Biosurfactant-producing Bacteria Research Articles

Isolation and screening of potential biosurfactant-producing bacteria from used engine oil-contaminated soil

Biosurfactants are compounds with surface activity and emulsification ability that are produced by an extensive variety of microorganisms such as bacteria, actinobacteria, algae and fungi. This study was done in order to isolate and screen for potential biosurfactant-producing bacteria from soil contaminated with used engine oil. Mineral salt medium (MSM) with used engine oil as the sole carbon source was used to isolate potential biosurfactant producing bacteria. The potential isolates were screened through drop collapse test, oil spread test and emulsification index test. There were 26 isolates that have been recovered from the enrichment culture. Three isolates namely 2c, 2k and 2m were further identified as the potential biosurfactant producers based on drop collapse, oil displacement and emulsification index tests. Then, the selected isolates were grown in MSM supplemented with used engine oil as sole carbon source to determine the production of biosurfactant. Isolate 2k produced 15.1 g/L biomass which was the highest as compared to the other isolates. In contrast, the highest yield of biosurfactant (0.029 g biosurfactant/g biomass) was produced from isolate 2m eventhough the biomass was lower than isolate 2k.

Comparison of Biosurfactants Yield Produced by Pseudomonas fluorescens Strain PC20 Isolated from Hydrocarbon Contaminated Soil using Different Agricultural Waste

Biosurfactants are amphipathic compounds produced extracellularly by microorganisms on cell surfaces, or excreted extracellularly. They contain hydrophilic and hydrophobic moieties that reduce surface and interfacial tension between molecules at the surface and interface respectively. The present study focused on isolation, screening, identification of biosurfactant producing bacteria and also development of economical methods for biosurfactant production by the use of unconventional substrates. The research investigated the potential of utilizing agro industrial (Pineapple and cassava) wastes to replace synthetic media for biosurfactant production. Eighteen isolates obtained were screened for biosurfactant activity using Emulsification Index, haemolysis and oil displacement method. The bacteria Pseudomonas fluorescens Strain PC20 isolated was able to grow and produce biosurfactant 10ml and 5ml per 200ml of cassava and pineapple peel respectively.

Screening and Identification of Efficient Biosurfactant Producing Bacteria for some Medical Applications

Screening and Identification of Efficient Biosurfactant Producing Bacteria for some Medical Applications

Isolation of biosurfactant producing bacteria from Potwar oil fields: Effect of non-fossil fuel based carbon sources

AbstractBiosurfactants are surface-active metabolites produced by microorganisms. Biosurfactants tend to solubilize hydrocarbons in the surrounding environment, by reducing surface tension and increasing carbon uptake. In this study, isolation of biosurfactant producing bacteria and effect of renewable, non-fossil fuel based carbon sources on production were assessed. Oil field produced water was collected from Potwar oil fields and thirteen strains were isolated. Strains were screened for biosurfactant production by hemolysis test, emulsification index test, emulsification assay, oil displacement test, drop collapse test, tilted glass slide test and oil coated agar emulsification test. Strains were further screened for maximum production on cost effective renewable carbon source e.g. glucose, waste glycerol and animal fat. Promising strains were identified as Bacillus subtilis (MH142143), Pseudomonas aeruginosa (MH142144), Bacillus tequilensis (MH142145) and Bacillus safensis (MH142146) by 16S rRNA gene sequencing. Among all isolates, highest biosurfactant production on glucose (37%), glycerol (48%) and animal fat oil (49%), after 24 h cultivation was by Pseudomonas aeruginosa. Biosurfactant showed similarity to rhamnolipids by Thin Layer Chromatography (TLC). Assessment of bioactive propertiaes of rhamnolipid showed strong antimicrobial activity against Bacillus spp. Future investigations can be focused on application of these strains in environmental as well as industrial sectors.

