Growth Medium Conditions Research Articles

Carbon–nitrogen ratio and in vitro assimilate partitioning patterns in Cyrtanthus guthrieae L

In response to variations in nutritional composition of the growth medium, plants often adjust their metabolism and progressively alter their growth patterns. Carbon (C) and nitrogen (N) constitute the major plant nutritional components influencing plant growth and development patterns. This study examined the growth dynamics and patterns of assimilate partitioning to primary and secondary metabolites in response to varying levels and combinations of C and N in the culture media of Cyrtanthus guthrieae. In vitro callus-derived C. guthrieae plantlets were cultured on solid Murashige and Skoog (MS) media with different concentrations and combinations of C and N. Relative growth rate (RGR) increased proportionally with an increase in C concentrations up to 88 mM sucrose (0.58 d−1) beyond which it was hardly influenced by further increases in C. Growth was also significantly favoured in media with high concentrations of N at all C concentrations tested. In C-limited media regimes with growth saturating N conditions, alkaloid accumulation became favoured while polyphenol content increased with an increase in C levels in the medium, a characteristic pattern that appeared to be less influenced by the amount of N. Of the primary metabolites, only proteins showed small significant variations across different media treatments, with starch and soluble sugars increasing proportionately with C levels. In the medium with a high sucrose concentration (175 mM), soluble sugars, amino acids and polyphenols increased markedly, possibly as an adaptive response to the reduced osmotic potential in the media and/or a storage mechanism for excess C and N reserves in the media. From a medicinal perspective, with regard to polyphenolic compounds in C. guthrieae, growth medium conditions that allow for high levels of C pools in the tissue would thus be favourable for the enhanced synthesis of this group of compounds. The medium conditions with 175 mM sucrose and 10.3 mM NH4NO3 gave the highest total polyphenols, flavonoids and proanthocyanidins with a moderate growth rate.

Combining Genotype Improvement and Statistical Media Optimization for Isoprenoid Production in E. coli

Isoprenoids are a large and diverse class of compounds that includes many high value natural products and are thus in great demand. To meet the increasing demand for isoprenoid compounds, metabolic engineering of microbes has been used to produce isoprenoids in an economical and sustainable manner. To achieve high isoprenoid yields using this technology, the availability of metabolic precursors feeding the deoxyxylulose phosphate (DXP) pathway, responsible for isoprenoid biosynthesis, has to be optimized. In this study, phosphoenolpyruvate, a vital DXP pathway precursor, was enriched by deleting the genes encoding the carbohydrate phosphotransferase system (PTS) in E. coli. Production of lycopene (a C40 isoprenoid) was maximized by optimizing growth medium and culture conditions. In optimized conditions, the lycopene yield from PTS mutant was seven fold higher than that obtained from the wild type strain. This resulted in the highest reported specific yield of lycopene produced from the DXP pathway in E. coli to date (20,000 µg/g dry cell weight). Both the copy number of the plasmid encoding the lycopene biosynthetic genes and the expression were found to be increased in the optimized media. Deletion of PTS together with a similar optimization strategy was also successful in enhancing the production of amorpha-1,4-diene, a distinct C15 isoprenoid, suggesting that the approaches developed herein can be generally applied to optimize production of other isoprenoids.

Exposure of Escherichia coli O157:H7 to soil, manure, or water influences its survival on plants and initiation of plant defense response

