Basal Salts Research Articles

Development of a strategy induction AOX1 promoter methylotrophic yeast Komagataella phaffii

Currently, there is a significant increase in interest in the industrial production of enzyme preparations (and other recombinant proteins) using various microorganisms, including methylotrophic yeasts such as Komagataella phaffii. At the same time, the most significant productivity of the target proteins is achieved by methanol induction of heterologous genes cloned under the control of the AOX1 promoter. Thus, the efficiency of biosynthesis is largely determined by the metabolism of methanol. In this connection, the aim of the work is to develop an optimal strategy for methanol induction of the AOX1 promoter of Komagataella phaffii. The object of the study is the culture of the recombinant phospholipase A2 producing strain Komagataella phaffii. The studies were carried out in a laboratory fermenter Infors Minifors (Switzerland) on a liquid nutrient medium BSM (Basal Salt Medium) We used the generally accepted methods of studying the characteristics of metabolic activity, including the calculation of specific characteristics and productivity of the strain. The result of the study is the determination of the specific rate of consumption of methanol used as a carbon source, which was 19.2±1.8 mg/g*h. Also, the specific growth rate of Komagataella phaffii was determined and amounted to 0.24 h-1.Based on the data obtained during the research, a strategy for the induction of the AOX1 promoter in the cultivation of the methylotrophic yeast Komagataella phaffii was developed by maintaining the methanol concentration in the range of 0.6 to 2% based on the concentration of dissolved oxygen in the medium. The developed strategy of induction of the AOX1 promoter made it possible to obtain at least 1.87 g / l of recombinant protein (phospholipase A2) during cultivation of Komagataella phaffii for 96 h, which is 3.7 times higher than the known results.

In vitro mutagenesis of pruacan (Pimpinella pruatjan Molk): effect of chemical mutagen EMS and lethal dose determination

Pruacan is a rare medicinal plant endemic to the Dieng Plateau of the Central Java, Indonesia which has properties as an aphrodisiac. Increasing genetic variability ofthis plant in order to support the breeding programs and increase the productivity and active ingredients of this plant is important. This study is aimed to evaluate the effect of chemical mutagen EMS on the growth and development of in vitro culture of pruacan and lethal dose determination. Embryogenic callus were soaked in 0, 0.1, 0.3, 0.5 and 0.7% EMS for 2 hours and then cultured on regeneration medium containing DKW basal salt medium + IBA 5 mg/l. EMS suppressed the growth and development of pruacan somatic embryos culture. The lowest explant fresh weight, percentage of explants forming plantlets and number of plantlets per explant were obtained from 0.7 % EMS (2.38 g, 91.67 % and 6.22, respectively). Lethal dose 50 was obtained from EMS 0.61, 0.645 and 0.53% based on the explants fresh weight, the percentage of explants forming plantlets. And the number of plantlets per explant, respectively. EMS 0.53% can then be applied to induce mutation in the in vitro culture of pruacan.

Methyl tertiary butyl ether biodegradation by the bacterial consortium isolated from petrochemical wastewater and contaminated soils of Imam Khomeini Port Petrochemical Company (Iran)

Methyl tertiary butyl ether (MTBE) is a high soluble oxygenated supplement to fuel that may rapidly contaminate water and soil. Low biodegradability of this compound in the environment has led to more extensive efforts for finding powerful MTBE degrading microbial populations to be used in biological treatment of polluted waters and soils. The present study aimed to isolate MTBE degrading bacteria from petrochemical wastewater and soil of Mahsahar MTBE production site (Iran), and increasing their biodegradation potential in a bacterial consortium. Enrichment of degrading bacteria was done in basal salt media (BSM) containing 50–400 mg/l MTBE. The MTBE degradation pattern was assessed via gas chromatography (GC). The isolates were identified molecularly based on the sequence of 16S rRNA gene. The growth and degradation potential of bacterial consortium was optimized in different pH and temperatures. The best MTBE degrading bacteria were identified as Pseudomonas putida, Pseudomonas aeruginosa, and Enterobacter oryzendophyticus. The first order kinetics for MTBE degradation showed that the bacterial consortium was able to degrade 99.93% of MTBE during 15 days that was 6.08% higher than the most putative monoculture bacterium. The highest MTBE degradation by bacterial consortium was obtained at 30 °C and pH value of 7. The isolated bacterial consortium that involved novel MTBE degrading bacterial strains, decomposed high amounts of MTBE during 15 days of incubation in different temperatures and pH values without addition of co-catalytic substrate is proposed for cost effective removal of this pollutant from the environment.