Isolation and identification of the same type LMG1242 of biosurfactant-producing strain of Pseudomonas aeruginosa from different oil contaminated soils

Emulsification is a major rate limiting step in any biological conversion of heavy hydrocarbons. Here, thirty bacterial strains were isolated from seven native consortia and screened for biosurfactant-producing activities which could enhance emulsification of heavy oils. The consortia were obtained by sampling from oil contaminated soils of different petroleum refineries of Iran. The oil spreading test, drop collapse test, emulsification index (E24) and surface tension measurements were used to evaluate the biosurfactant producing activities of the strains. A total number of 5 strains out of 30 were finally selected as the best biosurfactant-producing bacteria. The clear zone diameters in their oil spreading test were 4 to 5 cm, and E24 were 48 to 92%. The selected strains also properly lowered the surface tensions of the supernatants to 23.5-32 mN/m when grown on heavy diesel. All the selected strains were identified by 16S rRNA sequencing analysis as Pseudomonas aeruginosa LMG1242 (99.9% homology) which is a newly isolated type in oil polluted soils. The results suggest P. aeruginosa LMG1242 as a predominant and highly active biosurfactant producing bacterium which could be further evaluated in petroleum bioremediation and bioprocessing applications.

Screening of biosurfactant producing bacteria from hydrocarbon contaminated soil

Biosurfactants are surface-active molecule produced by microorganisms and have several advantages over the chemical surfactants. The objective of this research was to obtain biosurfactant-producing bacteria from hydrocarbon contaminated soils in Jakarta Bay and to observe their biosurfactant activity through emulsification capacity and surface tension reduction capability. Overall, 19 isolates were screened for biosurfactant production by hemolysis assay, drop collapse assay and oil spreading assay. The result showed that 13 strains gave positive results on the screening tests and were determined as biosurfactant-producing bacteria. Three strains (TKY 3, TKY 14 and TKY 19) had the best biosurfactant activity. The lowest surface tension value was achieved by two strains (TKY 19 and TKY 14) with the same value of 37,84±1,45 mN/m.

Enhanced biodegradation of styrene vapors in the biotrickling filter inoculated with biosurfactant-generating bacteria under H2O2 stimulation

Biotrickling filters (BTFs) applied to hydrophobic volatile organic compounds (VOCs) suffer from limited mass transfer. Phase transfer kinetic and equilibrium effects limit the biodegradation of hydrophobic VOCs especially at high concentrations. This study evaluates two strategies for overcoming the problem. First, a natural process was used to enhance the aqueous availability of styrene, a hydrophobic VOC model, by inoculating the BTF with a mixture of biosurfactant–generating bacteria. This method achieved a maximum elimination capacity (ECmax) of 139 g m-3h−1 in the BTF at an empty bed residence time (EBRT) of 60s. The highest concentrations of the biosurfactants surfactin and rhamnolipid were 205 and 86 mg L-1, respectively, in this step. Sequencing 16S rRNA confirmed the presence of biosurfactant-producing bacteria capable of biodegrading styrene in the BTF including Bacillus sonorensis, Bacillus subtilis, Lysinibacillus sphaericus, Lysinibacillus fusiformis, Alcaligenes feacalis, Arthrobacter creatinolyticus, and Kocuria rosea. Second, the effect of adding H2O2 to the recycle liquid on the BTF performance was determined. The biodegradation and mineralization of styrene in the BTF operated at a loading rate of 266 g m-3h−1 and H2O2/styrene molar ratio of 0.05 with EBRT as short as 15 s were 94% and 53%, respectively, with the EC of 250 g m-3h−1. High concentrations of antioxidant enzymes (peroxidase and catalase: 56 and 7 U gbiomass-1, respectively) were produced and biosurfactant generation was increased in this step, contributing to enhanced styrene biodegradation and mineralization. The styrene biodegradation and mineralization values in the BTF in the last day operated under similar conditions but without H2O2 were 11.4% and 5.3%, respectively. The bacterial population had no considerable change in the BTF after adding H2O2. Accordingly, stimulating the BTF inoculated with biosurfactant-generating bacteria with H2O2 is a promising strategy for improving the biodegradation of hydrophobic VOCs.

