Shake Flask Experiments Research Articles

Comparison of bioleaching of a sulfidic copper ore (chalcopyrite) in column percolators and in stirred-tank bioreactors including microbial community analysis

Chalcopyrite is the most abundant Cu-sulfide and economically the most important copper mineral in the world. It is known to be recalcitrant in hydrometallurgical processing and therefore chalcopyrite bioleaching has been thoroughly studied for improvement of processing. In this study, the microbial diversity in 22 samples from the Sarcheshmeh copper mine in Iran was investigated via 16S rRNA gene sequencing. In total, 1063 species were recognized after metagenomic analysis including the ferrous iron- and sulfur-oxidizing acidophilic genera Acidithiobacillus, Leptospirillum, Sulfobacillus and Ferroplasma. Mesophilic as well as moderately thermophilic acidophilic ferrous iron- and sulfur-oxidizing microorganisms were enriched from these samples and bioleaching was studied in shake flask experiments using a chalcopyrite-containing ore sample from the same mine. These enrichment cultures were further used as inoculum for bioleaching experiments in percolation columns for simulating heap bioleaching. Addition of 100 mM NaCl to the bioleaching medium was assessed to improve the dissolution rate of chalcopyrite. For comparison, bioleaching in stirred tank reactors with a defined microbial consortium was carried out as well. While just maximal 32% copper could be extracted in the flask bioleaching experiments, 73% and 76% of copper recovery was recorded after 30 and 10 days bioleaching in columns and bioreactors, respectively. Based on the results, both, the application of moderately thermophilic acidophilic bacteria in stirred tank bioreactors, and natural enrichment cultures of mesoacidophiles, with addition of 100 mM NaCl in column percolators with agglomerated ore allowed for a robust chalcopyrite dissolution and copper recovery from Sarcheshmeh copper ore via bioleaching.

Mechanistic approach on comparative biosorption of dyes by extracellular polymer of Ochrobactrum pseudintermedium C1 utilizing waste mineral lubricating oil

Dyes are widely used in manufacturing of various consumer products for their ability to impart characteristic color. However, dyes are also one of the most important hazardous pollutants in this present day. Presence of hydroxyl, phosphate, ether, carbonyl and alkyl groups also enable bacterial extracellular polymers to function as biosorbent. In this study, negatively charged biosorbent EP-C1 produced by Gram-negative bacterium Ochrobactrum pseudintermedium C1 utilizing waste mineral lubricating oil has given different decolorization percentages for triphenylmethane cationic Malachite Green and azoic anionic Congo Red at neutral pH. Time wise absorbances from Ultraviolet-Visible Spectrophotometry has estimated maximum biosorption of Malachite Green after 300 min. Biosorption capacities have differed with initial dye concentrations, biosorbent dosages and contact time. Time taken by each dye to reach saturation has depended upon their charged bearing groups of each dye and biosorbent. Kinetic (pseudo first and pseudo second order) and diffusion (intra particle and liquid film) parameters in presence of different dye concentrations and biosorbent dosages were studied for understanding the biosorption mechanism. Morphological differences in chemical structure, chemical functional groups of EP-C1 and EP-C1 loaded dye was studied by Fourier Transform Infrared Spectroscopy, Scanning Electron Microscopy and Zeta potential measurements. A compiled overview on comparative mechanistic biosorption of Malachite Green and Congo Red dyes by a bacterial extracellular polymer in miniature laboratory shake flask experiments was done in this study. Newer advents on biological remediation of dyes with mineral lubricating oil as sole carbon source can add a new feather to the already available bioremediation techniques in mitigation of hazardous wastes.

Development, optimization, and scale-up of suspension Vero cell culture process for high titer production of oncolytic herpes simplex virus-1.

