Rotary Shaker Research Articles

Bioremediation Efficiency of Saccharomyces cerevisiae on Cadmium and Lead from Groundwater Obtained from Mining Community

Water is a vital requirement for life and it is also an effective vehicle for the transmission of diseases if contaminated. Pollution caused by heavy metals is one of the major environmental problems that are imperative to be solved. Mining of solid minerals has been identified as an entry point of heavy metals into the environment consequently polluting various components of the environment such as soil and water. Bioremediation offers a promising means to reclaim such contaminated environment in an economical and eco friendly way. The focus of this study is to evaluate the bio sorption efficiency of cadmium and lead-resistant yeast from well water samples collected from Angwan Magiro, one of the lead-contaminated villages of Niger State, North Central Nigeria. Microbial enumeration of the water samples were carried out using pours plate technique, while physicochemical parameters were done by standard methods. Tolerance ability of the yeast isolates to the heavy metals was determined by cultivating on yeast broth supplemented with synthetic solutions of 1.50 mg/L cadmium concentration and 5.50 mg/L lead concentration. Based on the result of heavy metal tolerance assay, Saccharomyces cerevisiae was then selected to determine its efficiency in bio sorption of cadmium and lead in a rotary shaker incubated at an ambient temperature for a period of 28 days. Yeast cells were separated from solutions by centrifugation and the supernatants were analyzed for residual metals in solution using Atomic Absorption Spectrophotometer (AAS). Bio sorption experiment was carried out as function of solution pH. The results of this investigation reveal that Saccharomyces cerevisiae was efficient in the removal of lead with 99.54% and cadmium with 88.24% at pH 8.20. These findings suggest that Saccharomyces cerevisiae present in heavy metalcontaminated water could be an effective measure for remediation of the ecosystem.

Modified Over-the-Row Machine Harvesters to Improve Northern Highbush Blueberry Fresh Fruit Quality

Improved blueberry mechanical harvesting (MH) equipment that maintains fresh market quality are needed due to rising costs and decreasing availability of laborers for harvesting by hand. In 2017, a modified over-the-row (OTR) blueberry harvester with experimental catch surfaces and plates designed to reduce fruit bruising was evaluated. The catch surfaces were made of neoprene (soft catch surface; SCS) or canvas (hard catch surface; HCS) and compared to hand-picked fruit (control). Early- and early/mid-season ‘Duke’ and ‘Draper’, respectively, were evaluated in Oregon, while late-season ‘Elliott’ and ‘Aurora’ were evaluated in Washington. Harvested berries were run through commercial packing lines with fresh pack out recorded and bruise incidence or fresh fruit quality evaluated during various lengths of cold storage. The fresh pack out for ‘Duke’ and ‘Draper’ were 83.5% and 73.2%, respectively, and no difference was noted between SCS and HCS. ‘Duke’ fruit firmness was highest among MH berries with SCS, but firmness decreased in storage after one week. Firmness was highest among hand harvested ‘Draper’ followed by MH with SCS. For ‘Elliott’ and ‘Aurora’, fruit firmness was the same across harvesting methods. ‘Draper’ exhibited more bruising than ‘Duke’, but bruise ratings and the incidence of bruising at ≤10% and ≤20% were similar between hand and MH ‘Draper’ with SCS after 24 h of harvest. ‘Aurora’ berries had similar bruise ratings after 24 h between hand harvesting and MH with SCS, while ‘Elliott’ showed more bruise damage by MH with both SCS and HCS than hand harvested fruit. Although our studies showed slightly lower fresh market blueberry pack outs, loss of firmness, and increased bruise damage in fruit harvested by the experimental MH system compared to hand harvested fruit, higher quality was achieved using SCS compared to HCS. We demonstrated that improved fresh market quality in northern highbush blueberry is achievable by using modified OTR harvesters with SCS and fruit removal by either hand-held pneumatic shakers or rotary drum shakers.