English

This study aims at characterizing new chicken feather-degrading bacteria isolated from soils of keratinic waste collected from four dumping sites of Ngaoundere. Fifteen proteolytic bacteria were isolated with three (D1, D2 and F1) showing the capacity to degrade feather in Basal Salt Media. Highest degradation rate (71.11 ± 3.05%) of feather was obtained from isolate D2 from Municipal slaughterhouse. The partial characterization of the strain D2 based on morphological and biochemical tests revealed that it is a Bacillus sp. The keratinolytic activity was positively correlated to the bacterial growth with the highest value (13.76 U.ml-1) obtained at the stationary phase after 120 h of incubation. The optimal conditions for the keratinolytic activity of this isolate were determined to be pH 8.0 and temperature of 45°C. Thus, the Bacillus strain (D2) isolated is a promising strain for the management and valorization of feather wastes.   Key words: Chicken feather-degrading bacteria, keratinolytic enzyme activity, characterization, Ngaoundere, Cameroon.

Factors Influencing Somatic Embryo Maturation in Sugi (Japanese Cedar, Cryptomeria japonica (Thunb. ex L.f.) D. Don).

This paper presents the results of several experiments identifying basal salts (BS) contained in maturation medium, polyethylene glycol (PEG) concentration, abscisic acid (ABA) concentration, additional supplementation with potassium chloride (KCl), amino acid (AA) concentration, and proliferation culture medium (PCM) as the main culture factors affecting somatic embryo maturation in sugi (Japanese cedar, Cryptomeria japonica, Cupressaceae). Highly efficient embryo maturation was achieved when embryogenic cell lines (ECLs) were cultured on media supplemented with a combination of PEG, ABA, and AAs. More than 1000 embryos per gram of fresh weight (FW) can be produced on EM maturation medium supplemented with 175 g L−1 PEG, 100 µM ABA, 2 g L−1 glutamine, 1 g L−1 asparagine, and 0.5 g L−1 arginine.

Purification of Wickerhamomyces anomalus Keratinase and Its Prospective Application in Poultry Feed Industries

Animal wastes emanating from cow horns, hooves and feathers are keratinous in nature which can only be degraded by keratinolytic microorganisms. Consequently, the pollution resulting from the accumulation of these wastes in response to growing livestock demand is posing a significant threat to human health and the environment. This study was carried out using cow hooves as the substrate for the production of keratinase from fungal identified as Wickerhamomyces anomalus 9 (18S rDNA gene sequencing) was isolated from soil rich hooves using basal salt agar medium and potato dextrose agar. The keratinase of the isolate was assessed using skim milk agar and the enzyme was produced by solid-state fermentation. The crude enzyme was purified using ammonium sulphate precipitation, ion exchange and gel-filtration chromatography. The specific activity of the enzyme was 0.29 U/mg with a yield of 45% and a 7.25 purification fold. The optimal pH and temperature of the enzyme were 8.0 and 60 oC respectively. The enzyme was observed to be thermo-stable at 50oC between for 30 minutes. The kinetics revealed that the Vmax was 0.384U/min with Km 86.95mg/ml. The native molecular weight of the enzyme was found to be 34KDa. There were significant differences at 95% confidence with poultry feed treated with Wickerhamomyces anomalus keratinase in moisture, ash content, crude fibre, crude fat, nitrogen content, crude protein and carbohydrate compare to the untreated feed. These results suggest an environment-friendly approach for biodegradation of cow hooves wastes for the production of keratinases, animal waste management as well as a promising tool for chicken feed additives. Keywords: Biodegradation, Chromatography, Cow hooves, Keratinase, Pollution