This study evaluated whether growth medium or exposure conditions influence the production of capsular polysaccharides (CPS) by Escherichia coli O157:H7, and whether changes in CPS impact plant defense responses, consequently affecting survival on plants. E. coli O157:H7 grown in Luria–Bertani (LB) broth supplemented with manure extracts showed an approximately 58% increase in CPS production compared to cells grown in LB medium alone. Levels of CPS were significantly higher for E. coli O157:H7 cells exposed to soil or manure extracts as compared to the non-exposed LB cultured control. Arabidopsis thaliana plants expressing β-glucuronidase (GUS) under the control of the β-1,3-glucanase (BGL2) promoter were used to investigate whether E. coli O157:H7 induces defense-related gene expression. Plants inoculated with E. coli O157:H7 grown in LB containing manure extracts or cells exposed to manure extracts exhibited 3-fold and 2-fold lower GUS activity, respectively, suggesting a limited plant defense response compared to plants inoculated with cells grown in LB. On day 5 post inoculation the population of E. coli O157:H7 grown in LB supplemented with manure on plants was significantly greater than the population of E. coli O157:H7 grown in LB medium alone. E. coli O157:H7 cells exposed to soil or manure exhibited greater survival on plants compared to LB-grown E. coli O157:H7. The results of this study underscore the need to consider medium composition and cultural conditions when conducting crop challenge studies.

PdeB, a Cyclic Di-GMP-Specific Phosphodiesterase That Regulates Shewanella oneidensis MR-1 Motility and Biofilm Formation

Shewanella oneidensis MR-1, a gammaproteobacterium with respiratory versatility, forms biofilms on mineral surfaces through a process controlled by the cyclic dinucleotide messenger c-di-GMP. Cellular concentrations of c-di-GMP are maintained by proteins containing GGDEF and EAL domains, which encode diguanylate cyclases for c-di-GMP synthesis and phosphodiesterases for c-di-GMP hydrolysis, respectively. The S. oneidensis MR-1 genome encodes several GGDEF and EAL domain proteins (50 and 31, respectively), with a significant fraction (∼10) predicted to be multidomain (e.g., GGDEF-EAL) enzymes containing an additional Per-Arnt-Sim (PAS) sensor domain. However, the biochemical activities and physiological functions of these multidomain enzymes remain largely unknown. Here, we present genetic and biochemical analyses of a predicted PAS-GGDEF-EAL domain-containing protein, SO0437, here named PdeB. A pdeB deletion mutant exhibited decreased swimming motility and increased biofilm formation under rich growth medium conditions, which was consistent with an increase in intracellular c-di-GMP. A mutation inactivating the EAL domain also produced similar swimming and biofilm phenotypes, indicating that the increase in c-di-GMP was likely due to a loss in phosphodiesterase activity. Therefore, we also examined the enzymatic activity of purified PdeB and found that the protein exhibited phosphodiesterase activity via the EAL domain. No diguanylate cyclase activity was observed. In addition to the motility and biofilm phenotypes, transcriptional profiling by DNA microarray analysis of biofilms of pdeB (in-frame deletion and EAL) mutant cells revealed that expression of genes involved in sulfate uptake and assimilation were repressed. Addition of sulfate to the growth medium resulted in significantly less motile pdeB mutants. Together, these results indicate a link between c-di-GMP metabolism, S. oneidensis MR-1 biofilm development, and sulfate uptake/assimilation.

Su1782 Sucrose Increases Expression of Antimicrobial Peptides Cryptdin-5 and Cryptdin-7 in Rat Small Intestine

by 1-by-1 Y2H interaction assay on solid growth under restrictive growth medium conditions. The interaction between hSVCT1 and hGRHPR was further confirmed by in vitro GST-pulldown assay and in vivo co-immunoprecipitation and mammalian two-hybrid firefly luciferase assays. In addition, hSVCT1-YFP and DsRed-hGRHPR co-expressing human liver HepG2 cells demonstrated co-localization of these two proteins in intracellular trafficking vesicles. Co-expression of hGRHPR with hSVCT1 led to a marked increase in 14C-AA uptake. On the other hand, knock down of endogenous hGRHPR with gene-specific siRNA led to a marked inhibition in 14C-AA uptake. This effect was dependent upon a marked reduction in GRHPR mRNA, while, hSVCT1 and hSVCT2 mRNA levels were unaffected. Conclusion: This study demonstrates hGRHPR is an hSVCT1 interacting partner in human liver cells and this interaction is important for the function and cell biology of hSVCT1. (Supported by grants from the DVA and NIH DK 71538, DK 84094, DK 56061).