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, and four OVs have been approved for cancer immunotherapy. However, high-yield and cost-effective production processes remain to be developed for most OVs. Here suspension-adapted Vero cell culture processes were developed for high titer production of an OV model, herpes simplex virus type 1 (HSV-1). Our study showed the HSV-1 productivity was significantly affected by multiplicity of infection, cell density, and nutritional supplies. Cell culture conditions were first optimized in shake flask experiments and then scaled up to 3L bioreactors for virus production under batch and perfusion modes. A titer of 2.7×108 TCID50mL-1 was obtained in 3L batch culture infected at a cell density of 1.4×106 cellsmL-1 , and was further improved to 1.1×109 TCID50mL-1 in perfusion culture infected at 4.6×106 cellsmL-1 . These titers are similar to or better than the previously reported best titer of 8.6×107 TCID50mL-1 and 8.1×108 TCID50mL-1 respectively obtained in labor-intensive adherent Vero batch and perfusion cultures. HSV-1 production in batch culture was successfully scaled up to 60L pilot-scale bioreactor to demonstrate the scalability. The work reported here is the first study demonstrating high titer production of HSV-1 in suspension Vero cell culture under different bioreactor operating modes.

Strain improvement for enhanced erythritol production by Moniliella pollinis Mutant-58 using jaggery as a cost-effective substrate.

Erythritol has been produced by various microorganisms including Yarrowia, Moniliella, Aureobasidium, and Candida strains. Due to its relatively high price, erythritol sweetener is used lesser than other polyols despite having many advantages. Therefore, in this study, Moniliella pollinis strain was improved for erythritol production by chemical mutagenesis and subsequently screening for cost-effective carbon sources for the enhanced erythritol yield. M. pollinis was subjected to N-methyl N-nitro N-nitroso guanidine (NTG), ethyl methyl sulfonate (EMS), and UV mutagenesis for improved erythritol production. The fmutant strains were evaluated for enhanced erythritol production medium optimization by using different carbon substrates at the shake flask level. To enhance the production of erythritol and statistical media, optimization was carried out using a central composite design (CCD). Among 198 isolated mutants, Mutant-58 strain generated by EMS mutagenesis was selected for further assessment. The Mutant-58 strain showed significant morphological changes as compared to the parent strain. Furthermore, statistically optimized media composition resulted in the higher production of erythritol (91.2 ± 3.4 g/L) with a yield of 40.7 ± 3.4 % in shake flask experiments. The optimized medium composition for erythritol production constitutes (g/L) 225 jaggery, 4.4 yeast extract (YE), 4.4 KH2PO4, 0.31 MgSO4, and pH 5.5. The present study demonstrated strain improvement, media, and process optimization resulting in a 30% increase in the erythritol production in the Mutant-58 as compared to the parent strain. This is also the first instance where jaggery has been used as a cost-effective carbon source alternative to glucose for industrial-scale erythritol production.

High yield of heat-resistant spores of Bacillus megaterium in bioreactors

Microbial inoculants have been proposed as a sustainable and cost-effective alternative to conventional agricultural practices. However, the effectiveness of these bio-based products can be negatively impacted by reduced cell viability during product development, storage, and commercialization. To address these limitations, it is critical to identify and optimize the cultivation conditions that can lead to the production of highly resistant spores for use as inoculants. In this study, optimal cultivation conditions for obtaining Bacillus megaterium B119 spores in bioreactors were reached, with increases in the sporulation efficiency and heat resistance. The best nutrient medium and optimal temperature and initial pH conditions were obtained on shake flask experiments and subsequently validated in two bench-scale bioreactor models (stirred and aerated tank and the bubble column). The optimized temperature and initial pH were found to be 37 °C and 5.6. The highest concentration of heat-resistant Bacillus spores of 1.31·1012 CFU/mL was obtained in the bubble column bioreactor with a sporulation efficiency of 100.0%, a heat resistance of 78.4%, and an energetic efficiency on spores production of 4.83·1010 CFU/J. The production of highly resistant spores can significantly improve the survival rate of microorganisms during inoculant production and application, thereby enhancing their effectiveness in the field.

Changes in microbial community structure during the biooxidation of iron and inorganic/organic sulfur provide prediction of acid mine drainage from coal spoil

To thoroughly investigate the formation mechanism of acid mine drainage (AMD) from coal mine spoil, this study conducted microbial shake flask experiments on gangue possessing inorganic and organic sulfur to predict the future generation of AMD. The results revealed that microbial-mediated sulfur oxidation effectively lowered the pH of leachate and increased metal ion and sulfate concentrations. The oxidation of pyrite sulfur and thiophene sulfur contributed to 70 % and 30 % of the total acid production, respectively, highlighting the importance of both processes in coal mine acidification. The abundance and diversity of the microbial community increased, with “new” iron and sulfur oxidizing bacterial species during acidification, with “new” iron and sulfur oxidizing bacterial species, such as Sulfobacillus sp. and Acidibacillus sp., emerging and constituting approximately one-third of the bacterial population. These bacteria survived desiccation and proliferated faster than A. ferrooxidans YQ-N3 when conditions favored growth.

