Inoculation Volume Research Articles

Study on Chlorpyrifos Residues Degradation by Mutagenic Bacteria DX1 in Soil

In this study, it was carried out by designing PH, soil fertilization content, humidity and mutagenic bacteria DX1 inoculation concentration of orthogonal experiment to research chlorpyrifos degradation rate in the soil, which was affected by mutagenic bacteria DX1 in the soil environment. SPSS software was taken visualized analysis and variance analysis to evaluate the impact of different levels of the test results, to determine the factors which had significant impact on the experimental results. It showed that the best environmental factors of chlorpyrifos degradation rate in soil were the pH 7, organic fertilizer content 5mg/kg, the soil moisture 70%, inoculation volume 50ml/kg. The pH value and inoculation volume had a significant impact on chlorpyrifos degradation rate in soil; the soil humidity had little effect and the organic fertilizer content had almost no effect.

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus for acetic acid production

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus was carried out for high yield of acetic acid. Acetic acid production process was divided into three stages. The first stage was the growth of S. cerevisiae and ethanol production, fermentation temperature and aeration rate were controlled at 32°C and 0.2vvm, respectively. The second stage was the co-culture of S. cerevisiae and A. pasteurianus, fermentation temperature and aeration rate were maintained at 34°C and 0.4vvm, respectively. The third stage was the growth of A. pasteurianus and production of acetic acid, fermentation temperature and aeration rate were controlled at 32°C and 0.2vvm, respectively. Inoculation volume of A. pasteurianus and S. cerevisiae was 16% and 0.06%, respectively. The average acetic acid concentration was 52.51g/L under these optimum conditions. To enhance acetic acid production, a glucose feeding strategy was subsequently employed. When initial glucose concentration was 90g/L and 120g/L glucose was fed twice during fermentation, acetic acid concentration reached 66.0g/L.

Fermentation optimization and antioxidant activities of mycelia polysaccharides from Morchella esculenta using soybean residues

The mycelia polysaccharides from Morchella esculenta are active ingredients in a number of medicines that play important roles in immunity improvement and tumor growth inhibition. So far, the production of polysaccharides from M. esculenta mycelia has not been commercialized. The aims of this work were to screen and optimize the fermentation conditions to produce mycelia polysaccharides from using soybean residues as basic substrates in the composition of the medium, and to evaluate the antioxidant activities of mycelia polysaccharides from M. esculenta. Our results demonstrate that M. esculenta mycelia made good use of soybean residues. The optimal media contained the following components (g/l): soybean residue, 22.2; glucose, 20.1; KH2PO4 2.0 and MgSO4·7H2O 1.5. The optimum parameters of liquid culture were identified as the following: initial fermentation pH 7.0, inoculation volume 10%, temperature 28°C, and fermentation time 56 h. Under these optimized conditions, the values of dry cell weight (DCW) and the production rates of mycelia polysaccharides were 36.22 g/l and 68.23 mg/g, respectively. Furthermore, the mycelia polysaccharides from M. esculenta demonstrated strong antioxidant activities, suggesting their potential therapeutic use as antioxidants. Our study provides insights to the commercial production of mycelia polysaccharides from M. esculenta. Our study also provides a new route for utilization of soybean residues. Key words: Morchella esculenta, liquid fermentation, mycelia polysaccharides, soybean residues, antioxidant activity.

Optimization of Culture Medium and Conditions for <i>Bacillus coagulans</i> T242 Producing Thermostable Lactase

A thermotolerant lactase-producing strain Bacillus coagulans T242 was obtained in our previous work, its media and culture conditions were investigated and optimized for enhanced production of lactase in the present work. Results showed medium containing 1.5% lactose, 1.0% peptone, 1.5% yeast extract, 0.7% MgSO4, and pH 8.0, was the optimum medium; And its optimum culture conditions were the age of bacterial culture 36 h, inoculation volume 2%, liquid medium volume loaded 30 mL/250 mL flask, 60 °C, 36 h. When cultured at the optimum condition Bacillus coagulans T242 yielded the maximum of 1.21U/mL lactase activity.