1165 HCV INFECTION REPROGRAMS THE HEPATIC GLUCOSE AND GLUTAMINE METABOLISM

Background and Aims: Hepatitis C virus (HCV) is the only virus that is known to perturb hepatic glucose and lipid metabolism with important patho-physiological consequences. Chronic carriers often develop steatosis, insulin resistance and type 2 diabetes, which resolve with successful antiviral treatment. Therefore it is thought that HCV interferes directly with the lipogenic and glycolytic pathways and requires these changes for its replication. However, the exact circumstances of this metabolic reprogramming still remain vague and require further analysis. Here we investigate some fundamental changes in glucose and glutamine metabolism linked to HCV infection. Methods: RNA derived from Huh7.5 cells infected or not with JFH1 and from biopsies of chronic HCV patients were used for RT-qPCR analysis with primers targeting metabolic genes. Nutrient deprivation and biochemical and NMR-based metabolic flux analysis were performed with JFH1 infected Huh7.5 cell culture extracts. Results: We show that HCV modulates the transcript levels of some key regulators of glucose metabolism (HIF-1 a, PKM2, G6PD) in the hepatocyte-derived cell-line Huh7.5 as well as liver biopsies of patients with chronic hepatitis C, which hinted at changes to glycolytic fluxes. In addition, we found enzymes and factorsregulating glutamine metabolism (MYC, SLC1A5, SLC7A5, GLS) to be upregulated by HCV. Indeed, cell proliferation rates of HCV infected and uninfected cells in conditioned growth media showed that infected cells become dependent on glutamine and lose their glucose dependence. NMR-based metabolomic assays further corroborated these findings. We then showed that silencing of MYC, an oncogene and metabolic transcription factor known to induce glutamine addiction, as well as silencing of GLS, considerably reduced HCV infection. Conclusions: Altogether, these data suggest that HCV reprograms the hepatocyte metabolism and establishes glutamine dependence. This HCV-induced metabolic reprogramming is similar to that commonly found in many types of tumor cells. Because these changes seem to be required for viral replication, we are currently investigating their roles in the various steps of the viral life cycle, and their impact on the pathological features associated with chronic hepatitis C.

The effects of growth medium and partial shade on early growth of milkweed (Calotropis procera L.) under drought stress

The use of growth medium is often recommended milkweed seedlings to grow and develop after emergence, and it is affected by growth medium and local habitat conditions. The effects of growth medium and partial shade on early growth of milkweed under drought stress (Calotropis procera L.) were studied in a field experiment. A split-split plot experimental design with three replications was carried out in the nursery. The main treatment plot was divided into two levels of shade; (no shading and partial shading). Sub treatment plot1 included growth medium at four levels (G1 = clay [suitable for milkweed growth], G2 = clay + sand, G3 = clay + perlite, G4 = clay + perlite + sand) and sub treatment plot2 included drought (irrigation intervals) at six levels (D1 = 2 [control], D2 = 4, D3 = 6, D4 = 8, D5 = 10, and D6 = 12 days per for three month). The results showed that drought stress significantly decreased emergence percentage, shoot length, shoot dry weight (SDW1), root dry weight (RDW), seedling dry weight (SDW2) and vigor index (VI). The use of growth medium increased all seedling characteristics. The G3 (clay + perlite) growth medium showed the highest performance, especially in terms of emergence percentage and seedling dry weight. Partial shade improved shoot length, shoot dry weight, and vigor index. Our results showed that the best treatment for high-vigor milkweed seedlings under drought stress was G3 (clay + perlite) growth medium and partial shade.