Liquid-liquid chromatography isolation of Petasites hybridus sesquiterpenes and their LC-HR-MS/MS and NMR characterization

Petasites hybridus L. (butterbur, Asteraceae) is a well-known medicinal plant traditionally used as a remedy for neurological, respiratory, cardiovascular, and gastrointestinal disorders. Eremophilane-type sesquiterpenes (petasins) are considered to be the major bioactive constituents of butterbur. However, efficient methods to isolate high-purity petasins in sufficient amounts for further analytical and biological testing are lacking. In this study, various sesquiterpenes were separated from a methanol rootstock extract of P. hybridus with liquid-liquid chromatography (LLC). The appropriate biphasic solvent system was selected using the predictive thermodynamic model COSMO-RS and shake-flask experiments. After the selection of the feed (extract) concentration and operating flow rate, a batch LLC experiment was performed with n-hexane/ethyl acetate/methanol/water 5/1/5/1 (v/v/v/v). For those LLC fractions containing petasin derivatives with purities < 95%, a preparative high-performance liquid chromatography purification step followed. All isolated compounds were identified by state-of-the-art spectroscopic methods, i.e., liquid chromatography coupled with high-resolution tandem mass spectrometry and nuclear magnetic resonance techniques. As a result, six compounds were obtained, namely 8β-hydroxyeremophil-7(11)-en-12,8-olide, 2-[(angeloyl)oxy]eremophil-7(11)-en-12,8-olide, 8α/β-H-eremophil-7(11)-en-12,8-olide, neopetasin, petasin, and isopetasin. The isolated petasins can be further used as reference materials for standardization and pharmacological evaluation.

Effects of potassium monopersulfate on nitrification activity and bacterial community structure of sponge biocarrier biofilm in Litopenaeus vannamei aquaculture system

ABSTRACT Effects of potassium monopersulfate (KMPS) on the nitrification activity, aquacultural water quality and bacterial community structure of sponge biocarriers with pre-cultured biofilm (SBBF) were analysed through shaking flask experiments and L. vannamei aquaculture experiments. Changes in the ammonia oxidation rate (AOR) and nitrite oxidation rate (NOR) of SBBF under six KMPS concentration treatments (0, 1, 2, 3, 4 and 5 mg/L) were studied. The results showed that the AOR and NOR of SBBF treated with high concentrations of KMPS (3, 4 and 5 mg/L) were significantly lower than those of the control group (CK) (p < 0.05). However, compared with the first dosing of NH4Cl and NaNO2, the inhibition of AOR and NOR by KMPS on AOR and NOR was weakened after the second and third dosing times. That is, AOR and NOR can recover partially or completely over time. The L. vannamei aquaculture experiment was performed using four concentrations of KMPS (0, 2, 4 and 8 mg/L). The results showed that with increasing KMPS dosage, the average and peak concentrations of NH4 +-N and NO2 –-N in each treatment significantly increased (P < 0.05), and the final body weight of shrimp significantly decreased (P < 0.05). Furthermore the highest dose (8.0 mg/L) of KMPS reduced the survival rate by 9.33% compared to the CK. High-throughput sequencing analysis of the biofilm structure showed that the relative abundances of Nitrospirota, Nitrosomonas and Nitrococcus, which are related to nitrogen cycling, and beneficial bacteria including Firmicutes and Bacilli decreased with the addition of KMPS (p < 0.05).