Production of antioxidant exopolysaccharide from Pseudomonas aeruginosa utilizing heavy oil as a solo carbon source

Aim/Background: Pseudomonas aeruginosa is capable of utilizing heavy oil hydrocarbons as a sole carbon source. P. aeruginosa produce exopolysaccharide (EPS) in an inorganic medium in the presence of crude oil. Several environmental factors affect the majority of EPS production. Materials and Methods: Strain of P. aeruginosa were managed under various media (maintenance medium, inoculum, and basal media). Different heavy petroleum oil concentrations (5, 10, 20, and 30 ml/L) were used as solo carbon source to the basal medium. Various conditions of bacterial growth were monitored. The growth of cells was estimated by measuring the absorbance of the mixture of 1 ml of the basal medium diluted with 1 ml of distilled water at 600 nm spectrophotometrically. P. aeruginosa was grown aerobically in a production medium at 37°C and 150 rpm on a rotary shaker. The culture broth was centrifuged to separate the cells. The precipitated polysaccharide was separated by centrifugation and washed with ethanol, acetone, and ether, and then dried under reduced pressure oven at 45°C. The DPPH test was carried out as described by Burits and Bucar to monitor the free radical scavenging activities of the extracts. Results: The preferable culture conditions for EPS production were at 10 ml/L heavy oil, with 0.5 g/L NaNO3as best N sources at pH 6.0 after 5 days incubation. The net weight of purified EPS production was 0.5 g/L. Conclusion: The obtained polysaccharide showed antioxidant activity that possesses DPPH radical scavenging activity, with an EC50 =0.201.

Preliminary Studies on the Microbial Degradation of Plastic Waste Using <i>Aspergillus niger</i> and <i>Pseudomonas</i> sp.

The possibility of microbial degradation of plastic waste was investigated by isolating microorganisms present in dumpsite containing low-density polyethylene (LDP). Aspergillus niger (fungi) and Pseudomonas sp. (bacteria) were identified and subsequently used to biodegrade plastic waste. The medium was made up of 0.2 g of MgSO4, 1.0 g of KH2PO4, 1.0 g of K2HPO4, 1.0 g of NH4NO3, 0.02 g of CaCl2, 0.05 g of FeCl3 in 1000 ml water. 10 ml of the medium containing the bacteria and/or fungi was poured into test tubes and 0.1 g of the plastic sample (Pure water sachet) pre-treated with ethanol was introduced into the tubes. The pH of the medium was adjusted to 7.2, 5.4 and 6.0 for Pseudomonas sp., Aspergillus niger and the mixed culture respectively. Each experiment was carried out aerobically at room temperature and incubated on a rotary shaker at 120 rpm. The weight loss in each experiment was monitored at 10 days interval for 60 days. The total weight loss after 60 days was 7.2%, 12.4%, 15% for degradation with Pseudomonas sp., Aspergillus niger and the mixed culture respectively. From this study it can be inferred that Pseudomonas sp. and Aspergillus niger have the ability to degrade plastics. It can also be inferred that Aspergillus niger degraded plastics better than Pseudomonas sp. and there was synergy between the two microorganisms since the mixed culture gave a higher degradation.

Screening and Evaluation of Various Carbon Sources on the Ability of Trichoderma harzianum InaCC to Solubilize Insoluble Phosphate

ABSTRACT Our previous works revealed that Trichoderma harzianum InaCC (Indonesian Culture Collection) produced volatile organic compounds that inhibited the growth of Fusarium oxysporum. The objective of this study was to evaluate the ability of T. harzianum InaCC to solubilize insoluble phosphate and optimize the carbon sources condition. Screening was conducted to 10 isolates of T. harzianum on Pikovskaya’s broth in 100-mL conical flasks. The cultures were incubated at 30 ± 1°C for 7 days on a rotary shaker at 80 r/min. All T. harzianum isolates were observed for the solubility of insoluble tricalcium phosphate with the highest ability was performed by T. harzianum InaCC F88. Using this isolate, the optimization of insoluble phosphate solubilization was conducted on modifed Pivoskaya medium in 500-mL conical flasks in similar screening incubation condition with applying of different combination of phopshate sources (tricalcium phosphate and rock phosphate) and carbon sources (dextrose, lactose, and sucrose). When tricalcium phosphate was substituted by rock phosphate, less soluble phosphate was produced. Glucose was the best carbon source used for solubilization of both tricalcium phosphate and rock phosphate, then followed by lactose and sucrose. Acidification of medium was not the major mechanism of phosphate solubilization by T. harzianum. Based on the ability of high phosphate solubilization, T. harzianum InaCC F88 is the most potential strain as a plant growth promoting fungus among the 10 isolates of T. harzianum Ina CC. Key words: biocontrol, Pikovskaya medium, phosphate solubilization, Trichoderma harzianum