Mass propagation of Juniperus procera Hoechst. Ex Endl. From seedling and screening of bioactive compounds in shoot and callus extract

BackgroundJuniperus procera Hoechst. ex Endl. is a medicinal tree in Saudi Arabia, primarily in the Enemas region, but it is locally threatened due to die-back disease and difficulties regarding seed reproduction (seed dormancy and underdeveloped embryonic anatomy, and germination rate < 40%). Hence, the alternative methods for reproduction of Juniperus procera are really needed for conservation and getting mass propagation for pharmaceutical uses.ResultsIn this manuscript, we articulated the successful in vitro shoot multiplication and callus induction of J. procera by using young seedling as explants and detected an important antibacterial and antitumor product. Explants were grown on different types of media with the supplement of different combinations of Plant Growth Regulators (PGRs) at different concentrations. The best media for shoot multiplication was Woody Plant Media (WPM) supplemented with PGRs (0.5 μM of IAA and 0.5 μM BAP or 0.5 μM IBA and 0.5 μM BAP). Whereas for callus induction and formation Woody Plant Media (WPM) with the addition of PGRs (0.5 μM 2,4-D and 0.5 μM BAP) was better than the Chu Basal Salt Mixture (N6), Gamborg’s B-5 Basal Medium (B5), and Murashige and Skoog media. The possibility of multiplication of J. procera in vitro creates significant advantages to overcome the difficulties of seeds dormancy for the reproduction of plants, conservation of trees, and getting mass propagation material for pharmaceutical studies. The shoot and callus extract of J. procera was detected using gas chromatography-mass spectrometry analysis and revealed more than 20 compounds related to secondary metabolites, which contained antibacterial and antitumor agents, such as ferruginol, Retinol, and Quinolone as well as confirmed by Direct Analysis in Real Time, Time of Flight Mass Spectrometry (DART-ToF-MS). Podophyllotoxin (PTOX) was detected in callus material by HPLC with sigma standard and confirmed by DART-ToF-MS and UV spectra.ConclusionWe successfully conducted in vitro shoot multiplication and callus induction from J. procera seedlings using WPM and a different combination of PGRs and, detected an important antibacterial and antitumor product such as ferruginol and podophyllotoxin. According to our findings, J. procera has become a new natural source of novel bioactive compounds.

Biodegradation of alicyclic amines by a newly isolated hypersaline tolerant strain Paenarthrobacter sp. TYUT067.

Alicyclic amines are widely used in several types of industries, and considerable attention has been devoted to possible environmental pollution by alicyclic amines in hypersaline industrial wastewater. In this study, a new hypersaline tolerant bacterial TYUT067 capable of growing in liquid basal salt medium with cyclohexylamine (CHAM) as the sole carbon source and energy source, was isolated from soil, and discovered with highly efficient CHAM degrading ability. The strain TYUT067 was identified as Paenarthrobacter sp. based on 16S rDNA gene sequence, and its degradation characteristic was examined. The results revealed that the isolated TYUT067 could grow well under pH range of 6.5-10.0, temperature from 20 °C to 30 °C. For degradation of 60 mM of cyclohexylamine, 100% degradation could be finished within 120 h. The TYUT067 could degrade 10 mM CHAM under hypersaline conditions (3-5% NaCl, w/v), revealed the hypersaline tolerance of TYUT067. Different type of amines was also tested with TYUT067, the degradations of >90% were achieved toward several alicyclic amines. The current results suggested that TYUT067 was a potential species could be efficiently used for the degradation of alicyclic amines and might be applicable to a hypersaline wastewater treatment system for the removal of alicyclic amines.