Abstract 1229: Fibroblast growth factor signaling in ovarian cancer

Abstract Epithelial ovarian cancer (OC) is a rapidly progressive, highly lethal disease. It is the fifth leading cause of cancer-related death among women. Over the last decades, there has been only modest improvement in the treatment of OC. Nevertheless, recent clinical trials with inhibitors of transmembrane receptor tyrosine kinases such as fibroblast growth factor (FGF) receptors (FGFRs) yielded some promising results in OC. FGFRs are involved in malignant transformation, angiogenesis and chemoresistance. Inhibition of FGFR-dependent signaling can overcome resistance to standard therapies. Thus, FGFRs represent potential targets for cancer therapy. Unfortunately, however, the biological and therapeutic relevance of the FGF-FGFR system has not been elucidated yet in OC. The aim of this study was to characterize the FGF-FGFR system, its influence on malignancy-related cell properties and its potential role as a druggable signaling pathway in OC cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that expression of FGFs and FGFRs, leading to autocrine signaling loops with a strong mitogenic potential, is a common event in OC. Immunoblotting proved that the FGF signaling system is functionally active in these cells. Conditioned growth medium was able to induce phosphorylation of ERK1/2. Moreover, recombinant FGF-2 significantly elevated the growth rate in approximately 50% of the OC cell lines as demonstrated by formazan dye assay, whereas FGF-1 markedly induced migration in 66% of the cell lines. The dependency of OC cells on FGFR signaling was further evaluated by growth inhibition assays using the two small molecular FGFR inhibitors PD173074 and dovitinib (TKI-258, CHIR-258). A-2780 cells were found to be extremely sensitive to FGFR inhibition, while HEY and SKOV-3 cells showed moderate sensitivity. In contrast, OVCAR-3 cells were not inhibited by these compounds. Together, our data suggest that FGFR inhibitors reveal anticancer activity in a significant proportion of OC cells even as single drugs. Thus, combination of conventional cytotoxic intervention with blockade of the FGF-FGFR system might represent a promising future strategy for the treatment of OC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1229. doi:1538-7445.AM2012-1229

Abstract 5342: FC-IBC-02: A new in vitro-in vivo model of inflammatory breast cancer (IBC)

Abstract Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast can[[Unsupported Character - Codename ­]]cer and it is associated with a poor prognosis in spite of appropriate multidisciplinary treatments. The disease is characterized by peculiar molecular and clinical features and usually affects younger patients, mostly under the age of 50 years at diagnosis. IBC has shown the capacity to spread early, primarily through lymphatic channels and secondarily through blood vessels causing the inflammatory signs and development of early metastasis. The high incidence of metastatic disease at presentation, the persistence of residual disease after induction chemotherapy associated with significant risk of disease recurrence immediately surgery strongly support our hypothesis that these patients develop micro metastatic disease early in the disease course (Cristofanilli et al., Cancer 2007;110:1436-44). Moreover, the lower survival rate of IBC patients may be due to the early activation of metastatic process associated with a peculiar phenotype of cancer stem cells (CSCs) (Cristofanilli et al., Oncologist 2003;8:141-8). Although IBC research has been ongoing for over 15 years, very few molecular alterations have been associated specifically with IBC and few preclinical models are currently available to evaluate the peculiar biology of IBC. The majority of IBC studies have been performed using the cell lines SUM149 and SUM 190 which were developed from the primary tumor and, KPL-4 isolated from the pleural effusion of IBC patients. Although both the SUM149 and KPL-4 injected into immuno- compromised mice form primary tumors, there are currently only two in vivo xenograft models of IBC, the Mary-X and the WIBC-9 models that recapitulate the tumor emboli that are the signature of IBC in humans. We have developed another IBC model, FC-IBC-02, derivate from the pleural effusion of a 49 years old IBC triple negative patient. Tumor cells from the pleural effusion were ER(-) Pgr(-) and Her2(-) and strongly positive for tetraspanin CD151 and E-cadherin. Cells from the pleural effusion of this patient were growth under non-adherent conditions in serum-free mammary epithelial growth medium (MEGM). These tumor spheroids have Aldhehyde Dehydrogenase 1 (ALDH-1) activity and characteristics of cancer stem cells (CSC) and they rapidly developed tumors when they were injected in the mammary fat pad of SCID mice. These tumors were poorly differentiated carcinomas ER-negative PgR-negative Her2-negative and the mice developed micro metastasis in the lungs. In contrast, cells isolated under adherent conditions were unable to produce tumor in SCID mice. In summary, our studies demonstrated that IBC is a disease enriched for cells with a stem cell phenotype and these cells are highly tumorigenic. IBC may represent an ideal model to evaluate stem cell targeting therapeutic modalities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5342. doi:1538-7445.AM2012-5342