Dehalogenase enzyme activity of Bacillus sp. D1 isolated from pharmaceutical waste

Dichloromethane (DCM) is widely used in the pharmaceutical industry as an intermediate solvent and active reagent. Most of the pharmaceutical industry waste contains of DCM which is a toxic compounds in the environment. Microorganism plays an important role in maintaining environmental ecosystems by degrading organic pollutants. Dichloromethane degrading microorganisms are known have dehalogenase enzyme activity. Bacillus sp. D1 isolated from pharmaceutical industry waste was used in this study. This study aims to determine the activity of Bacillus sp. D1 dehalogenase enzyme in various incubation time and pH condition. This research is an experimental study that used a completely randomized design with three replications. Bacillus sp. D1 was cultured in the mineral salt medium (MSM) added by DCM in rotary shaker 130 rpm until 7 days incubation. Evaluation of enzyme activity was carried out at various incubation times and pH conditions using the Bergman method. The best enzyme activity was 0.285 U/mL reached at 48 hours of incubation. The highest dehalogenase enzyme activity was 0.913 U/mL at pH 9. The activity of dehalogenase enzyme from Bacillus sp. D1 optimum at alkaline condition.

Aktivitas Enzim Inulinase dan Laju Pertumbuhan Spesifik Isolat Bakteri IS-1 pada Medium Tepung Umbi Dahlia

Dahlia bulbs (Dahlia variabilis) contains carbohydrates in the form of inulin. Inulin is a form of fructose polymer from fructose monomers. Inulin hydrolysis produces a product in the form of fructose.Inulinase enzyme is an enzyme able to purely hydrolyze inulin. This study aims to determine the activity of inulinase enzymes and the specific growth rate of IS_1 bacterial isolates producing inulinase in medium of Dahlia tuber flour. Isolates of inulinase-producing bacteria were isolated directly from decay dahlia tubers and used as an inoculum in creating starter.The measurement of cell growth was carried out by inoculating a 20-hour starter on the production medium. Incubation is done for 24 hours using a rotary shaker with a speed of 120 rpm. Sampling was carried out every 4 hours at 0-hour (T0), 4 hours (T4), 8 hours (T8), 12 hours (T12), 16 hours (T16), 20 hours (T20) and 24 hours (T24) intervals with a sample of 5 mL measured for absorbance of λ 520 nm using spectrophotometer. Production medium culture in each sample was taken 1 mL and centrifuged at 3000 rpm for 10 minutes to obtain supernatant of crude enzyme. Measurement of inulinase enzyme activity was carried out using 3,5-dinitrosalicylic acid (DNS) method and spectrophotometric method at λ 570 nm. IS_1 bacterial isolate showed optimum growth at incubation time of 8 hours with OD of 0.647 and had a specific growth rate (µ) of 0.02 / hour with a generation time of 34.65 hours. The highest enzyme activity produced by IS_1 bacteria at 8 hours incubation time was 0.490 IU. The conclusion of this study is that IS_1 bacteria indicates good growth in medium of dahlia tuber flour and has inulinase enzyme activity which is able to hydrolyze inulin into fructose.  Key words: inulin, bacterial isolate, inulinase, productionÂ

Blue Diode and Red He-Ne Lasers Affect the Growth of Anthocyanin Producing Suspension Cells of Apple