Investigation of a Broad-Bean Based Low-Cost Medium Formulation for Bacillus subtilis MSCL 897 Spore Production

Bacillus subtilis (Bs) is a bacterium that benefits plants and is used in the production of bio-fungicides. The cultivation of Bs is a crucial step in bio-control preparation production, as it greatly impacts the quality and price of the final product. In a series of shake flask experiments, we investigated the economically feasible broth composition for spore production of Bacillus subtilis MSCL 897, a Latvian soil isolate. Our study investigated the impact of utilizing legume-based flours (such as broad bean, grey pea, and soybean) as the primary nitrogen source, along with sugar-beet molasses, sucrose, or glucose as the carbon source, and yeast extract, peptone, and corn-steep liquor as growth factor additives. Additionally, we examined the effect of using (NH3)2HPO4 or urea as supplementary nitrogen sources, as well as previously established media formulations, on spore yield. Our results showed that a culture medium composed of broad bean flour (10 g/L) and molasses (10 g/L) led to spore productivity of 1.35 ± 0.47 × 108 CFU/mL at 48 h. By enriching the culture medium base constituents with a minor (0.5–1.0 g/L) yeast extract or corn-steep liquor additive, a notable increase in spore productivity was observed, with values of 2.00 ± 0.28 × 108 and 2.34 ± 0.18 × 108 CFU/mL at 48 h, respectively, and sporulation efficiency &gt; 80–90%. As a result, we achieved a high spore yield of the Bacillus subtilis MSCL 897 strain, demonstrating the competitiveness of our approach, which relied on a low-cost medium made mainly from locally available and renewable raw materials.

Investigating the effect of electrosprayed alginate/PVA beads size on the microbial growth kinetics: Phenol biodegradation through immobilized activated sludge

The presence of cyclic organic compounds, including phenol, in the wastewater of many industries has made phenol removal an important issue. Meanwhile, the biological methods of removing phenol have attracted the attention of researchers in recent years. Recently, the use of immobilized microbial cells is proposed as a new approach in industrial wastewater treatment. In this research, the aim is to study the effect of immobilized beads size on the phenol biodegradation efficiency and specific microbial growth rate. For this purpose, electrospray technique was used to immobilize activated sludge in hybrid matrix of alginate and polyvinyl alcohol (PVA). The fabricated alginate/PVA beads were characterized using Fourier transform infrared spectroscopy (FTIR). Evaluation of the results related to the free and immobilized cell systems in the shake flask experiments showed that at low phenol concentrations the immobilized cell system had the same performance as the free cell system, while the immobilized cell system at higher concentrations had a better performance in removing phenol so that at a concentration of 2000 mg/L, removal percentage has increased from 15% to 25–34%. On the other hand, in this survey, the kinetic behavior of activated sludge was in good agreement with Haldane's equation. Moreover, the maximum specific growth rate was measured 0.033 and 0.041 (h−1) beside 544 and 636 mg/L substrate inhibition constant, for free and immobilized cell systems, respectively. This result shows that the phenol biodegradation has been improved by using the cell immobilization technique especially with applying the smaller beads, which is due to improved mass transfer and microbial cell protection from harsh environments.

Extra-cellular production of uricase through the sec-type secretion system in Escherichia coli

A sec-type protein secretion is an alternative approach to recombinant protein production, where protein expression and purification are simultaneously achieved. In this study, uricase (urate oxidase, EC 1.7.3.3) was fused with a sec-type Epr signal peptide to achieve the extra-cellular uricase production in Escherichia coli MG1655. In shake-flask experiments, the enzyme activity and the specific activity in the extra-cellular fraction reached 0.73 ± 0.05 U/mL and 3.17 ± 0.22 U/mg-protein, respectively. When a 3-L bioreactor was used, the extra-cellular fraction exhibited the enzyme activity of 0.74 ± 0.00 U/mL and the specific activity of 2.75 ± 0.35 U/mg-protein. It was estimated that 89 % and 72 % of total enzyme activity as produced were found in the extra-cellular fraction in the shake flask and the 3-L bioreactor, respectively. The uricase secretion in E. coli was further supported by SDS-PAGE. In contrast, no uricase activity was found when Bacillus subtilis 168 was used as the host. This study demonstrates the efficacy of protein secretion using the Epr signal peptide in E. coli MG1655. Finally, this study presents that acidic pH results from the acetate production could be detrimental to extra-cellular uricase production.

Construction of the advanced flavin mononucleotide producers in the flavinogenic yeast Candida famata.