Micropropagation and clonal fidelity assessment of acclimatized plantlets of Crotalaria longipes Wight &amp; Arn. using ISSR markers

An efficient protocol for micropropagation of Crotalaria longipes Wight & Arn., an endemic and endangered Indian legume, was developed. Nodal explants cultured on Murashige and Skoog (MS) supplemented with Thidiazuron (TDZ) 1.0 mg L−1 and NAA 0.5 mg L−1 showed maximum shoot induction. The multiplied shoots were subsequently sub-cultured every 3 weeks on the same medium. The separated microshoots cultured on half-strength MS basal salts supplemented with 0.5 mg L−1 indole-3-butyric acid (IBA) showed maximum root induction. After 4 weeks of culture, the rooted shoots (> 6 cm) were planted in pots containing coco peat and garden soil (1:1) covered with plastic domes and maintained at 25 oC for 2 weeks. After 2 weeks, the potted plants were transferred to a glasshouse, maintained at 25 °C with a relative humidity of 80%. The acclimatized plants were then successfully established in soil with an 80% survival rate. Clonal fidelity assessment of acclimatized plantlets was confirmed using inter simple sequence repeat (ISSR). All the in vitro developed plants showed monomorphic banding pattern similar to the mother plant, thus ascertaining the true-to-type nature of the in vitro raised plants.

Promotion of polylactic acid biodegradation by a combined addition of PLA-degrading bacterium and nitrogen source under submerged and soil burial conditions

This article investigated an efficient process for accelerating polylactic acid (PLA) biodegradation. A combined use of bioaugmentation and biostimulation to faster PLA biodegradation under submerged and soil burial conditions was studied. Under submerged condition, PLA films in basal salt medium were inoculated with either Pseudomonas geniculata WS3 (PLA-degrading bacterium), manure extract, or wastewater sludge extract, with and without the addition of different nitrogen sources. The results revealed that soytone addition with the cultures of P. geniculata WS3, manure extract and wastewater sludge extract significantly increased PLA weight loss percentage and lactic acid content, indicating that soytone is an excellent nitrogen source for enhancing PLA biodegradation. These conditions were selected to evaluate PLA biodegradation under soil burial exposure. It was found that soytone with manure extract addition increased microbial activity and PLA biodegradation in soil. Moreover, the addition both soytone and P. geniculata WS3 speeded up biodegradation rate, resulting in almost 100 percent of PLA mass loss within 60 days in non-sterile soil burial. In conclusion, a combined use of P. geniculata WS3 with soytone is an efficient process for accelerating PLA biodegradation under both submerged and soil burial exposure conditions.

In vitro somatic embryogenesis of Texas ebony (Ebenopsis ebano [Berland.] Barneby &amp; J.W. Grimes)

Texas ebony (Ebenopsis ebano [Berland.] Barneby & J.W. Grimes) is a member of the Fabaceae that is native to Mexico. Its wood has multiple uses, and its natural propagation is limited and only by seed. The present investigation describes the first report of Texas ebony somatic embryogenesis with the objective of identifying medium, growth regulator components, and culture conditions. Immature cotyledons were cultured on semisolid and in liquid media containing Murashige and Skoog (MS) basal salts plus 0.0 to 9.06 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The most frequent generation of embryogenic callus occurred with cotyledons cultured in the dark in liquid medium containing 9.06 μM 2,4-D. For the maturation phase, the embryogenic calluses were subcultured on semisolid MS medium with 3.78 to 30.24 μM abscisic acid (ABA), 3% and 6% polyethylene glycol (PEG) 4000, or 3 to 5% sucrose. Sucrose at 4% and 5% induced the greatest total number of somatic embryos; however, the maturation frequency and embryo quality were both higher with 6% PEG. Histological analysis revealed the ontogeny, morphology, and development of somatic embryos. Finally, the mature somatic embryos were subcultured for germination in half-strength MS medium plus 6-benzylaminopurine (BAP; 2.22 μM and 4.44 μM), gibberellic acid (GA3; 1.45 μM and 2.89 μM), or BAP (2.22 μM) and GA3 (1.45 μM). Embryo germination was superior with 2.22 μM BAP; however, radicle generation was poor in all of the treatments. This study demonstrated that for each phase of somatic embryogenesis, specific combinations of culture medium components and incubation environment are required.

Gelatin expression from an engineered Saccharomyces cerevisiae CUP1 promoter in Pichia pastoris.