Light is an effective factor in cell suspension culture and must be controlled for optimizing cell growth. Growth of anthocyanin producing suspension cells of a red-fleshed genotype of apple (RFA) was assessed in response to blue diode laser (BDL) and red He Ne (RHNL) laser. The suspension cells in L-shaped test tubes were exposed to short-term laser radiation for 20 min in a rotary shaker. The fresh cell weight (FCW), dry cell weight (DCW), cell volume after sedimentation (CVS), cell number (CN) and cell viability (CV) as criteria of cell growth were recorded at 0, 4, 8, 12 days during cell culture. The cell growth was negatively affected in response to BDL and RHNL compared to control and darkness, respectively. The FCW and DCW was enhanced by BDL whereas were not affected by RHNL. Also, only 30.4 mWcm-2 intensity of BDL could increase CVS in RFA cells. Changes in CN were not displayed by RHNL and BDL. BDL more than RHNL decreased CV. Cell death rates observed due to BDL and RHNL were 40.42% and 33.67%, respectively. All these results showed that these lasers had diverse effects on FRA cell growth, however, these cells were more sensitive to BDL than RHNL especially in higher intensities, presumably because of its damage to cell membrane leading to cell death.

ANTI-PSEUDOMONAS AERUGINOSA DRUG

Objectives: In this research we assess in-vitro susceptibility of Pseudomonasaeruginosa (P. aeruginosa) using the ethanolic extract of medicinal plant Tabebuia impetiginosa(dried inner bark). To evaluate the synergistic effect of ethanolic extract of Tabebuia impetiginosacombine with ciprofloxacin In-vitro anti-Pseudomonas aeruginosa activities of the extracts andciprofloxacin were confirmed, and synergism was verified for this combine extracts. StudyDesign: Experimental study. Period: October 2016 to February 2017. Place of Experiment:Rashid Latif Medical College, Lahore. Method: Diffusion method tests are mostly qualitativemethods that are used to identify the antimicrobial activity, resistance and synergistic effect. Thefresh plants inner bark was grinded and soaked in 95% ethanol for extraction. The antibacterialsensitivity of this compound against P. aeruginosa was assessed using the diffusion method.About 1000mg of grinded menstruum was added in 800ml of petroleum ether in a conicalflask and adjust in rotary shaker at 100 rpm for 12 hours and then the final extract was filteredwith 0.45mμ filter membrane and centrifuged at 2000rpm for 15 minutes. The final extract wasredissolved in ciprofloxacin solution (10 for bioassay analysis. Results: We concludedthat the fresh ethanolic extract of Tabebuia impetiginosa with ciprofloxacin has high antibacterialpotency against P. aeruginosa which is prominent then a single. However this was not pureextract and if it is refined then it might gives significant antibacterial activity at low concentration.There is still need to test Tabebuia impetiginosa extract for antibacterial activity and to checksynergistic effect with other drugs in-vivo against Pseudomonas aeruginosa. Extract presentedthe highest synergism rate with antimicrobial drug. Conclusion: In-vitro study showed Tabebuiaimpetiginosa fresh inner bark extract with ciprofloxacin dilution have significant antibacterialactivity against Pseudomonas aeruginosa with P.value <0.001. Isolates were susceptible tothis combine solution with mean zone diameter of 16.15 ± 0.95 mm and no regrowth wasnoticed. In the present research the synergistic effect of ciprofloxacin antibiotic with Tabebuiaimpetiginosa ethanolic extract were observe against Pseudomonas aeruginosa bacteria.

QUANTITATIVE ESTIMATION OF MAGNESIUM CARBONATE IN PAN MASALA AND GUTKA

Objective: The objective of present study is to detect and quantitatively estimate Magnesium Carbonate, a potential carcinogen used for its anti-caking property in various brands of Pan Masala and Gutka by the simple, economic and effective method.Methods: The water-soluble extract of various brands of Pan Masala and Gutka was prepared by a wrist action rotary shaker for 60 min. The extract was filtered, and the filtrate was titrated against 0.1N EDTA using Eriochrome Black T indicator at pH 10 and using Murexide indicator at pH 12.Results: The presence of Magnesium Carbonate was found to be less than 2% in all brands of Pan Masala and less than 3% in all brands of Gutka samples used in the analysis.Conclusion: There is Magnesium Carbonate used in all brands of Pan Masala and Gutka used in the present study.