Approaching Electrodes Configurations in Bio-Electrokinetic Deoiling of Petrochemical Contaminated Soil

The expeditious growth of the petroleum industry and its products in the last three decades has led to increased pollution of soil which is of great concern considering the effects it poses to the environment. Oil waste usually considered as hazardous due to its composition presents the same problems as other petroleum-based products to the environment. An electrokinetic reactor with an inoculum of biosurfactant producing bacteria was studied at a bench scale with different electrode spacing configurations. A DC powered electrokinetic reactor consisting of electrode/electrolyte compartments, and a matrix chamber was operated under a constant voltage of 30 V with configurations of fixed electrodes spacing’s of 335 mm, 260 mm,185 mm and continuous approaching electrodes at 335 mm, 260 mm and 185 mm. The biosurfactant producing microbes and cell-free biosurfactant were introduced in the matrix chamber after which the reactor was left to run for 10 days under the electric field. The electroosmotic flow, electrical current, pH and biodegradation of the hydrocarbons in the soil were observed and assessed. The current in the reactor was highest with the least electrode distance of 185 mm. The increase in current led to a directly proportional increase in the electroosmotic flow towards the cathode leading to the increased coalescence of the oil from the soil as compared to the other electrode distances. The analysis of the results showed a reduction in the total carbon content in the soil with viable oil recovery rates for all the electrode distances with 185 mm being the most effective. The viable cell counts in the reactor showed significant growth of the bacteria in the first 6 days under the applied voltage of 30 V enabling the biodegradation of the petrochemical pollutants by the strain for all the four different electrode configurations.

Identification and optimization of biosurfactant producing bacteria isolated from rag layer crude oil emulsion

Biosurfactants are surface active compounds that synthesized as secondary metabolite by wide range of bacteria and have characteristic in lowering surface and interfacial tension. This study aimed to isolate and identify biosurfactant producing bacteria from rag layer crude oil emulsion. Rag layer is considered as undesirable material as it is difficult to be separated because of the stable interaction between different components. Mineral salt medium supplemented with glucose and crude oil was used to screen the ability of isolates to produce biosurfactant. Five bacterial strains that successfully isolated from rag layer crude oil emulsion sample were screened for hydrocarbon degradation and biosurfactant production. Two isolates shown positive results in drop collapse test, surface tension measurement and emulsification index, namely P3b and P4. 16S rRNA analysis revealed P3b and P4 to be closely related to Enterobacter xiangfangensis while P4 was Shewanella chilikensis, respectively. Only isolate P3b was selected for further study. Enterobacter xiangfangensis SSP3b16 was found to grow optimally at 37°C, pH 7.0 and 10mM glucose. The highest reduction of surface tension was recorded when culture medium supplemented with 7 % (v/v) glucose and 2 g/L ammonium nitrate. From this study, the biosurfactant production by Enterobacter xiangfangensis SSP3b16 can potentially be exploited to enhance oil recovery as well as in solving the rag layer problem in oil industries.

The genome of Alcaligenes aquatilis strain BU33N: Insights into hydrocarbon degradation capacity.

Environmental contamination with hydrocarbons though natural and anthropogenic activities is a serious threat to biodiversity and human health. Microbial bioremediation is considered as the effective means of treating such contamination. This study describes a biosurfactant producing bacterium capable of utilizing crude oil and various hydrocarbons as the sole carbon source. Strain BU33N was isolated from hydrocarbon polluted sediments from the Bizerte coast (northern Tunisia) and was identified as Alcaligenes aquatilis on the basis of 16S rRNA gene sequence analysis. When grown on crude oil and phenanthrene as sole carbon and energy sources, isolate BU33N was able to degrade ~86%, ~56% and 70% of TERHc, n-alkanes and phenanthrene, respectively. The draft genome sequence of the A. aquatilis strain BU33N was assembled into one scaffold of 3,838,299 bp (G+C content of 56.1%). Annotation of the BU33N genome resulted in 3,506 protein-coding genes and 56 rRNA genes. A large repertoire of genes related to the metabolism of aromatic compounds including genes encoding enzymes involved in the complete degradation of benzoate were identified. Also genes associated with resistance to heavy metals such as copper tolerance and cobalt-zinc-cadmium resistance were identified in BU33N. This work provides insight into the genomic basis of biodegradation capabilities and bioremediation/detoxification potential of A. aquatilis BU33N.

Ralstonia pickettii Enhance the DDT Biodegradation by Pleurotus eryngii.