Optimization of Fungal Rennet Production by local isolate of Rhizomucor nainitalensis Under Solid Substrate Fermentation system

In this study the production of substitute rennet by a newly isolated thermophilic fungus, Rhizomucor nainitalensis, using optimized solid substrate fermentation system have been explored. The results revealed that enzyme activity is at its highest level at 40˚C, 50 % humidity, inoculation volume of 10 3 spore /gr wheat bran, and exoenzyme biosynthesis induction by casein at a rate of 1.5 %. Enzyme activity reached its highest (8400 SU/gr wheat bran) after 110 hours of incubation under the stated conditions. Precipitated enzyme, by addition of ammonium sulfate with milk coagulating activity of 115000 SU/gr enzyme powder, was free of toxin and microorganisms, with market's favorable aroma. Keywords- Rhizomucor, Solid Substrate Fermentation, rennin, fungal proteases

Physical stress for overproduction of biomass and flavonoids in cell suspension cultures of Boesenbergia rotunda

Flavonoid, a bioactive compound isolated from the rhizomes of Boesenbergia rotunda, exhibited antibiotic and anti-inflammatory properties and has also shown high inhibitory activity of dengue-2 virus protease. Several factors are responsible for the production of flavonoid in cell cultures. In the present study, the effects of initial inoculation volume, temperature and speed of agitation on cell growth, total and selected flavonoid in suspension cultures of B. rotunda were determined. High performance liquid chromatography analysis showed that a 2 % inoculation volume induced a significantly high accumulation of biomass and of flavonoid in the cells. The cells cultured at 25 °C showed significantly high biomass and selected flavonoid accumulation while differences in medium agitation significantly affected the yield of selected flavonoid.

Parameters Optimization of Two-Phase United Anaerobic Fermentation Hydrogen-Methane Production

The two-phase united anaerobic fermentation has been paid more and more attention in recent years. But how to increase the biogas production yield and the degradation rate of organic matter needs more research. The batch anaerobic fermentation studies demonstrated the feasibility of H2 and CH4 generation utilizing kitchen wastewater as substrate, and the vaccination rate, dilution rate, substrate concentration were optimized. The conclusion showed that dilution rate is the most important factor, following by kitchen wastewater concentration and inoculum volume. The most excellent program of the organic load, the inoculation volume and the dilution volume are 150 +300 ml, 135 ml, 225 ml, respectively. Under these conditions the hydrogen and methane production rate were the highest of 4.15 ml/ml，2.55 ml/ml (calculated as the wastewater volume), respectively. The acid-forming pathways of kitchen wastewater was typical butyric- propionic acid mixed fermentation type. The appropriate dilution could decrease the concentration of the total acids and increase the biogas production yield.

Plasmodium liver load following parenteral sporozoite administration in rodents

One of the bottlenecks in the development of a whole sporozoite malaria vaccine is the route and method of sporozoite administration. Immunization and challenge of human volunteers by mosquito bites is effective, but cannot be used as a vaccine. Intravenous immunization with sporozoites is effective in rodents and non-human primates, and being studied in humans, but is not yet used for licensed vaccines for infectious diseases. Intradermal and subcutaneous immunization regimens show a strong decrease in protective efficacy, which in rodents, is associated with a decreased degree of parasite liver infection during immunization. The objective of this study was to explore alternative routes of sporozoite administration to increase efficiency of liver infection. Using in vivo imaging, we found that IM injection of sporozoites resulted in a greater parasite liver load compared to ID and SC injection. The use of small inoculation volumes and multiple injections further increased the subsequent liver load. These observations were corroborated in a Plasmodium yoelii model using cryopreserved sporozoites administered ID. Our findings provide a rationale for the design of clinical trials to optimize needle and syringe administration of Plasmodium falciparum sporozoites.

Biodegradation of phenol from a synthetic aqueous system using acclimatized activated sludge

Phenol is one of the aromatic hydrocarbons. Phenol and its derivatives are highly toxic. These pollutants can be observed in the effluents of many industries. This research investigates the removal of phenol by the use of activated sludge in a batch system. The effects of influencing factors on biodegradation efficiency have been evaluated. The main factors considered in this study were the volume of acclimatized activated sludge inoculation, pH, temperature, and initial concentration of phenol. The inoculation volumes of 1, 3, and 5 mL of acclimatized activated sludge were taken into account. Different pH values of 3, 5, 7, 9, and 11 were examined. The experiments were conducted for temperatures of 25, 30, 35, and 40 °C and initial phenol concentrations of 400, 800, 1,000, and 1,500 ppm. The results show that the acclimatized activated sludge has a high capacity for the removal of phenol. From a 100-mL aqueous solution was removed 1,500 ppm of phenol after 80 h. Furthermore, maximum phenol removal was observed for an inoculation volume of 5 mL for three different phenol concentrations of 100, 400, and 800 ppm. The best pH was 7 for the biodegradation process, and the optimum temperature was 30 °C. It was further found that an increase in the phenol concentration increased its removal time. Moreover, the activated sludge could effectively remove about 99.9 % of phenol from a synthetic aqueous solution in a batch system.