English

Trichoderma species is a heterotrophic saprobion organism, which competes with other microorganisms, preventing them from developing. This fungus has mechanisms of action, among which are: production of antibiotics and metabolites, and hyperparasitism, which also promotes the induction of systemic resistance in plants. In this study, five native Trichoderma strains which were collected from agricultural land in Tamaulipas, Mexico were evaluated. The objective of this research was to identify the secondary metabolites produced by native strains of Trichoderma spp. under in vitro conditions and evaluation of the effect of these compounds on the growth of the pathogen Fusarium oxysporum and the germination of tomato seeds (Solanum esculentum). The production of Trichoderma's metabolites was performed by culturing in 150 ml of potato broth supplemented with sucrose (5%) and yeast extract (5%). Inoculation was performed by the addition of a 4 mm disk of active mycelium and allowed to grow at 25C ± 2°C and 12 h light/dark on a rotary shaker at 150 rpm for three weeks. Subsequently, the extraction of metabolites was performed using liquid: liquid phase which consisted of the filtrate and methylene chloride in a ratio of 1:3, and then the organic phase was recovered. The organic fraction was evaporated at 40°C with the aid of a rotary evaporator (Buchi®); the sample was recovered in 1 ml of methylene chloride. Analysis by GC-MS indicates that Trichoderma isolates produced 41 secondary metabolites of volatile and semi-volatile molecules and was observed, such that the number of compounds varies from the species analyzed and the collection site. The evaluation of the antagonistic activity of the extracts of Trichoderma spp. on F. oxysporum showed no significant differences between treatments (P=0.05); however, it was observed that pigmentation decreased in the mycelium of the pathogen. Regarding the evaluation of the effect of the extracts on the seeds of tomato, it was observed that the compounds of Trichoderma asperellum (TV1) induced the germination and the development of the seedling. Key words: Volatile compounds, Trichoderma, gas chromatography-mass spectrometry secondary metabolites, Solanum esculentum.

REGENERASI IN VITRO Eucalyptus pellita F. Muell MENGGUNAKAN KULTUR MATA TUNAS

Plantation of Eucalyptus pellita F. Muell in the Industrial Plantation Forest (HTI) progam have applied a clonal forestry system to improve productivity of mean annual increament and homogenous plants on each rotation. However, the productivity ha s been low and not meet the need of raw materials for pulp industry. The objectives of the research is to develope of E . pellita in vitro by multiple node culture to increase shoots growth at each step in vitro propagation . Combination of in vitro and macro propagation in establishing hedge orchard (mini cutting) is the best technique and efficient to propagate and optimize of the growth of the species. The method to increase the successful of E . pellita in vitro at the induction step was using mutiple-node culture and application of rotary shaker. Four treatments were applied at multipication, rooting and acclimatization steps to obtain the best combination of media for E . pellita in the optimum condition. The results showed that application of rotary shaker increased the shoots growth at the induction step and accelerated the shoots growth at the multiplication step.The procedure applied in this study was also resulting an optimal and efficient response of shoots and roots growth in each step (induction, multiplication, rooting) of E . pellita in vitro until acclimatization in the greenhouse.

Purification and characterization of alkaline soda-bleach stable protease from Bacillus sp. APP-07 isolated from Laundromat soil.

The detergent-compatible alkaline protease was produced from the bacterial strain Bacillus sp. APP-07 isolated from Laundromat soil of Solapur, Maharashtra, India. The culture was grown in 1000 ml capacity baffled flask with a working volume of 100 ml and incubated at 55 °C for 33 h on a rotary shaker. After incubation, alkaline protease was partially purified by the sequential method of acetone precipitation followed by nominal molecular weight limit (NMWL) cut-off ultrafiltration using 50 K and 10 K filters. Finally, Sephadex G-100 gel filtration chromatographic purification was performed to obtain 3.12 fold purified alkaline protease enzyme with a 66.67% final yield. The purified enzyme showed 31907.269 units (U) of enzyme activity containing 8741.718 U/mg of specific enzyme activity. The molecular weight of the enzyme was confirmed about 33.0 kDa (kDa) by the SDS-PAGE analysis. The purified enzyme was stable at higher pH and temperature range, with an optimum pH 10.5 and temperature 55 °C. The enzyme showed excellent stability and compatibility in various detergents, surfactants, bleach, and oxidizing agents. The enzyme activity enhanced in the presence of Ca2+, Cu2+, and surfactants, whereas; the phenylmethylsulphonyl fluoride (PMSF) and Diisopropyl fluorophosphate (DFP) completely inhibit the enzymatic activity, which pointed out that the enzyme affiliated to serine-centered metalloproteases family.In conclusion, the remarkable tolerance and stability of the enzyme explored the promising candidature for the several potential applications in the laundry detergents. The sustainability of the enzyme might serve several possible applications in the laundry detergents, leather industries, and other harsh industrial processes.