Structural and Functional Characterization of Monomeric EphrinA1 Binding Site to EphA2 Receptor

The EphA2 receptor is overexpressed in glioblastoma multiforme and has been to shown to contribute to cell transformation, tumor initiation, progression, and maintenance. EphrinA1 (eA1) is a preferred ligand for the receptor. Treatment with monomeric eA1, the form of eA1 found in the extracellular environment, causes receptor phosphorylation, internalization, and down-regulation with subsequent anti-tumor effects. Here, we investigated the structure-function relationship of a monomeric eA1 focusing on its G-H loop ((108)FQRFTPFTLGKEFKE(123)G), a highly conserved region among eAs that mediates binding to their receptors. Alanine substitution mutants of the G-H loop amino acids were transfected into U-251 MG glioblastoma multiforme cells, and functional activity of each mutant in conditioned media was assessed by EphA2 down-regulation, ERK and AKT activation and cellular response assays. Alanine substitutions at positions Pro-113 Thr-115, Gly-117, Glu-122, and also Gln-109 enhanced the EphA2 receptor down-regulation and decreased p-ERK and p-AKT. Substitution mutants of eA1 at positions Phe-108, Arg-110, Phe-111, Thr-112, Phe-114, Leu-116, Lys-118, Glu-119, and Phe-120 had a deleterious effect on EphA2 down-regulation when compared with eA1-WT. Mutants at positions Phe-108, Lys-18, Lys-121, Gly-123 retained similar properties to eA1-WT. Recombinant eA1-R110A, -T115A, -G117A, and -F120A have been found to exhibit the same characteristics as the ligands contained in the conditioned media mainly due to the differences in their binding to the receptor. Thus, we have identified variants of eA1 that possess either superagonistic or antagonistic properties. These new findings will be important in the understanding of the receptor/ligand interactions and in further design of anti-cancer therapies targeting the eA/EphA system.

Ethanol production by Zymomonas mobilis PTCC 1718 using low cost substrates

We report our attempts to increase ethanol production from Zymomonas mobilis PTCC 1718, by optimization of the components of growth medium and culture conditions. First, low cost substrates, carbon and nitrogen sources, temperature and initial pH were optimized by one-factor-at-a-time method. Then, nutritional improvement was performed using Taguchi method. Amongst the different selected sources, sucrose and wheat bran have significant effects on ethanol production. The optimum temperature, inoculum-size and pH were 50°C, 12% (v/v) and 5.5, respectively. The optimal concentrations of nutritional components for improved ethanol production were 2.5 g/L sucrose, 1.5 g/L whey, 0.25 g/L wheat bran extract, 0.5 g/L yeast extract, 0.25 g/L peptone, 0.7 g/L KH2PO4 , 0.1 g/L (NH4)2SO4 and 0.05 g/L MgSO4. Ethanol production, which was 14.49 g/L (w/v) in the basal medium, increased to 41.67 g/L (w/v) and 86.26 g/L (v/v); about 2.8 and 5.9-fold higher than that in the basal medium, after optimization with one-factor-at-a-time and Taguchi method, respectively. Bioreactor fermentation was carried out in a 15 L stirred fermentor under optimal condition. Maximum ethanol production in 15 L fermentor indicated 91.22 g/L (w/v), about 6.3 fold higher than the basal medium. Key words: Ethanol, medium components, Zymomonas mobilis PTCC 1718, optimization, Taguchi method.

