose could attain better rhamnolipid yield, while 50 mM NH4NO3 appeared to be the most preferable nitrogen source. Meanwhile, the effect of carbon to nitrogen ratio (C/N ratio) on rhamnolipid yield was also investigated, and the optimal C/N ratio was identified as approximately 11.4. Moreover, response surface methodology (RSM) was applied to optimize the trace element concentration for rhamnolipid production. Results from two-level design indicate that concentrations of MgSO4 and FeSO4 were the most significant factors affecting rhamnolipid production. Using steepest ascent method and RSM analysis, an optimal medium composition was determined, giving a rhamnolipid production yield of 2.37 g/L in 100 h at 37 degrees C and 200 rpm agitation. Scale-up production of rhamnolipid in a well-controlled 5 L jar fermentor using the optimal medium and operating condition (at 37 degrees C and pH 6.8) further elevated the biosurfactant production yield to 5.31 g/L (in 97 h), which is over 2-fold higher than the best results obtained from shake-flask tests.

An attempt was made to further improve the purity of surfactin in the fermentation broth of Bacillus subtilis ATCC 21332 culture by adsorption or ion exchange after the broth had been treated by a two-stage ultrafiltration process. The commonly used neutral adsorption resin (macroporous XAD-7) and charged ion-exchange resin (AG1-X4) were adopted. Under the conditions tested the impurities in the treated broth such as macromolecules (proteins, polysaccharides, peptides) or smaller molecules (glycine, serine, threonine, alanine) were adsorbed or exchanged more quickly and preferably within 5 h of operation, purification of surfactin was hence achieved by adsorption removal of the impurities. It was shown that XAD-7 resins gave higher purity of surfactin than AG1-X4 resins. The recovery of surfactin from the remaining solution after adsorption using XAD-7 resin exceeded 95% at pH 6.5 and the purity increased from 76% (the feed) to 88%; however, the purity of surfactin reached up to 80% only after ion exchange using AG1-X4 resin. Finally, the breakthrough phenomena of surfactin solutions in a XAD-7 column were tested and discussed.

A newly isolated indigenous strain Pseudomonas aeruginosa J16 was able to produce a glycolipid biosurfactant (i.e., rhamnolipid). Analyses with 13C NMR and mass spectrometry show that the biosurfactant consisted mainly of mono-rhamnolipid (RL1) and di-rhamnolipid (RL2). Since RL2 possesses higher surface activity than RLl and is of better applicability and commercial benefits, the present work attempted to enhance the production of RL2 over RL1 in P. aeruginosa J16 from the perspective of medium improvement. The results show that the abundance and composition of RL1 and RL2 varied with the type of carbon and nitrogen sources used. When glycerol and NH4Cl were used as the carbon and nitrogen sources, respectively, an RL2 production of 448.3 mg/L was obtained with a volumetric productivity of 4.67 mg/(L h) and a RL2/RL1 ratio of 4.22 (by mass). Replacing NH4Cl by (NH4)2SO4, the RL2 concentration and volumetric productivity were improved to 2121 mg/L and 22.1 mg/(L h), respectively, with the RL2/RL1 ratio of 3.0. To further enhance RL2 production, statistical experimental design methodology was applied to optimize the culture medium composition favoring RL2 synthesis. Three key parameters (glycerol, (NH4)2SO4, and MgSO4·7H2O) were selected by two-level factorial design. Response surface methodology was then used to identify the optimal composition of the three key parameters, giving an optimal concentration of 0.38 M, 33.3 mM, and 577 μM for glycerol, (NH4)2SO4, and MgSO4·7H2O, respectively. With this optimal medium, the RL2 production could be markedly elevated to a maximum concentration of 3190 mg/L and a volumetric productivity of 44.3 mg/(L h), while the RL2/RL1 ratio maintained nearly constant at a value of 3.0.