The methylotrophic yeast Pichia pastoris (reclassified as Komagataella phaffii) is a versatile protein expression system, yet many commonly used promoters have attributes undesirable for fermentation or its optimization. Hence, the copper-inducible CUP1 gene promoter from the related yeast Saccharomyces cerevisiae was used to express human gelatin. Multimerization of a potential copper response element in the CUP1 promoter, a S.cerevisiae Ace1p binding site, significantly increased gelatin expression. Expression was induced by copper in a dose-dependent fashion and was not dependent on cell density. Gelatin was additionally induced in standard copper-containing fermentation basal salts media. Removal of a S.cerevisiae heat shock factor (Hsf1p) binding site reduced copper-dependent gelatin induction suggesting that a similar protein may regulate this promoter in P.pastoris. This engineered copper inducible promoter expands the yeast recombinant protein production tool kit.

Nitrogen supplementation ameliorates product quality and quantity during high cell density bioreactor studies of Pichia pastoris: A case study with proteolysis prone streptokinase

Streptokinase is a well-established cost-effective therapeutic molecule for thrombo-embolic complications. In the current study, a tag-free variant of streptokinase with a native N-terminus (N-rSK) was developed using the Pichia expression system. A three-copy clone was screened that secreted 1062 mg/L of N-rSK in the complex medium at shake flask level. The biologically active (67,552.61 IU/mg) N-rSK recovered by anion exchange chromatography was predicted to contain 15.43% α-helices, 26.43% β-sheets. The fermentation run in a complex medium yielded a poor quality product due to excessive N-rSK degradation. Therefore, modified basal salt medium was also employed during fermentation operations to reduce the proteolytic processing of the recombinant product. The concomitant feeding of 1 g/L/h soya flour hydrolysate with methanol during the protein synthesis phase reduced the proteolysis and yielded 2.29 g/L of N-rSK. The fermentation medium was also supplemented with urea during growth and induction phases. The combined feeding approach of nitrogen-rich soya flour hydrolysate and urea during bioreactor operations showed significant improvement in protein stability and resulted in a 4-fold increase in N-rSK concentration to a level of 4.03 g/L over shake flask. Under optimized conditions, the volumetric productivity and specific product yield were 52.33 mg/L/h and 33.24 mg/g DCW, respectively.

Direct Shoot Organogenesis from Lycium chinense Miller Leaf Explants and Assessment of Genetic Stability Using ISSR Markers

An efficient in vitro direct shoot regeneration system has been described for Lycium chinense Miller using leaf explants. Influence of various parameters such as growth regulator concentration, explant type, effect of basal salt type, Murashige and Skoog (1962) medium (MS), Schenk and Hildebrandt (1972) medium (SH), Gamborg et al. (1968) medium (B5), and carbon sources (sucrose, maltose, and fructose) on the regenerating shoots has been studied. Micromorphological studies and genetic fidelity of regenerated shoots were assessed and compared with those of the donor plants. Among the different concentrations of plant growth regulator (PGRs) tested, MS supplemented with lower concentration of 6-benzylaminopurine (BAP) (0.5 mgL−1) and thidiazuron (TDZ) (0.5 mgL−1) increased the frequency of shoot. Comparatively, indole-3-butyric acid (IBA) was more effective in the regeneration and growth of the root system. A higher number of root formation (6.67 ± 1.25) was observed when the rooting medium comprised half-strength MS salts supplemented with 3% sucrose. The surviving plantlets were gradually transferred to the greenhouse and natural soil. More than 90% of the plantlets survived and matured within 85 days. Similarity in the band patterns produced by inter simple sequence repeat (ISSR) primers confirmed the genetic stability and uniformity between the regenerated and donor plants. The present optimized direct shoot regeneration system may be useful for mass propagation and improving the genetic traits in L. chinense.