Flavin mononucleotide (FMN, riboflavin-5'-phosphate) is flavin coenzyme synthesized in all living organisms from riboflavin (vitamin B2 ) after phosphorylation in the reaction catalyzed by riboflavin kinase. FMN has several applications mostly as yellow colorant in food industry due to 200 times better water solubility as compared to riboflavin. Currently, FMN is produced by chemical phosphorylation of riboflavin, however, final product contains up to 25% of flavin impurities. Microbial overproducers of FMN are known, however, they accumulate this coenzyme in glucose medium. Current work shows that the recombinant strains of the flavinogenic yeast Candida famata with overexpressed FMN1 gene coding for riboflavin kinase in the recently isolated by us advanced riboflavin producers due to overexpression of the structural and regulatory genes of riboflavin synthesis and of the putative exporter of riboflavin from the cell, synthesized elevated amounts of FMN in the media not only with glucose but also in lactose and cheese whey. Activation of FMN accumulation on lactose and cheese whey was especially strong in the strains which expressed the gene of transcription activator SEF1 under control of the lactose-induced LAC4 promoter. The accumulation of this coenzyme by the washed cells of the best recombinant strain achieved 540 mg/L in the cheese whey supplemented only with ammonium sulfate during 48 h in shake flask experiments.

Spectrophotometric analysis of bioactive metabolites and fermentation optimisation of Streptomyces sp. HU2014 with antifungal potential against Rhizoctonia solani

Streptomyces spp. produce a range of secondary metabolites that can inhibit phytopathogen growth. This study aimed to optimise the environmental and nutritional parameters for improving the production of antifungal components by Streptomyces sp. HU2014 through statistical approaches. Spectrophotometry and bioassay revealed that two fractions, F6 and F8, from the cell-free filtrates of HU2014 culture broth were rich in antifungal compounds. Subsequently, single-factor experiments were conducted using glucose-peptone-yeast extract medium as basal medium, and the effects of different parameters on the production of bioactive metabolites by Streptomyces sp. HU2014 were evaluated and optimised using shake-flask experiments as follows: incubation time, 10 days; temperature, 25 °C; agitation speed, 150 rpm; inoculum size, 5% (v/v); initial pH, 7.0; and medium composition, dextrin, yeast extract and KNO3. The central composite face-centred design from response surface methodology predicted 39.6 g/L dextrin, 8.5 g/L yeast extract and 2.5 g/L KNO3 as the optimum medium composition. The validation experiment confirmed that a 50-fold dilution of the optimised filtered fermentation broth of the strain culture showed an increase of 14.9% in the inhibition of Rhizoctonia solani YL-3 when compared with the basal medium. A good correlation between measured and predicted values of the model validated both the responses. Thus, the present study used statistical optimisation to improve the bioactive metabolites productivity in Streptomyces sp. HU2014.

Accelerated Adaptive Laboratory Evolution by Automated Repeated Batch Processes in Parallelized Bioreactors

Adaptive laboratory evolution (ALE) is a valuable complementary tool for modern strain development. Insights from ALE experiments enable the improvement of microbial cell factories regarding the growth rate and substrate utilization, among others. Most ALE experiments are conducted by serial passaging, a method that involves large amounts of repetitive manual labor and comes with inherent experimental design flaws. The acquisition of meaningful and reliable process data is a burdensome task and is often undervalued and neglected, but also unfeasible in shake flask experiments due to technical limitations. Some of these limitations are alleviated by emerging automated ALE methods on the μL and mL scale. A novel approach to conducting ALE experiments is described that is faster and more efficient than previously used methods. The conventional shake flask approach was translated to a parallelized, L scale stirred-tank bioreactor system that runs controlled, automated, repeated batch processes. The method was validated with a growth optimization experiment of E. coli K-12 MG1655 grown with glycerol minimal media as a benchmark. Off-gas analysis enables the continuous estimation of the biomass concentration and growth rate using a black-box model based on first principles (soft sensor). The proposed method led to the same stable growth rates of E. coli with the non-native carbon source glycerol 9.4 times faster than the traditional manual approach with serial passaging in uncontrolled shake flasks and 3.6 times faster than an automated approach on the mL scale. Furthermore, it is shown that the cumulative number of cell divisions (CCD) alone is not a suitable timescale for measuring and comparing evolutionary progress.