The isolation and the purification of bacterial polyhydroxyalkanoates are the key step of the process profitability in the fermentation system. That is why many scientists have studied this field for the production of this biodegradable polymer. The ideal method should lead to a high purity and recovery level at a low production cost. This paper reviews four isolation methods, i.e. solvent extraction of halosolvent and nonhalosolvent, digestion of non-polyhydroxyalkanoate cell material involving surfactants, sodium hypochlorite or enzyme, mechanical cell disruption methods like using bead mills and high pressure homogenization, and new methods like spontaneous liberation of poly(3-hydroxybutyrate), dissolved air flotation, air classification, or by using supercritical CO2. The pretreatment of cell disruption and the purification methods and analytical methods of polyhydroxyalkanoates are also presented.

Bacteria isolated from marine sediments were screened for their ability to accumulate polyhydroxyalkanoates. Among the isolates, four Vibrio spp. (strain M11, M14, M20 and M31) were studied in detail. All synthesized intracellular lipid inclusions during growth on diverse carbon sources including acetate, glycerol, succinate, glucose and sucrose. The inclusions were identified to be poly-beta-hydroxybutyrate (PHB) using gas chromatography and nuclear magnetic resonance analysis. No other type of polyhydroxyalkanoates (PHAs) was found to be accumulated by these marine isolates, suggesting that the diversity of PHAs produced in marine environments may be not as versatile as found in other environments. Strain M11 accumulated PHB in concentrations as high as 41% of cell dry weight when grown in medium containing 4% of sodium chloride. One of the Vibrio spp. was identified to be closely related to Vibrio natriegens (98% identity) by partial 16S rDNA sequence homology. V. natriegens has the shortest generation time (9.8 min) of any bacterium and this characteristic may be an exploitable trait for the industrial production of PHB.

Rhamnolipid is one of the most commonly used biosurfactants with the ability to reduce the surface tension of water from 72 to 30 mN/m. An indigenous isolate Pseudomonas aeruginosa S2 possessing excellent ability to produce rhamnolipid was used as a model strain to explore fermentation technology for rhamnolipid production. Using optimal medium and operating conditions (37 degrees C, pH 6.8, and 250 rpm agitation) obtained from batch fermentation, P. aeruginosa S2 was able to produce up to 5.31 g/l of rhamnolipid from glucose-based medium. To further improve the rhamnolipid yield, a pH-stat fed-batch culture was performed by maintaining a constant pH of 6.8 through manipulating glucose feeding. The effect of influent glucose concentration on rhamnolipid yield and productivity was investigated. Using the pH-stat culture, a maximum rhamnolipid concentration (6.06 g/l) and production rate (172.5 ml/h/l) was obtained with 6% glucose in the feed. Moreover, combining pH-stat culture with fill-and-draw operation allowed a stable repeated fed-batch operation for approximately 500 h. A marked increase in rhamnolipid production was achieved, leading to the best rhamnolipid yield of approximately 9.4 g/l during the second repeated run.

In this work, optimizing trace element composition was attempted as a primary strategy to improve surfactin production from Bacillus subtilis ATCC 21332. Statistical experimental design (Taguchi method) was applied for the purpose of identifying optimal trace element composition in the medium. Of the five trace elements examined, Mg 2+ , K + , Mn 2+ , and Fe 2+ were found to be more significant factors affecting surfactin production by the B. subtilis strain. In the absence of Mg 2+ or K + , surfactin yield decreased to 0.4 g/l, which was only 25% of the value obtained from the control run. When Mn 2+ and Fe 2+ were both absent, the production yield also dropped to ca. 0.6 g/l, approximately one-third of the control value. However, when only one of the two metal ions (Fe 2+ or Mn 2+ ) was missing, the B. subtilis ATCC 21332 strain was able to remain over 80% of original surfactin productivity, suggesting that some interactive correlations among the selected metal ions may involve. Taguchi method was thus applied to reveal the interactive effects of Mg 2+ , K + , Mn 2+ , Fe 2+ on surfactin production. The results show that interaction of Mg 2+ and K + reached significant level. By further optimizing Mg 2+ and K + concentrations in the medium, the surfactin production was boosted to 3.34 g/l, which nearly doubled the yield obtained from the original control.