Immobilization of Rubia tinctorum L. suspension cultures and its effects on alizarin and purpurin accumulation and biomass production

The present study is investigating the immobilization of Rubia tinctorum L. suspension cultures. The effects of three inoculation volumes and three immobilization materials (loofa, sisal and jute) on fresh and dry weights of biomass as well as on alizarin and purpurin production were determined in this study. Two grams of four-week old callus tissue were transferred to liquid medium to establish suspension cultures. After four weeks, suspension cultures of R.tinctorum at concentration of 8 × 105 living cells/ml were immobilized with lignocellulosic materials and the cells were attached to all immobilization materials at the end of the first week and started to form aggregates on them. At the fourth week of these batch systems, biomass was measured approximately three times higher than the starting suspension cultures. The highest fresh weight was obtained (339.40 g/l) from sisal with ½ inoculation ratio. Immobilization materials and inoculation volumes had an effect on dry weights, and accordingly, the most effective combinations were jute with ¼ (J3) and ½ (J1) inoculation volumes with 7.86 and 7.82 g/l dry weights, respectively. Alizarin and purpurin contents of immobilized cells, analyzed with U-HPLC method, were 6.05 and 22.91 times higher than inoculated cells. All immobilization materials used in this study had no negative effect on to cells and biomass accumulation was enhanced. Concomitantly with rapid biomass increase, alizarin and purpurin production was ascended.

The Development and Evaluation of Blood Volume Measuring System for Blood Culture Quality Improvement

Abstract The blood volume is the most important factor affecting the sensitivity of blood cultures. However, there has not been a system for measuring blood volume prior to starting the blood culture process. The aim of this study is to develop a blood volume measuring system and to evaluate its usability in a clinical setting. A system for measuring blood volume in a culture bottle using digital scales was designed. The suggested system was installed and evaluated in the Department of Laboratory Medicine. During the 13-month period, the weight of blood bottles before inoculation and blood volumes in 113 564 blood culture bottles were analyzed. The weight differences between bottles were up to 12 g and about 9 g for bottles distributed by BACTEC and bioMérieux, respectively. Of the 113 564 aforementioned bottles, 96 661 cultures were analyzed from patients over the ages of 18. After the application of blood volume measuring systems, the adequate blood volume submission ratio increased from 4.9 % to about 10 %. After the intervention, the proportions of bottles for adequate submissions showed an increasing trend. In routine clinical practice, providing regular feedback and education regarding the adequate blood volumes for culture to clinicians and nurses would be a good way to maintain quality of the blood culture results and to reduce culture bottles being submitted with an inadequate amount of sample.

Statistical Optimization of Process Variables for Antibiotic Activity of Xenorhabdus bovienii

The production of secondary metabolites with antibiotic properties is a common characteristic to entomopathogenic bacteria Xenorhabdus spp. These metabolites not only have diverse chemical structures but also have a wide range of bioactivities of medicinal and agricultural interests. Culture variables are critical to the production of secondary metabolites of microorganisms. Manipulating culture process variables can promote secondary metabolite biosynthesis and thus facilitate the discovery of novel natural products. This work was conducted to evaluate the effects of five process variables (initial pH, medium volume, rotary speed, temperature, and inoculation volume) on the antibiotic production of Xenorhabdus bovienii YL002 using response surface methodology. A 25–1 factorial central composite design was chosen to determine the combined effects of the five variables, and to design a minimum number of experiments. The experimental and predicted antibiotic activity of X. bovienii YL002 was in close agreement. Statistical analysis of the results showed that initial pH, medium volume, rotary speed and temperature had a significant effect (P<0.05) on the antibiotic production of X. bovienii YL002 at their individual level; medium volume and rotary speed showed a significant effect at a combined level and was most significant at an individual level. The maximum antibiotic activity (287.5 U/mL) was achieved at the initial pH of 8.24, medium volume of 54 mL in 250 mL flask, rotary speed of 208 rpm, temperature of 32.0°C and inoculation volume of 13.8%. After optimization, the antibiotic activity was improved by 23.02% as compared with that of unoptimized conditions.