Super-Resolution Study of the Dynamics and Spatial Distribution of Ribosomes in Live E. Coli Cells

Little is known about the spatial organization and dynamics of the translational machinery in bacteria. We have examined the distribution and diffusion of ribosomes in live E. coli cells by localizing and tracking single ribosomes labeled by an S2-eYFP construct expressed from the chromosome. Fast reversible photobleaching of eYFP was used to image single ribosome molecules and obtain the time-averaged spatial distribution of ribosomes with ∼30-nm resolution. In medium growth conditions (doubling time 62 minutes) the ribosomes are highly segregated from the nucleoid. In DNA rich regions the concentrations of ribosomes is less than 20% of that in the ribosome-rich regions in the end caps and space between the two nucleoid lobes. This is in reasonable agreement with a Monte Carlo model using realistic parameters for the number of ribosomes and the amount of plectonemic DNA. We have studied dynamics of single ribosomes by using time lapse imaging. The mean diffusion constant is Dribo = 0.04 μm2/s. Halting of transcription with Rifampicin resulted in about 10 fold increase in the diffusion constant and a homogeneous distribution of labels throughout the cytoplasm. The drastic change in dynamics can be explained in part by conversion of polysomes to monomeric ribosomes and 30S subunits in the absence of new mRNA synthesis. Expansion of the nucleoid after rifampicin treatment might be responsible for eliminating ribosome/DNA segregation and may further enhance diffusion. Halting of translation by Chloramphenicol treatment increased the ribosome-DNA segregation as the DNA compacted. The diffusion constant remained the same. Studies of the spatial distribution of different size fluorescent proteins in the presence and absence of drugs suggest that the degree of segregation from DNA is largely size dependent.

Temporal changes in species interactions in simple aquatic bacterial communities

BackgroundOrganisms modify their environment and in doing so change the quantity and possibly the quality of available resources. Due to the two-way relationship between organisms and their resource environment, and the complexity it brings to biological communities, measuring species interactions reliably in any biological system is a challenging task. As the resource environment changes, the intensity and even the sign of interactions may vary in time. We used Serratia marcescens and Novosphingobium capsulatum bacteria to study how the interaction between resource environment and organisms influence the growth of the bacterial species during circa 200 generations. We used a sterile-filtering method to measure how changes in resource environment are reflected in growth rates of the two species.ResultsChanges in the resource environment caused complex time and species composition-dependent effects on bacterial growth performance. Variation in the quality of the growth medium indicated existence of temporally fluctuating within-species facilitation and inhibition, and between-species asymmetric facilitation.ConclusionsThe interactions between the community members could not be fully predicted based only on the knowledge of the growth performance of each member in isolation. Growth dynamics in sterile-filtered samples of the conditioned growth medium can reveal both biologically meaningful changes in resource availability and temporally changing facilitative resource-mediated interactions between study species. This is the first study we are aware of where the filter-sterilization – growth assay method is applied to study the effect of long-term changes in the environment on species interactions.

Knockdown of RAGE expression inhibits colorectal cancer cell invasion and suppresses angiogenesis in vitro and in vivo