Bacillus subtilis ATCC 21332 was used to produce a lipopeptide-type biosurfactant (namely, surfactin) in an innovative bioreactor tailored to solve the problems of severe foaming arising from production of the biosurfactant. To cope with the rapid foam generation, a conventional jar fermentor was integrated with a foam collector, a cell recycler, and a surfactin precipitation unit. Meanwhile, solid carriers (e.g., activated carbon) were added into the fermentation broth to increase cell mass concentration and surfactin yield. The proposed bioreactor allowed stable and efficient surfactin fermentation under intensive foaming conditions without the need of adding antifoam agents. The effect of oxygen transfer rate and mass transfer efficiency on surfactin production was also explored by employing various combinations of aeration and agitation rates. The best combination was 1.5 vvm and 300 rpm, giving an excellent maximum production rate, overall production rate, surfactin concentration, and surfactin yield of 190 mg L−1 h−1, 106 mg L−1 h−1, 6.45 g L−1, and 161 mg surfactin (g glucose)−1, respectively. It was also found that the oxygen volumetric mass transfer coefficient (KLa) was highly correlated with the performance of surfactin production. The highest surfactin productivity was achieved when the fermentation was carried out at a KLa value of 0.0132 s−1.

Pseudomonas aeruginosa J4, isolated from wastewater of a petrochemical factory located in southern Taiwan, was used to produce rhamnolipid from a variety of carbon substrates, including hydrophilic substrates, vegetable oils, and mineral oils. The P. aeruginosa J4 strain was able to assimilate the seven carbon substrates examined (namely, glucose, glycerol, olive oil, sunflower oil, grape seed oil, diesel, and kerosene), whereas it grew less efficiently in mineral oils (esp., kerosene). Rhamnolipid production from the J4 strain was affected by temperature and agitation rate, as 30 °C and 200 rpm agitation were favorable for rhamnolipid production. The rhamnolipid concentration (CRL) and production rate ( v RL ) was also influenced by the carbon sources used to grow the J4 strain. Similar v RL (10–12 mg/h/L) and CRL (1400–2100 mg/L) were obtained from using glycerol, glucose, grape seed oil, and sunflower oil as the sole carbon substrate, while using olive oil delivered the best rhamnolipid production. Maximum CRL (3600 mg/L) and v RL (26 mg/h/L) were attained at 10% olive oil. P. aeruginosa J4 also utilized diesel and kerosene for rhamnolipid production but with much lower CRL and v RL values Rhamnolipid was purified (nearly 90% pure) from the culture broth. Mass spectrometry and NMR analysis indicate that the purified product contained two types of commonly found rhamnolipids: l -rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (RL1) and l -rhamnosyl l -rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (RL2). The rhamnolipid product can reduce the surface tension of water to 31 mN/m with a critical micelle concentration of nearly 50 mg/L. The biosurfactant also achieved a maximum emulsion index of 70 and 78%, for diesel and kerosene, respectively, at a low concentration of about 300 mg/L.

Serratia marcescens SMdeltaR, an SpnR-defective isogenic mutant of S. marcescens SS-1, was used to produce a prodigiosin-like pigment (PLP). Luria-Bertani (LB) broth, frequently used for prodigiosin biosynthesis with S. marcescens strains, was modified by increasing the concentrations of tryptone and yeast extract while completely removing NaCl from the medium. The resulting modified LB (MLB) medium achieved an almost 3.0-fold increase in PLP yield (152 mg l(-1)) when compared with the original LB broth. The addition of vegetable oils (2-6% [v/v]) to the fermentation broth markedly enhanced PLP production. PLP yields of 525, 579, and 790 mg l(-1) were obtained when the MLB medium was supplemented with 4% soybean oil, 4% olive oil and 6% sunflower oil, respectively. PLP production was found to be positively correlated with extracellular surface emulsification activity, suggesting a link between the PLP production and the presence of biosurfactant. This work shows that the optimal medium for PLP yield was sunflower oil (6%)-supplemented MLB medium, which resulted in an approximately 14-fold higher PLP yield than that in LB broth. Mass spectrometry and NMR analysis indicated that the PLP product is a prodigiosin derivative, called undecylprodigiosin.