Optimization of Fermentation Conditions for Antibiotic Production by Actinomycetes YJ1 Strain against Sclerotinia sclerotiorum

In order to improve the productivity of activities substance of actinomycetes YJ1, the effects of the medium components, inoculation volume, medium capacity, rotary speed, fermentation time, temperature and initial pH on the biological activity of YJ1 were investigated by detecting the mycelium growth inhibition rate of Sclerotinia sclerotiorum. The results show that the inhibition rate is the highest under the optimum medium composition (g/L), containing sucrose 10.0, soluble starch 10.0, soybean meal 20.0, K2HPO4 0.5, MgSO4 0.5, NaCl 1.0 and FeSO4 0.01. The maximum antibiotic activity is obtained at the inoculation volume 5%, medium volume in 250 ml flask 75 ml, rotary speed of 180 r/min, fermentation time 4 days, temperature 28°C and initial pH 7.0. The inhibition rate has been increased by 6.71% under the optimized condition.

Influenza Virus Respiratory Infection and Transmission Following Ocular Inoculation in Ferrets

While influenza viruses are a common respiratory pathogen, sporadic reports of conjunctivitis following human infection demonstrates the ability of this virus to cause disease outside of the respiratory tract. The ocular surface represents both a potential site of virus replication and a portal of entry for establishment of a respiratory infection. However, the properties which govern ocular tropism of influenza viruses, the mechanisms of virus spread from ocular to respiratory tissue, and the potential differences in respiratory disease initiated from different exposure routes are poorly understood. Here, we established a ferret model of ocular inoculation to explore the development of virus pathogenicity and transmissibility following influenza virus exposure by the ocular route. We found that multiple subtypes of human and avian influenza viruses mounted a productive virus infection in the upper respiratory tract of ferrets following ocular inoculation, and were additionally detected in ocular tissue during the acute phase of infection. H5N1 viruses maintained their ability for systemic spread and lethal infection following inoculation by the ocular route. Replication-independent deposition of virus inoculum from ocular to respiratory tissue was limited to the nares and upper trachea, unlike traditional intranasal inoculation which results in virus deposition in both upper and lower respiratory tract tissues. Despite high titers of replicating transmissible seasonal viruses in the upper respiratory tract of ferrets inoculated by the ocular route, virus transmissibility to naïve contacts by respiratory droplets was reduced following ocular inoculation. These data improve our understanding of the mechanisms of virus spread following ocular exposure and highlight differences in the establishment of respiratory disease and virus transmissibility following use of different inoculation volumes and routes.

Optimization of Fermentation Conditions of &lt;I&gt;Paraconiothyrium variabile&lt;/I&gt; GHJ-4 for Laccase Production by Response Surface Methodology*

In order to enhance the production of laccase by Paraconiothyrium variabile GHJ-4,response surface methodology was applied to optimize the fermentation conditions.Plackett-Burman design was used to investigate the main factors affecting laccase yield of Paraconiothyrium variabile GHJ-4.The results showed that four factors played important roles in the fermentation conditions,including inoculation volume(X1),culture time(X2),broth content(X3) and rotating speed(X4).After steepest ascent experiment approaching the optimal level of the four factors,central composite design and response surface analysis were applied to establish the second-order regression equation model,Y = 698.72 –13.64X1 + 3.46X2 + 25.45X3 – 11.64X4 + 44.94X1X3 + 0.31X2X3 + 23.14X2X4 – 19.55X3X4 – 44.65X12 – 45.07X22 – 36.52X32 – 21.60X42,for the yield of laccase.Based on this model,the optimal fermentation conditions were obtained as following: 6.43%(φ) of inoculation,28 ℃ of culture temperature,initial pH value 4,182 h of culture time,72.5 mL of broth content,157 r/min of rotating speed,and 96 h of seed age.Under the optimum conditions,the activity of laccase increased nearly 1.73 times to 710.44 U/mL and the experimental values argreed with the predicted values.These results suggested that the predicted model was reliable and available for the optimization of laccase fermentation conditions.Fig 1,Tab 6,Ref 20

Factors affecting microbial contamination rate of cord blood collected for transplantation