The receptor for advanced glycation end-products (RAGE) is a transmembrane receptor in cells, and the interaction of RAGE with ligands results in pro-inflammatory gene activation. Aberrant RAGE activation was reported to promote the pathogenesis of colorectal cancer. This study aimed to investigate the effects of RAGE on the regulation of cell viability, invasion, and angiogenesis, as well as the underlying molecular mechanisms regulating these interactions in colorectal cancer cells. The RAGE mRNA and protein were evaluated in five colorectal cancer cell lines and in 45 cases of colorectal cancer tissue specimens (using immuohistochemistry). RAGE expression was then knockdown using RAGE shRNA for assessing cell viability and invasion assays as well as for tube formation and CAM assays in human umbilical vein endothelial cells and chick embryos, respectively. RAGE was highly expressed in colorectal cancer tissues, and was associated with increased microvessel density. Two of the four RAGE shRNA constructs were able to significantly knockdown RAGE expression in SW480 cells. RAGE knockdown inhibited invasion capacity of SW480 cells, but did not significantly affect cell viability. Furthermore, the conditioned growth medium from stable RAGE shRNA-transfected cells suppressed tube formation of human umbilical vein endothelial cells and angiogenesis of chicken embryos. Knockdown of RAGE inhibited expression of VEGF and SP1 protein in colorectal cancer cells. In summary, these data suggest that silence of RAGE expression could effectively inhibit colorectal cancer angiogenesis in vitro and in vivo.

Isolation and characterization of a Pichia anomala strain: a promising candidate for bioethanol production

A yeast strain designated as Y-1 was isolated and characterized from wine yeast ("Jiuqu"). Based on the morphological and biochemical results, along with the rDNA internal transcribed spacer region (ITS), Y-1 was identified to be a Pichia anomala strain. Y-1 is an ethanol-tolerant strain, enduring ethanol concentrations of up to 14 %. Y-1 growth medium conditions were optimized, results showing good growth in medium with pH ranges from 3.5-6.5, temperature ranges from 25-30 °C, and inoculums range of 8 %-12 %, while optimum growth conditions were reached at a temperature of 30 °C, pH 5.0, and inoculums of 10 %. Furthermore, when the alkaline hydrolyzed Shatian pummelo peel solutions were inoculated with 10 % Y-1 and fermented at 30 °C for 6 d, 4.7 % pure ethanol (w/w) was produced, as evidenced by gas chromatography analysis. Our present study shows potential for the Y-1 strain to be a promising candidate for bioethanol production.

Factors of Osteogenesis Influencing Various Human Stem Cells on Third-Generation Gelatin/β-Tricalcium Phosphate Scaffold Material

Human bone marrow-derived stem cells (hBMSCs) and adipose-derived stem cells (hASCs) have been used to regenerate bone. Both sources are claimed to have comparable osteogenic potential, but few comparative studies are available. Third-generation biomaterials have been developed to reduce steps in regenerating tissues. For osteogenesis gelatin/β-tricalcium phosphate (β-TCP) scaffolds with incorporated controlled-release bone morphogenetic protein-2 (BMP-2) as third-generation biomaterials were recently developed. So far, few studies on protein-induced osteogenesis versus chemical-induced osteogenesis have been performed. This study evaluates the osteogenic potential of hBMSCs versus hASCs derived on gelatin/β-TCP scaffolds in vitro under four different conditions. Gelatin/β-TCP scaffolds with and without incorporated controlled-release BMP-2 were seeded with hBMSCs or hASCs under oscillating fluid conditions in osteogenic (OS) medium or growth medium (GM). All were evaluated radiologically (computed tomography [CT] scan), histologically, biomechanically, and for gene expression at 1, 2, 4, and 6 weeks. The highest radiological densities were seen in specimens at 6 weeks with controlled-release BMP-2, close to native bone. HBMSCs, hASCs, OS, and GM conditions resulted in similar bone formation with gelatin/β-TCP scaffolds and incorporated controlled-release BMP-2. This was confirmed histologically by Toluidine Blue and van Kossa staining and biomechanically. Gene expression studies of these specimens showed the presence of preosteoblasts, transitory osteoblasts, and secretory osteoblasts. Specimens comprised of gelatin/β-TCP scaffolds without incorporated controlled release BMP-2 in OS medium showed lesser bone formation. hASCs and hBMSCs have similar osteogenic potential. hASCs are an attractive alternative to hBMSCs for bone regeneration using third-generation gelatin/β-TCP scaffolds with incorporated controlled-release BMP-2.