Supplement of Fe(2+) into fermentation medium was utilized as a tool to optimize the iron-mediated enhancement of surfactin production from Bacillus subtilis ATCC 21332. Up to 3000 mg L(-)(1) of surfactin was produced using an iron-enriched minimal salt (MS) medium amended with an optimal Fe(2+) dosage of 4.0 mM, leading to 8-fold and 10-fold increase in cell concentration and surfactin yield, respectively, as compared to those without Fe(2+) supplement. In addition to resulting in an optimal production yield of surfactin, a supplement of 4.0 mM of Fe(2+) also propelled maximum overall surfactin production rate to a highest value of 24 mg L(-)(1) h(-)(1). Our results also show that production of surfactin followed a growth-associated kinetic model. The best yield coefficient estimated from the model was ca. 162 mg surfactin (g dry cell)(-)(1). The supernatant of the iron-enriched culture of B. subtilis ATCC 21332 exhibited the ability to emulsify kerosene and achieved a maximum emulsion index (E(24)) of 80% for culture supplemented with 4.0 mM of Fe(2+). Comparison of emulsion index and the corresponding surfactin production indicates that the emulsification activity was essentially contributed by surfactin.

A simple procedure for surface modification of polyurethane by the Langmuir-Blodgett (LB) method using the amphiphile 1,2-distearoyl-sn-glycero-3-phosphocholine dihydrate (DSPC) was developed. The polyurethane containing the fluorinated moiety was prepared via the perfluoro-containing chain extender 2,2,3,3-tetrafluoro-1,4-butanediol. The fluorinated polyurethane (FPU) films were prepared by spin coating and dipping methods. The spin-coated FPU films exhibited hydrophobic characteristic and, thus, enhanced the transferability of DSPC. Held at constant pressure of 45 mN/m, the DSPC monolayer was transferred successfully to FPU films with a near-unity transfer ratio. The in vitro platelet adhesion assay revealed that the FPU modified with DSPC monolayer was more platelet compatible than the unmodified FPU substrates with no pseudopods and flattening of adherent platelets as well as lower platelet adhesion density. Moreover, the DSPC monolayer remained intact after platelet adhesion testing. In addition, the platelet compatibility of the unmodified FPU was affected by the film preparation methods. This might be attributed to the distinctive surface micromorphology formed. This simple DSPC deposition scheme by a LB technique would be very useful to further enhance the platelet compatibility of hydrophobic substrate and can be utilized for biomedical application in which the flow shear rate is not too high.

Bacillus subtilis is able to synthesize a lipopeptide biosurfactant (known as surfactin), which is one of the most effective biosurfactants available. In our previous study, B. subtilis ATCC 21332 was used to produce surfactant and it was found that the addition of iron at an appropriate amount significantly improved the biosurfactant production. However, it also showed that excess addition of iron led to a sharp decrease in pH of the culture and surfactin in the broth disappeared rapidly once the pH fell below 5.0. This study reveals that the disappearance of surfactin at the acidic pH was due to the precipitation of surfactin triggered by the accumulation of extracellular acidic metabolites. The acidic material was isolated and purified from the broth and was characterized by GC-MS and IR spectroscopy. The results of GC-MS and IR analysis show that the molecular weight and molecular structure of the acidic metabolite resembled those of phthalic anhydride.