DDT is a hydrophobic organic pollutant, which can be bio-accumulated in nature and have adverse consequences on the physical condition of humans and animals. This study investigated the relationship between the white-rot fungus Pleurotus eryngii and biosurfactantproducing bacterium Ralstonia pickettii associated with the degradation of DDT. The effects of R. pickettii on fungal development were examined using in vitro confrontation assay on a potato dextrose agar (PDA) medium. R. pickettii culture was added to the P. eryngii culture at 1, 3, 5, 7, and 10 ml (1 ml ≈ 1.44 × 1013 CFU). After 7 d incubation, about 43% of the initial DDT (12.5 μM) was degraded by the P. eryngii culture only. The augmentation of 7 ml of R. pickettii culture revealed a more highly optimized synergism with DDT degradation being approximately 78% and the ratio of optimization 1.06. According to the confrontational assay, R. pickettii promoted the growth of P. eryngii towards the bacterial colony, with no direct contact between the bacterial cells and mycelium (0.71 cm/day). DDD (1,1-dichloro-2,2-bis(4- chlorophenyl) ethane), DDE (1,1-dichloro-2,2-bis(4-chlorophenyl) ethylene), and DDMU (1- chloro-2,2-bis(4-chlorophenyl) ethylene) were identified as metabolic products, indicating that the R. pickettii could enhance the DDT biodegradation by P. eryngii.

Complete Genome Sequence of Hydrocarbon-Degrading Halotolerant Acinetobacter radioresistens DD78, Isolated from the Aconcagua River Mouth in Central Chile.

Acinetobacter radioresistens strain DD78 (= CCUG 69565) is a soil hydrocarbon-degrading and biosurfactant-producing bacterium isolated from chronically crude oil-polluted soil of the Aconcagua River mouth in Chile. The 3.25-Mb A. radioresistens DD78 genome (41.8% GC content) was completely sequenced, with 4 replicons, 2,970 coding sequences, and 77 tRNAs.

Stability test of biosurfactant produced by Bacillus licheniformis DS1 using experimental design and its application for MEOR

Microbial enhanced oil recovery (MEOR) is a proven tertiary recovery technique. Biosurfactant is a microbial bioproduct that plays an important role in MEOR applications. This study aimed to test biosurfactant stability using a design experiment based on response surface methodology. First, isolation and screening for potential biosurfactant-producing bacteria from crude oil samples was performed, followed by their characterization. A biosurfactant core flooding experiment was also conducted to examine bacterial activity on MEOR. Thirty-one sequential isolates of bacteria were screened based on qualitative and semi-qualitative parameters. One selected biosurfactant-producing bacterium was identified as Bacillus licheniformis DS1 based on phylogenetic analysis of the 16S rRNA gene. This bacterium had the highest emulsification activity (Ei24 = 65.19%) in light crude oil and could reduce the interfacial tension between oil and water with an effective critical-micelle concentration of 157.5 mg/L. The biosurfactant was observed as a growth-associated metabolite type and the Fourier transform infrared spectrum revealed that the biosurfactant produced belonged to a group of lipopeptides. The biosurfactant has good stability in maintaining emulsification activity at pH 4–10, high temperatures up to 120 °C, and with an NaCl concentration up to 10% (w/v). Based on response surface methodology using the Box–Behnken experimental design, the optimum condition for the most stable biosurfactant is pH 12, a 40 °C temperature and 10% salinity, with an Ei24 value of 94.28%. Core flooding experiments with biosurfactant resulted in 5.4% additional oil recovery. Therefore, this biosurfactant shows a high potential application for MEOR.

Assessment of biosurfactant-producing bacteria from oil contaminated soils and their hydrocarbon degradation potential

Biosurfactants (BSs) are amphiphilic compounds produced by microorganisms and have diverse applications in various industries. This study was aimed to identify and characterize BS producing bacteria from three oil-polluted sites from Pune, Maharashtra, India. Out of the total 48 bacterial isolates, 14 BS producing isolates were detected through drop collapse method (DCM) and oil spreading method (OSM), hemolytic activity, cetyl trimethyl ammonium bromide (CTAB) test, emulsification index (E24), and surface tension (SFT) measurement. The BS producing isolates were identified by matrix-assisted laser desorption/ionization time of flight mass spectrophotometry (MALDI-TOF MS) and 16S rRNA gene sequencing. Results indicated that the isolates belonged to six genera, including Bacillus, Agrococcus, Arthrobacter, Kocuria, Dietzia, and Lysinibacillus. We are reporting BS producing Agrococcus terreus for the first time. Out of 14 BS producing bacteria, four isolates showed β hemolysis in blood agar plates, while all isolates showed negative results on CTAB agar plate. A positive correlation was observed between DCM, OSM, and SFT measurements. Bacillus sp. (isolate PR170) presented the maximum diameter of the clear zone (32 mm) in OSM. The E24 was found to be in the range of 15–57%. Four isolates including PR170, PP151, PP168, and PG138 significantly reduced the SFT of the medium by up to 26.79, 27.81, 28.83, and 30.99 mN/m, respectively. Nine isolates were able to grow in media containing various polyaromatic hydrocarbons. Results gathered in this study demonstrated that isolates belonging to four genera from the contaminated sites had an outstanding potential for further application in biotechnological processes such as large-scale BS production or bioremediation of hydrocarbon polluted sites, and will contribute towards environmental sustainability.