Metabolic engineering of Escherichia coli to utilize methanol as a co-substrate for the production of (R)-1,3-butanediol

Due to its abundance, cost-effectiveness, and high reducibility, methanol has gained considerable attention in the biomanufacturing industry as a nonfood feedstock for the production of value-added chemicals. The range of chemicals that can be derived from methanol, however, remains constrained and is currently in the concept validation phase. This study aimed to develop and evaluate a hybrid methanol assimilation pathway in Escherichia coli to improve the production of (R)-1,3-butanediol ((R)-1,3-BDO) by utilizing methanol and sugars as co-substrates. By combining the methanol dehydrogenase (MDH) from the prokaryotes with the dihydroxyacetone synthase (DAS) from the eukaryotes, the hybrid pathway facilitates methanol conversion into the central metabolism while generating NADH at the same time. Through pathway optimization and targeted gene deletions, we have successfully developed an E. coli strain capable of producing 5.79 g/L (R)-1,3-BDO in shake flask experiments and 13.71 g/L (R)-1,3-BDO with a yield of 0.35 C-mol/C-mol in batch fermentation using methanol and glucose as co-substrates. Our study also showed the incorporation of 13C-methanol into cellular intermediates and an increase in NAD(P)H concentration, confirming the role of methanol as a co-substrate and supplier of NADH. In addition, our study also demonstrated the co-utilization of methanol with xylose for the production of (R)-1,3-BDO, expanding the substrate spectrum for sustainable 1,3-BDO production.

Ethanol production by immobilized Saccharomyces cerevisiae cells on 3D spheres designed by different lattice structure types

Natural or synthesized materials can be used for cell immobilization to enhance cell viability and productivity in fermentation processes (batch, fed-batch or continuous fermentations). 3D printing is one of the newest technologies to produce unique immobilization support materials. In this research, the selection strategy of 3D lattice structure type for unique immobilization materials, effect of different lattice structure types on cell immobilization in a shake flask experiments and optimization of ethanol production in a laboratory scale bioreactor with 3D spheres were studied. Firstly, 3D designs were compared to determine the best type of 3D spheres with a higher area:volume ratio for cell immobilization. Then shake flask experiments were performed and showed that W, 20 mm is the best 3D sphere combination for ethanol fermentation. Finally, over 37 g/L ethanol (ethanol yield ranged from 41.91% to 46.36%) was produced at all repeated batch cycles by using 100 g/L initial sugar with at least 82% sugar utilization capacity in a lab-scale bioreactor successfully. All results showed that 3D-printed materials could be used as immobilization agents for microorganisms by selecting the proper geometry, size and printing technology.

Sugarcane Bagasse as Carbon Source and Filler to Enhance the Treatment of Low C/N Wastewater by Aerobic Denitrification Flora

Sugarcane bagasse as a biomass solid waste has good prospects as a carbon source for biological wastewater treatment. However, it has rarely been investigated for aerobic denitrification (AD). Herein, we investigated its carbon release performance and constructed a bagasse-loaded sequencing batch biofilm reactor (SBBR) to investigate the feasibility of sugarcane bagasse as a carbon source and filler for AD bacteria to treat wastewater with low carbon–nitrogen ratios (C/N). Results showed that chemical oxygen demand (COD) leaching from sugarcane bagasse was 418.07 ± 17.05 mg/g; high-temperature and high-pressure operation had no significant effect on the carbon release performance of the bagasse. A shake-flask experiment revealed that adding sugarcane bagasse significantly enhanced the AD flora denitrification effect in low C/N wastewater; the removal process of NO3−–N by AD flora followed first-order kinetics. In the SBBR inoculated with AD flora, biofilm formation on the bagasse surface was evident; the NO3−–N removal rate reached 20.20 ± 4.27% after 28 operation cycles, which was significantly higher than that of the control sequencing batch reactor during the same period, and the effluent COD stabilized below 50 mg/L. The results provide a reference for AD application in the practical wastewater treatment and biomass resource utilization of sugarcane bagasse.