Screening and Evaluation of Polyhydroxybutyrate-Producing Strains from Indigenous Isolate Cupriavidus taiwanensis Strains

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 NH4Cl, 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.

Influence of process parameters on the production of detergent compatible alkaline protease by a newly isolated Bacillus sp. Y.

Detergent compatible alkaline protease production by a newly isolated Bacillus sp.Y from laundry soil was studied under shake flask conditions in growth medium comprised (g/L): peptone, 1; NaCl, 5; skim milk, 100; Na_2CO_3, 4. Different environmental process parameters such as fermentation period, initial pH, incubation temperature, and nutritional parameters such as supplementation of different starch sources and nitrogen sources were standardized for the maximum yield of alkaline protease. Rice flour showed an almost 2.2-fold increase in protease activity followed by wheat flour, which showed a 1.85-fold increase over the basal media. However, supplementation of potato starch, corn starch, and ragi exerted an inhibitory effect on alkaline protease production. Protease production by Bacillus sp. Y was suppressed up to 80% in the presence of most of the organic and inorganic nitrogen sources tested in comparison to rice flour:peptone supplementation. Hence, based on the optimization studies, we achieved a yield of 88 PU/mL (2.2-fold increase) with the Bacillus sp. Y when cultivated for 72 h at pH 7.5, 37 °C in a medium containing: rice flour (1%, w/v), NaCl (0.5%, w/v), and skim milk (10%, v/v) with Na_2CO_3 (0.4%, w/v).

Isolation and characterization of a Pichia anomala strain: a promising candidate for bioethanol production

A yeast strain designated as Y-1 was isolated and characterized from wine yeast (“Jiuqu”). Based on the morphological and biochemical results, along with the rDNA internal transcribed spacer region (ITS), Y-1 was identified to be a Pichia anomala strain. Y-1 is an ethanol-tolerant strain, enduring ethanol concentrations of up to 14 %. Y-1 growth medium conditions were optimized, results showing good growth in medium with pH ranges from 3.5–6.5, temperature ranges from 25–30 °C, and inoculums range of 8 %-12 %, while optimum growth conditions were reached at a temperature of 30 °C, pH 5.0, and inoculums of 10 %. Furthermore, when the alkaline hydrolyzed Shatian pummelo peel solutions were inoculated with 10 % Y-1 and fermented at 30 °C for 6 d, 4.7 % pure ethanol (w/w) was produced, as evidenced by gas chromatography analysis. Our present study shows potential for the Y-1 strain to be a promising candidate for bioethanol production.

The effects of the composition of growth medium and fermentation conditions on the production of lipase by R. delemar

Lipases (triacylglycerol hydrolases) are hydrolytic enzymes that can catalyze the hydrolysis of the ester bond of long-chain acylglycerols at the oil-water interface. The present study investigated the effects of inducers, surface-active materials, activators, and inhibitors in the fermentation medium on lipase activity in Rhizopus delemar. In the presence of certain commercial oils and tributyrin as an inducer, lipase activity decreased in the order of sunflower oil > soybean oil > hazelnut oil > corn oil > tributyrin > olive oil. In addition, the effects of the stirring and aeration rates on lipase activity were investigated. To investigate the effect of surface-active materials on lipase activity 2 different surface-active materials, Tween-80 and rhamnolipid (biosurfactant), were used as comparatives with tributyrin. Maximum lipase activity was observed in the fermentation medium containing Tween-80 as both an inducer and surface-active material. The combined effects of molasses and sucrose on lipase production by R. delemar were also investigated. Lipase activity in the presence of activators decreased in the order of NaCl > KCl > CaCl_2 > MgCl_2 > gum arabic > EDTA, and maximum lipase activity of 964.55 U/L was obtained. The presence of heavy metal ions in the fermentation media severely inhibited lipase activity. Lipase activity in the presence of heavy metal ions decreased in the order of Fe^{2+} > Mn^{2+}> Co^{2+}> Ni^{2+}.