The potential of indigenous bacteria from oil sludge for biosurfactant production using hydrolysate of agricultural waste

Abstract. Ni’matuzahroh, Sari SK, Ningrum IP, Pusfita AD, Marjayandari L, Trikurniadewi N, Ibrahim SNMM, Fatimah, Nurhariyati T, Surtiningsih T, Yuliani H. 2019. The potential of indigenous bacteria from oil sludge for biosurfactant production using hydrolysate of agricultural waste. Biodiversitas 20: 1374-1379. Biosurfactants are amphipathic compounds which are useful in various fields of health, industry, and remediation. Biosurfactants are produced by bacteria that grow in hydrocarbon or sugar substrates. Hydrolysis product of agricultural waste can be used as a biosurfactant production medium. This research aims to obtain biosurfactant producing bacteria from Balongan oil sludge, Indonesia. The ability to grow and produce biosurfactant by indigenous bacteria was tested using a medium of Synthetic Mineral Water (SMW) added by 209.3 ppm of rice straw hydrolysis product (RSHP). The growth of bacteria was evaluated through Total Plate Count (TPC) and biosurfactant production was evaluated through measurement of emulsification activity and surface tension. Six indigenous bacteria were capable to produce biosurfactants in the RSHP. Emulsification activity was not detected, but surface tension reduction was founded. The best biosurfactant was indicated by surface tension value of 53.56 mN/m with TPC value of 20.07 CFU/mL at the 5th day of incubation by BP (1) 5. The indigenous bacteria were identified as Propionibacterium BP (1) 1, Propionibacterium BP (1) 3, Bacillus BP (1) 4, Corynebacterium BP (1) 5, Corynebacterium BP (1) 8, and Rothia BP (1) 6. Utilization of sugar as hydrolysis product of agricultural waste is an innovation of raw materials for biosurfactant production.

Microbial\xa0enhanced oil recovery: interfacial tension and biosurfactant-bacteria growth

Microbial enhanced oil recovery (MEOR) is a method that utilises bacteria or bioproducts to increase oil recovery at the tertiary stage. Clostridium sp. produces biosurfactant that alters rock–fluid properties and increases oil detachment. The interaction between bacteria and surfactant is interesting relation to study. We revisit and develop models for biosurfactant-producing bacteria’s growth and the interfacial tension (IFT) response. The biosurfactant-producing bacteria growth model (BBG model) mimics the predator–prey interaction and the IFT response model derived from analogy. Both models form an integrated model called coupled-simultaneous model. We deliver the suitability of these models to experimental datasets by conducting parameter estimation. The decreased number of parameter in BBG model is with the help of rate estimation model. It estimates the bacteria growth rate and biosurfactant production rate. This research introduces a graphical method to narrow parameters initial guess in the IFT model. The method comes with a proposed index to compare surfactant performance called as surfactant performance index (SPI). The paper exposes the logic of each parameter, physics behind the models, and addresses the mathematical artefacts. The significant findings are valuable to anticipate bacterial performance for MEOR.

Isolation and characterization of biosurfactant producing bacteria from groundnut oil cake dumping site for the control of foodborne pathogens