Efficient bioimmobilization of cadmium contamination in phosphate mining wastelands by the phosphate solubilizing fungus Penicillium oxalicum ZP6

In recent years, Cd contamination in phosphate mining wastelands has become increasingly serious, posing a great threat to ecological stability and human health. In this study, we aimed to investigate the bioremediation effect and mechanisms of phosphate-solubilizing fungi (Penicillium oxalicum ZP6) on Cd contamination in phosphate mining wastelands. A liquid shake-flask experiment showed that strain ZP6 was able to solubilize phosphate rock by reducing the pH, thus effectively removing Cd2+. Moreover, strain ZP6 adsorbed Cd2+ mainly through tight binding. Soil experiments showed that 81.64% Cd2+ removal was achieved after 21 d of inoculation with ZP6. FTIR and XRD analyses revealed that strain ZP6 achieved bioimmobilization of Cd2+ through the formation of Cd-phosphate and Cd-oxalate precipitates. Inoculation with strain ZP6 increased the microbial community diversity of Cd-contaminated soil from a phosphate mining wasteland, increasing the relative abundance of unclassified-d-unclassified and Actinobacteria. The activation of high-abundance genes (ATPF1A, atpD, GGT, cydA, and G6PD) was promoted, thereby enhancing the expression of signaling and metabolism-related functional genes. Our study indicates that strain ZP6 has enormous potential for bioremediation of Cd in phosphate mining wastelands. It also provides an efficient, green and economical biomaterial for remediating Cd contamination in phosphate mining wastelands.

Response of mixed bacterial culture towards dibenzothiophene desulfurization under the influence of surfactants and microscopically (SEM and TEM) characterized magnetic Fe3 O4 nanoparticles.

Excessive emission of sulfur dioxides from the combustion of coal and other fossil fuels for thermal and industrial purposes has been associated with serious environmental hazards. Biodesulfisation (BDS) can be an effective approach for reducing the impact of toxic gases to its inbuilt operational feasibility under ambient environmental conditions. In the present research, two strategies for BDS of a standard organosulfur compound such as dibenzothiophene (DBT) were investigated under laboratory conditions. In the first treatment, the role of different surfactants such as Tween-20, Tween-80, SDS, and EDTA on the desulfurization of DBT was investigated by the application of bacterial consortium IQMJ-5. In the second treatment, Iron oxidenanoparticles were synthesized and immobilized on the surface of bacteria cells. Shake flask experiments were conducted with immobilized cells, surfactant amended immobilized cells, and control or noncoated cells. Among different surfactant treatments, Tween-80 was found to be the most effective surfactant, showing maximum desulfurization activity at a concentration of 5g/L. The transmission electron microscopy and X-ray diffraction analysis indicated that produced nanoparticles were spherical in shape with a size of about 46 nm and had a stoichiometric ratio of 55.85% and 44.15% between O and Fe, respectively. The nanoparticle treatment enhanced the DBT desulfurization process up to 11.37% as compared to the control, specifically when immobilized cells were used. Therefore, it was concluded that nanoparticles treatments with immobilization of the bacterial cells enhanced the desulfurization rate of DBT under ambient reaction conditions and provide a sustainable alternative for commercial coal BDS.

Isolation of lanosterol and dihydrolanosterol from the unsaponifiable matter of lanolin by urea complexation and countercurrent chromatography in heart-cut recycling mode

4,4-Dimethyl-substituted sterols are bioactive minor sterols of most animal fats and plant oils, but higher shares are present in lanolin (wool grease). Here, the isolation of the 4,4-dimethyl-substituted sterols dihydrolanosterol and lanosterol from lanolin by countercurrent chromatography (CCC) is described. An initial examination of the hexane extract of saponified lanolin showed the presence of relatively high portions of fatty alcohols which were known to co-elute with the target analytes in CCC. Hence, fatty alcohols were precipitated by urea complexation. Unexpectedly, 4,4-dimethyl-substituted sterols were also found in the crystalline fraction, while cholesterol and other desmethylsterols were detected in the liquid phase. Urea complexation represented a useful preparative method for the separation of desmethylsterols and 4,4-dimethyl-substituted sterols from lanolin. Shake flask experiments of 4,4-dimethyl-substituted sterols and fatty alcohols with 14 biphasic solvent systems indicated suitable partition coefficients (K values) with n–hexane/ethanol/water (12:8:1, v/v/v) and n–hexane/benzotrifluoride/acetonitrile (20:7:13, v/v/v). After initial tests with conventional CCC, the application of CCC in heart–cut recycling mode provided 4,4-dimethyl-substituted sterols with purities of 99 % (dihydrolanosterol) and 95 % (lanosterol).