Production of a lipopeptide surfactant, surfactin, by Bacillus subtilis ATCC 21332 was highly enhanced when iron concentration in the medium was raised from 4 μ m to the few m m level. This iron enrichment of the medium also resulted in increased biomass concentration. With 1.7 m m iron sulfate added to the media of different initial iron concentrations or at different stages of growth, surfactin concentration can be raised to levels of several hundred mg l −1 in general. Acidification of the broth was usually observed following the iron addition, and surfactin in the broth disappeared rapidly due to precipitate as soon as the pH level dropped below 5.0; however, if this acidification phenomenon was delayed, avoided (for reasons still unknown), or reversed (by alkaline addition), surfactin production was highly enhanced to as high as 3,500 mg l −1 which was almost ten times over the previously reported level for this strain and higher than most reported values for genetically improved strains.

The feasibility of using genipin cross-linked type II collagen scaffold with rabbit bone marrow mesenchymal stem cells (RBMSCs) to repair cartilage defect was herein studied. Induction of RBMSCs into chondrocytic phenotype on type II collagen scaffold in vitro was conducted using TGF-β 3 containing medium. After 3-weeks of induction, chondrocytic behavior, including marker genes expression and specific extracellular matrix (ECM) secretion, was observed. In the in vivo evaluation experiment, the scaffolds containing RBMSCs without prior induction were autologous implanted into the articular cartilage defects made by subchondral drilling. The repairing ability was evaluated. After 2 months, chondrocyte-like cells with lacuna structure and corresponding ECM were found in the repaired sites without apparent inflammation. After 24 weeks, we could easily find cartilage structure the same with normal cartilage in the repair site. In conclusion, it was shown that the scaffolds in combination of in vivo conditions can induce RBMSCs into chondrocytes in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.

Chondroitin sulfate C (CSC) is a kind of glycosaminoglycans (GAGs) with molecular weights of 10,000 to 50,000 Da and a high charge density. GAGs are major components in extracellular matrix (ECM), which play important role in the regulation of cell proliferation, migration, and differentiation. In this study, we studied the effects of chondroitin sulfate C (CSC) on the differentiation of human mesenchymal stem cells (MSCs) toward the chondrocyte lineage. The MSCs were either cultured on type II collagen (COL II) scaffolds with high molecular weight CSC addition in the medium (free CSC) or with free oligosaccharide CSC. Special attention was given to the effects of MSCs cultured on CSC cross-linked type II scaffolds (cross-linked CSC). According to the analysis of histology stain, gene expression, and ECM secretion, our results showed that MSCs cultured with free CSC, free oligosaccharides CSC, and on the cross-linked CSC scaffolds all would be induced into chondrocytes. Moreover, free oligosaccharide CSC present in the microenvironment could significantly up-regulate MSC chondrogenesis gene expression and stimulate cartilage ECM accumulation more than free CSC with high molecular weight after 3-week induction. Importantly, cross-linked CSC had the most excellent effects on the MSC chondrogenesis. Thus, we believed that cross-linked CSC in the scaffold would play the similar roles with free oligosaccharide CSC in the medium. Cross-linked CSC would be a potential candidate for cartilage repair in the cell therapy and tissue engineering.

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P(BAD) promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.

A screening method was developed to evaluate the oil removal capability of biosurfactants for oil-contaminated soils collected from a heavy oil-polluted site. The ability of removing total petroleum hydrocarbon (TPH) from soil by two biosurfactants was identified and compared with that of synthetic surfactants. The results show that biosurfactants exhibited much higher TPH removal efficiency than the synthetic ones examined. By using 0.2 mass% of rhamnolipids, surfactin, Tween 80, and Triton X-100, the TPH removal for the soil contaminated with ca. 3,000 mg TPH/kg dry soil was 23%, 14%, 6%, and 4%, respectively, while removal efficiency increased to 63%, 62%, 40%, and 35%, respectively, for the soil contaminated with ca. 9000 mg TPH/kg dry soil. The TPH removal efficiency also increased with an increase in biosurfactant concentration (from 0 to 0.2 mass%) but it did not vary significantly for the contact time of 1 and 7 days.