Optimization of the 9α-hydroxylation of steroid substrates using an original culture of Rhodococcus erythropolis

To obtain inoculation material (cultivation stage 1), the biomass of Rhodococcus erythropolis VKPM AC-1740 was transferred from agar slants into 750 ml conic flasks containing 100 ml of vegetation media of the following composition (g/l): medium 1 – yeast extract, 10.0; glucose, 10.0; soybean flour, 10.0; КН2РО4, 2.0; Na2НРО4, 4.0 (рН 6.8–7.4); medium 2 – corn extract, 15.0; glucose, 10.0; КН2РО4, 2.0; Na2НРО4, 4.0 (рН 6.8–7.4). The culture was grown on a rotary shaker (220 rpm) for 68–72 h at 28–29 °С. To obtain a working biomass (cultivation stage 2), the inoculum obtained at the stage 1 was transferred into flasks containing the same media (the volume of seed material was 20% of the medium volume) and grown under the same conditions for 23–25 h. During a study of the effect of the inducer concentration on the rate of 9α-OH-AD formation, different concentrations (0.25, 0.50, and 1 g/l) of the AD solution in dimethylformamide (DMF) were added to the vegetation medium after 6 h of incubation. To perform AD transformation at a load of 5 g/l, 10 ml of Rh. erythropolis cells at the age of 23–25 h were transferred into 750 mL flasks with baffles containing 40 mL of vegetation medium supplemented with the steroid. AD was added in the form of microcrystals or suspension with a surfactant or DMF. The process was carried out at 28–29 ºC and with constant mixing (220 rpm). During AD transformation at a load of 10–30 g/l, the steroid was preliminarily precipitated from DMF solution. The resulting paste was mixed with a surfactant and transformation medium. The obtained homogeneous suspension was poured in equal amounts into the flasks with baffles, and then a concentrated cell mass was added (25 vol.%). To obtain a cell concentrate, cells were centrifuged for 1 h at 1500 rpm at the age of 23–25 h. The resulting biomass was homogenized, supplemented with a fresh medium to the required volume, and added into transformation flasks. The amount of a biomass required for AD transformation at a load of 10 g/l was 3.13 g/l (dry weight); in the case of a 30 g/l load, the biomass was added by two equal portions, and its total amount was 6.2 g/l (dry weight). The amount of 9α-OH-AD in a culture broth was evaluated by a thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Steroids were extracted by ethylacetate. To perform TLC, Sorbifil plates (Russia) and benzol: acetone mix (3 : 1) were used. HPLC was performed on a Gilson chromatographer (United States) equipped with a Silasorb C-18 column (10 μm, 4.0 × 250 mm); the flow rate was 0.8 ml/min. The mobile phase was МеОН : Н2О mix (70 : 30). The absorbance was measured at 260 nm. Replacement of corn extract, which has an unstable composition, by yeast extract and soybean flour and the use of glucose as an optimal carbon source for a Rh. erythropolis culture have provided a high-yield production of 9α-hydroxy-4-ene-3,17-dione with increased AD loads. Use of such techniques as the inoculum induction and application of surfactants have provided a positive effect on the AD transformation with a load exceeding 10 g/l. During 9α-hydroxylation of AD with a load of 30 g/l, a target product with the yield of 83% has been obtained.

Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti

BackgroundAs a very important coenzyme in the cell metabolism, Vitamin B12 (cobalamin, VB12) has been widely used in food and medicine fields. The complete biosynthesis of VB12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB12 production. High-yield VB12-producing strains are usually obtained by mutagenesis treatments, thus developing an efficient screening approach is urgently needed.ResultBy the help of engineered strains with varied capacities of VB12 production, a riboswitch library was constructed and screened, and the btuB element from Salmonella typhimurium was identified as the best regulatory device. A flow cytometry high-throughput screening system was developed based on the btuB riboswitch with high efficiency to identify positive mutants. Mutation of Sinorhizobium meliloti (S. meliloti) was optimized using the novel mutation technique of atmospheric and room temperature plasma (ARTP). Finally, the mutant S. meliloti MC5–2 was obtained and considered as a candidate for industrial applications. After 7 d’s cultivation on a rotary shaker at 30 °C, the VB12 titer of S. meliloti MC5–2 reached 156 ± 4.2 mg/L, which was 21.9% higher than that of the wild type strain S. meliloti 320 (128 ± 3.2 mg/L). The genome of S. meliloti MC5–2 was sequenced, and gene mutations were identified and analyzed.ConclusionTo our knowledge, it is the first time that a riboswitch element was used in S. meliloti. The flow cytometry high-throughput screening system was successfully developed and a high-yield VB12 producing strain was obtained. The identified and analyzed gene mutations gave useful information for developing high-yield strains by metabolic engineering. Overall, this work provides a useful high-throughput screening method for developing high VB12-yield strains.

Use of Monascus sp. NP1 for bioethanol production from Cladophora glomerata

The fungus Monascus sp. was able to produce ethanol instead of red pigment under glucose derepression with greater and more tolerance to ethanol than yeast. Algae are known as a polysaccharide resource for bioethanol production. This work was focused on the optimization of cellulase production through solid-state fermentation of straw and rice bran using Trichoderma viride TISTR 3161 for saccharification by the alga, Cladophora glomerata. The best conditions for alga enzymatic hydrolysis were proposed after varying crude enzyme and temperature, comparing the enzyme method to the physical method of saccharification from the alga, characterizing bioethanol production from the fungi at the laboratory scale, and scaling up to a 10-L fermenter, and fermentation of the algal extract at a variety of pH at the laboratory scale. Cellulase production from Sangyod Phatthalung rice using 21.19-mg crude enzyme at 35 °C provided the best conditions of all five varieties of Thai rice tested in terms of producing reducing sugar via algal hydrolysis. This method obtained nearly the same amount of reducing sugar as the physical method (acid and high temperature). At the laboratory scale, 20% glucose in liquid medium was the best condition for ethanol production (1384 μM) of all glucose concentrations tested (2, 10, 20, 30, and 50%) at room temperature on a rotary shaker (110 rpm) for 120 h. The time of production could be reduced to 72 h to produce the same amount of ethanol in a fermenter. The pH of 6 was the proper condition for bioethanol production in algal extract media. The promise of this technique for bioethanol production was shown.

Cancer-adipose tissue interaction and fluid flow synergistically modulate cell kinetics, HER2 expression, and trastuzumab efficacy in gastric cancer.

Early local tumor invasion in gastric cancer results in likely encounters between cancer cells and submucosal and subserosal adipose tissue, but these interactions remain to be clarified. Microenvironmental mechanical forces, such as fluid flow, are known to modulate normal cell kinetics, but the effects of fluid flow on gastric cancer cells are poorly understood. We analyzed the cell kinetics and chemosensitivity in gastric cancer using a simple in vitro model that simultaneously replicated the cancer-adipocyte interaction and physical microenvironment. Gastric cancer cells (MKN7 and MKN74) were seeded on rat adipose tissue fragment-embedded discs or collagen discs alone. To generate fluid flow, samples were placed on a rotatory shaker in a CO2 incubator. Proliferation, apoptosis, invasion, and motility-related molecules were analyzed by morphometry and immunostaining. Proteins were evaluated by western blot analysis. Chemosensitivity was investigated by trastuzumab treatment. Adipose tissue and fluid flow had a positive synergistic effect on the proliferative potential and invasive capacity of gastric cancer cells, and adipose tissue inhibited apoptosis in these cells. Adipose tissue upregulated ERK1/2 signaling in gastric cancer cells, but downregulated p38 signaling. Notably, adipose tissue and fluid flow promoted membranous and cytoplasmic HER2 expression and modulated chemosensitivity to trastuzumab in gastric cancer cells. We have demonstrated that cancer-adipocyte interaction and physical microenvironment mutually modulate gastric cancer cell kinetics. Further elucidation of the microenvironmental regulation in gastric cancer will be very important for the development of strategies involving molecular targeted therapy.