Infection and intoxication are two common types of foodborne illnesses throughout the world. The aim of the present work was to isolate and characterize biosurfactant producing bacteria from groundnut oil cake dumping sites and evaluate their biocontrol effects against foodborne pathogens. Bacteria were isolated by enrichment culture technique and preliminary screening method. Biosurfactant activity evaluation was carried out by oil displacement, drop collapse test, lipase activity, hemolytic activity, emulsification index and emulsification assay methods. 16s rRNA sequence analysis was used for the isolate identification. Crude biosurfactant was extracted by acid precipitation method and characterized using FTIR (Fourier Transform Infrared Spectroscopy). Antibacterial activity was investigated using disc diffusion method. CMC and surface reduction were analyzed by DuNouy tensiometer. Top 10 strains were selected for biosurfactant activity assessment from the total 30 isolates. In addition, 16s rRNA sequence identified that the potential isolate was Bacillus pseudomycoides OR 1. Then, FTIR result of the extracted biosurfactant established the extract as a lipopeptide based on the absorption peaks at 3500 to 3200 cm−1 and 2963 to 2854.68 cm−1, respectively. 50 μg/mL of lipopeptide showed the highest antibacterial activity. Critical Micelle Concentration (CMC) of the lipopeptide was 60 mg/L and it reduced the surface tension of water from 71.6 to 31.6 mN/m. Hence, this study widens the scope to employ the bacterial lipopeptide surfactant as a promising biocontrol agent against foodborne pathogens.

Screening and characterization of biosurfactant produced by Pseudoxanthomonas sp. G3 and its applicability for enhanced oil recovery

Biosurfactants are one of the microbial bioproducts that are in most demand from microbial-enhanced oil recovery (MEOR). We isolated and screened potential biosurfactant-producing bacteria, followed by biosurfactant production and characterization, and a simulation of the MEOR application to biosurfactants in a sand-packed column. Isolate screening was conducted based on qualitative (hemolytic blood assay and oil-spreading test) and semi-qualitative (emulsification assay and interfacial tension measurement) parameters. Bacterial identification was performed using 16S rRNA phylogenetic analysis. Sequential isolation yielded 32 bacterial isolates, where Pseudomonas sp. G3 was able to produce the most biosurfactant. Pseudomonas sp. G3 had the highest emulsification activity (Ei = 72.90%) in light crude oil and could reduce the interfacial tension between oil and water from 12.6 to 9.7 dyne/cm with an effective critical-micelle concentration of 0.73 g/L. The Fourier transform infrared spectrum revealed that the biosurfactant produced was a glycolipid compound. A stable emulsion of crude extract and biosurfactant formed at pH 2–12, up to 100 °C, and with a NaCl concentration of up to 10% (w/v) in the response-surface method, based on the Box–Behnken design model. The sand-packed column experiment with biosurfactant resulted in 20% additional oil recovery. Therefore, this bacterium and its biosurfactant show potential and the bacterium is suitable for use in MEOR applications.

Biosurfactant-Producing Capability and Prediction of Functional Genes Potentially Beneficial to Microbial Enhanced Oil Recovery in Indigenous Bacterial Communities of an Onshore Oil Reservoir.

Microbial enhanced oil recovery (MEOR) is a bio-based technology with economic and environmental benefits. The success of MEOR depends greatly on the types and characteristics of indigenous microbes. The aim of this study was to evaluate the feasibility of applying MEOR at Mae Soon Reservoir, an onshore oil reservoir experiencing a decline in its production rate. We investigated the capability of the reservoir's bacteria to produce biosurfactants, and evaluated the potentials of uncultured indigenous bacteria to support MEOR by means of prediction of MEOR-related functional genes, based on a set of metagenomic 16s rRNA gene data. The biosurfactant-producing bacteria isolated from the oil-bearing sandstones from the reservoir belonged to one species: Bacillus licheniformis, with one having the ability to decrease surface tension from 72 to 32mN/m. Gene sequences responsible for biosurfactant (licA3), lipase (lipP1) and catechol 2,3-dioxygenase (C23O) were detected in these isolates. The latter two, and other genes encoding MEOR-related functional proteins such as enoyl-CoA hydratase and alkane 1-monooxygenase, were predicted in the bacterial communities residing the reservoir's sandstones. Exposure of these sandstones to nutrients, consisting of KNO3 and NaH2PO4, resulted in an increase in the proportions of some predicted functional genes. These results indicated the potentials of MEOR application at Mae Soon site. Using the approaches demonstrated in this study would also assist evaluation of the feasibility of applying MEOR in oil reservoirs, which may be enhanced by an appropriate nutrient treatment.

Withdrawn notice: 'Isolation and preliminary screening of biosurfactant producing bacteria from oil contaminated soil' [Braz. J. Biol. Sci. (2016), vol. 3, no. 6, p. 285-292

Withdraw.