Collection and processing of cord blood (CB) is associated with significant risk of microbial contamination and hence relevant standards mandate microbial screening of the final product. This study aimed to determine the contamination rate and associated risk factors during 14 years of banking at the Sydney Cord Blood Bank. CB was collected and processed using a closed system and tested for contamination using blood culture bottles (BacT/ALERT, bioMérieux) incubated for a minimum of 5 days. Four microbial screening methods were used with different combinations of inoculated bottles (adult or pediatric) and associated sample volumes (10 or 1 mL). Of 13,344 CB units screened, 537 (4.0%) tested positive for contamination, with Bacteroides spp. (20.9%), Staphylococcus spp. (18.6%), and Propionibacterium spp. (13.7%) being the most common isolates. The contamination rate reduced from 10% in 1997 to 1.1% in 2009. Multivariate analysis demonstrated the following variables were independently associated with higher contamination rates: vaginal delivery, collection by obstetric staff, and use of an anaerobic bottle in addition to an aerobic bottle (which facilitated a larger sample inoculation volume than pediatric bottles). This study demonstrates that contamination rates of CB collected for transplantation can be substantially reduced by collection after cesarean delivery and utilizing trained CB collection staff. These data also indicate that the common practice of testing using a pediatric (aerobic) bottle with its attendant small volume of the final CB product may be suboptimal for sensitive detection of contaminating anaerobic microbes.

Improving the performance of culture‐based bacterial screening by increasing the sample volume from 4 mL to 8 mL in aerobic culture bottles

In the setting of bacterial detection of apheresis platelets (PLTs), the manufacturer recommended PLT inoculation volume for BacT/ALERT culture bottles (bioMérieux) ranges from 4 to 10 mL. This study compares the rate of capture of true-positive (TP) contaminations between aerobic culture bottles inoculated with either 4 or 8 mL of sample and assesses if a larger sample volume reduces time to detection. Detection of TP samples and mean time to detection were compared for 4- and 8-mL samples collected between September 1, 2003, and May 2, 2011. A total of 180,263 and 283,114 PLT collections were tested with an 8- and 4-mL sample, respectively. Analysis of TP rates by volume sampled show an increase in the rate of detection of TP with the 8-mL sample relative to the 4-mL sample (139 vs. 106 per million events; odds ratio, 1.31; 95% confidence interval, 0.77-2.23). Comparison of mean time to detection for TP shows a decrease in mean time to detection using 8 mL compared with 4 mL (12.36 ± 3.7 hr to 15.97 ± 6.3 hr, p = 0.012). Doubling the sample volume to 8 mL showed a trend in improvement for the rate of detection of TP and shortened the mean time of detection for TP by 23% when compared to a sample volume of 4 mL. The decrease in mean time to detection using a larger sample volume suggests that a shorter release time after inoculation could be achieved without significantly increasing patient risk.

Statistical optimization of medium components to improve the antibiotic activity of Streptomyces sp. 19G-317

In this paper, fermentation parameters for the production of antibiotics by Streptomyces sp.19G-317 to enhance the antibiotic activity were optimized using a sequential optimization strategy, including the single factor, Plackett-Burman (PB) design, steepest ascent experiment and response surface methodology (RSM). Firstly, the basic medium, suitable carbon and nitrogen sources were selected by single factor test. Millet medium, maltose and tryptone had significant influences on antibiotics production. Maltose and tryptone were then proved to have remarkable effects on antibiotic activity among seven parameter (the selected carbon, nitrogen, initial pH, medium volume, rotary speed, temperature and inoculation volume) using PB design. Finally, steepest ascent experiment and RSM were used to further optimize the significant factors, optimized values were maltose 5.03%, tryptone 0.96%, NaCl 0.25%, CaCO3 0.20%, initial pH 8.0, medium volume (250 ml flask 125 ml), rotary speed 195 r/min, temperature 28℃ and inoculation volume 7.0%. An overall 38.30% increase in antibiotic activity was achieved under the optimized parameter as compared with the unoptimized parameter, from 23.5 to 14.5 mm. Key words: Antibiotic activity, fermentation condition, medium component, response surface methodology,Streptomyces sp.19G-317.

Microbial Fermentation Extraction Processing of Protein from Soybean

Although hexane extraction of soybean oil has the high recovery，residual hexane in oil is noxious for consumer health. Crew pressing and extruding-expelling extraction processes denature protein to render functional properties poor for food and meal use. This processing employed microbial fermentation to extract protein from full fat soy flour so that denaturation degree of protein reduced. The results showed that low temperature flaking and extrusing soy flakes was beneficial to improving protein extraction, after optimizing the fermentation conditions with the Response Surface Methodology (RSM), total protein extraction yield achieved the highest value - 86.66% at optimal fermentation conditions - 6.6% extruded full fat soy flour, 5.4% inoculation volume, the initial pH8.1. These advances make the microbial fermentation extraction process of protein from soybeans attractive channel as the environmentally – friendly technology.

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 μg or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 μl of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate). Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.