STATISTICAL OPTIMIZATION OF LACCASE PRODUCTION BY ENTEROBACTER CLOACAE STRAIN T137 UTILIZING WHEAT BRAN AS SUBSTRATE IN SUBMERGED FERMENTATION (SmF) BY PLACKETT-BURMAN METHOD

The work aimed to produce laccase enzyme by using wheat bran as substrate by a statistical approach by Enterobacter cloacae strain T137. The study was conducted through the submerged fermentation process (SmF) by using the regression model of Plackett-Burman experiments. The significant factors influencing the laccase production was analyzed by ANOVA and Pareto method. Among the variables screened, inoculum size, trace element solution, magnesium sulfate, pH of the medium, agitation rate and incubation time found was significant when the basal salt medium was supplemented with wheat bran as the substrate. The aptness of the model was determined by the coefficient (R2) of the Plackett-Burman factorial model.

Valorization of biodiesel side stream waste glycerol for rhamnolipids production by Pseudomonas aeruginosa RS6.

Biodiesel side stream waste glycerol was identified as a cheap carbon source for rhamnolipids (RLs) production which at the same time could improve the management of waste. The present study aimed to produce RLs by using Pseudomonas aeruginosa RS6 utilizing waste glycerol as a substrate and to evaluate their physico-chemicals properties. Fermentation conditions such as temperature, initial medium pH, waste glycerol concentration, nitrogen sources and concentrations resulted in different compositions of the mono- and di-RLs produced. The maximum RLs production of 2.73g/L was obtained when P.aeruginosa RS6 was grown in a basal salt medium supplemented with 1% waste glycerol and 0.2M sodium nitrate at 35°C and pH 6.5. At optimal fermentation conditions, the emulsification index (E24) values of cooking oil, diesel oil, benzene, olive oil, petroleum, and kerosene were all above E24=50%. The surface tension reduction obtained from 72.13mN/m to 29.4-30.4mN/m was better than the surface activity of some chemical-based surfactants. The RLs produced possessed antimicrobial activities against Gram-negative and Gram-positive bacteria with values ranging from 37% to 77% of growth inhibition when 1mg/mL of RLs was used. Concentrations of RLs below 1500μg/mL did not induce phytotoxicity effects on the tested seeds (Vigna radiata) compared to the chemical-based- surfactant, SDS. Furthermore, RLs tested on zebrafish (Danio rerio) embryos only exhibited low acute toxicity with an LC50 value of 72.97μg/mL at 48h of exposure suggesting a green and eco-biochemical worthy of future applications to replace chemical-based surfactants.

Desarrollo in vitro de la orquídea epífita Erycina hyalinobulbon (Orchidaceae), endémica de México, para promover su conservación

Antecedentes y Objetivos: Las orquídeas en México están amenazadas principalmente por deforestación, cambios de uso de suelo, comercio ilegal, carencias en la legislación y política ambiental y por una falta de participación de las comunidades para la conservación de sus bosques. Erycina hyalinobulbon es una orquídea endémica epífita de ramilla con un ciclo de vida corto y grandes flores en relación a su tamaño, por lo que ha sido extraída de sus poblaciones silvestres. Los objetivos de este trabajo fueron evaluar el desarrollo in vitro de E. hyalinobulbon en medios de cultivo con suplementos orgánicos, comparar la sacarosa vs. N´Joy Stevia® como fuente de carbono en sus primeras etapas de desarrollo y evaluar el desarrollo de sus plántulas con medios enriquecidos con reguladores de crecimiento vegetal (PGR).Métodos: Para la siembra de semillas los medios de cultivo PhytamaxTM y MS, a 30% de sus sales basales, fueron utilizados con suplementos orgánicos (leche de coco, puré de piña y puré de plátano), utilizando el medio Phy como control. Para poder medir el efecto de la sacarosa vs. N´Joy Stevia®, se utilizaron estos dos tratamientos con el medio PhytamaxTM. Para evaluar el desarrollo de plántulas con PGR, se hicieron tres tratamientos: control de PhytamaxTM 100%, PhytamaxTM 30% con 1.166 ml/l de Maxi-grow y el medio Chiu.Resultados clave: El medio PhytamaxTM adicionado con plátano promovió la germinación en 9.3%, siendo una opción de bajo costo y fácil producción. A los noventa días de efectuada la siembra, N´Joy Stevia® como fuente de carbono promovió la germinación con 8%. El mejor desarrollo de las plántulas se obtuvo en el medio PhytamaxTM sin PGR.Conclusiones: Con este estudio se logró desarrollar un sistema de propagación in vitro de E. hyalinobulbon accesible para poder manejarla sustentablemente y favorecer su conservación.