PM 7/21 (2) Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex)

Specific scopeThis Standard describes a diagnostic protocol for Ralstonia solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii, i.e. phylotype/sequevar strain in the Ralstonia solanacearum Species Complex (RSSC).It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.Specific approval and amendmentApproved in 2003‐09. First revised in 2018‐02.

Preparation and in vitro/in vivo evaluation of PLGA microspheres containing norquetiapine for long-acting injection.

BackgroundNorquetiapine (N-desalkyl quetiapine, NQ) is an active metabolite of quetiapine with stable pharmacokinetic and pharmacological properties. However, its short half-life is a drawback for clinical applications, and long-acting formulations are required.PurposeThe objectives of this study were to prepare improved entrapment efficiency NQ freebase microspheres by the solvent evaporation method with poly(d,l-lactic-co-glycolic acid) (PLGA) as a release modulator and to evaluate their physicochemical and in vitro/in vivo release properties.MethodsNQ freebase PLGA (1:5 w/w) formulations were prepared by the oil-in-water (o/w) emulsion–solvent evaporation method. A solution of the drug and PLGA in 9:1 v/v dichloromethane:ethanol was mixed with 0.2% polyvinyl alcohol and homogenized at 2,800 rpm. The emulsion was stirred for 3 h to dilute and evaporate the solvent. After that, the resulting product was freeze-dried. Drug-loading capacity was measured by the validated RP-HPLC method. The surface morphology of the microspheres was observed by scanning electron microscopy (SEM), and the physicochemical properties were evaluated by differential scanning calorimetry, powder X-ray diffraction, and Fourier-transform infrared spectroscopy particle size distribution. The in vitro dissolution test was performed using a rotary shaking bath at 37°C, with constant shaking at 50 rpm in sink condition.ResultsThe NQ freebase microspheres prepared by o/w emulsion-solvent evaporation showed over 30% efficiency. NQ was confirmed to be amorphous in the microspheres by powder X-ray diffraction and differential scanning calorimetry. Special chemical interaction in the microspheres was not observed by FT-IR. The in vitro dissolution test demonstrated that the prepared microspheres’ release properties were maintained for more than 20 days. The in vivo test also confirmed that the particles’ long acting properties were maintained. Therefore, good in vitro–in vivo correlation was established.ConclusionIn this study, NQ freebase-PLGA microspheres showed potential for the treatment of schizophrenia for long-periods.

Rapid in vitro shoot multiplication of the recalcitrant species Juglans nigra L.

Black walnut (Juglans nigra L.) has long been prized for its timber, leading to commercial cultivation and significant breeding efforts for improving marketable traits. Vegetative and in vitro black walnut propagation techniques, however, are variable and highly genotype dependent. Optimizing plant growth regulator type and concentration are integral for developing a successful micropropagation protocol. The addition of meta-topolin (MT) combined with the novel use of a liquid medium has led to the development of a rapid shoot multiplication system. The objective of this research was to develop a reproducible and dependable micropropagation protocol for elite black walnut genotypes. In vitro shoot cultures were established from nodal explants cultured on semi-solid Driver and Kuniyuki walnut (DKW) medium supplemented with 8.9 μM benzyladenine, 0.005 μM indole-3-butyric acid (IBA), 200 mg L−1 casein hydrolysate, 50 mg L−1 adenine hemisulfate, 2 mL L−1 Plant Preservative Mixture™, and 4.1 μM MT. Long-term survival and proliferation of microshoots was achieved when nodal segments of in vitro grown shoots were cultured in liquid initiation medium in 3-L polycarbonate Fernbach-style flasks on a rotary shaker (100 rpm) under a 16-h photoperiod at 25°C. Elongated microshoots (5–7 cm in length) were rooted in a slurry-like medium composed of half-strength DKW medium with 0.11% (w/v) Phytagel™ and coarse vermiculite (2:1, v/v) supplemented with 50 μM IBA for 5 wk. Rooted shoots were acclimatized to ambient culture room conditions, but plantlets did not survive once transferred to the greenhouse.