DKW basal salts improve micropropagation and callogenesis compared with MS basal salts in multiple commercial cultivars ofCannabis sativa

Existing Cannabis sativa micropropagation protocols use a limited number of cultivars and the results are often not reproducible. Currently, Murashige and Skoog basal salt mixture (MS) + 0.5 μmol/L thidiazuron (TDZ) has been reported as the optimal medium for nodal micropropagation, yet our preliminary studies with this medium have resulted in abnormal morphology and high mortality rates in multiple cultivars. Following an initial screen of basal salt mixtures [MS, B5, BABI, and Driver–Kuniyaki–Walnut (DKW)], we determined that DKW produced the healthiest plants. In a second experiment, the multiplication rate and canopy area of explants grown on MS + 0.5 μmol/L TDZ and DKW + 0.5 μmol/L TDZ were compared using five drug-type cultivars. The combined multiplication average of explants grown on DKW + 0.5 μmol/L TDZ was 1.5× higher than explants grown on MS + 0.5 μmol/L TDZ. Similarly, the combined average of the canopy area was twice as large on DKW + 0.5 μmol/L TDZ. In the third experiment, callogenesis was compared using a concentration range of 2,4-dichlorophenoxyacetic acid (0–30 μmol/L) on both MS and DKW, and similarly, callus growth was superior on DKW. This study presents the largest comparison of basal salt compositions on the micropropagation of five commercially grown Cannabis cultivars to date.

Reductive Dechlorination of 1,2-dichloroethane to Ethylene by Anaerobic Enrichment Culture Containing Vulcanibacillus spp.

Bioremediation has been widely used for clean-up of 1,2-dichloroethane (DCA) at contaminated sites. Only a small number of specific anaerobes, halorespiring bacteria (HRB), have been reported to degrade DCA. The goals of this research were the screening and isolation of HRB with capable dechlorination of DCA. HRB were screened and isolated from 7 enrichment cultures (ES1-ES7), from DCA-contaminated soils, on bicarbonate-buffered basal salts medium containing 10 mM acetate and 250 µM DCA under anaerobic conditions and analyzed by gas chromatography. The results showed that the mixed cultures of ES3 and ES5 could reductively dechlorinate DCA to ethylene via a direct reductive dihaloelimination pathway. In particular, ES5 showed rapid transformation of 250 µM DCA to ethylene within 6 days at 30 °C. Specific microbial populations of Vulcanibacillus spp., elucidated by polymerase chain reaction-denaturing gradient gel electrophoresis, were found only in ES3 and ES5, which was related to reductive dihaloelimination activities on DCA. A 16S rRNA gene analysis of isolate es5d8 from mixed culture ES5 revealed 2 strains of Vulcanibacillus spp. (KKU-DCA1 and KKU-DCA2) concerned with dechlorinating DCA. These findings suggested that Vulcanibacillus spp. were HRB that have the potential for detoxifying DCA.

Medium optimization for high yield production of human serum albumin in Pichia pastoris and its efficient purification

ObjectiveTo improve the yield of recombinant human serum albumin (HSA) in Pichia pastoris by medium optimization and establish the related purification scheme. ResultsA simplified version of the generally used buffered glycerol complex medium (BMGY), which contained yeast extract, glycerol and potassium salts, was found to be applicable. By decreasing the salt concentration of basal salt medium (BSM) to half of the original formula further, we achieved a high yield of 17.47 g/L HSA in the supernatant within a 192 h induction, which is the highest rHSA yield ever reported as far as we know. Accompanied with a three-step purification procedure which recovered two thirds of the desired protein at high purity, our work lays a solid foundation for large-scale industrial production of HSA. ConclusionMedium optimization plays a significant role in improving the yield of desired protein, lowering the production cost and helping to explore the producing strain's character.