Isolation, Screening, and Characterization of Biosurfactant-Producing Microorganisms from Petroleum-Contaminated Soil and Further Optimization of Parameters for Biosurfactant Production
Introduction: Biosurfactants are amphiphatic in nature and are surface-active compounds produced by microorganisms. These molecules reduce interfacial surface tension between aqueous solutions and hydrocarbon mixtures. Unfortunately, oil spills and industrial discharges from petroleum-related industries have been identified as the major pollution sources. The hydrophobicity and low aqueous solubility of petroleum pollutant limit the biodegradation process. The features that make biosurfactants as an alternative to commercially synthesized surfactants are its low toxicity, higher biodegradability and, hence, greater environmental compatibility, better foaming properties, and stable activity at extreme pH, temperature, and salinity. Objective: Therefore, in this study, hydrocarbon-degrading bacteria were screened from petroleum-contaminated soil, characterized and optimization of the physical and nutrient parameters were done to enhance the production of biosurfactants. Results: Petroleum-contaminated soil was collected from different petrol pumps in Pune and screening was done on minimal salt medium media containing palm oil as carbon source using hemolytic activity, emulsification index, drop-collapse test, and oil displacement method. The most promising strain was isolated and identified using Bergey’s Manual of Determinative Biology and 16s rRNA sequencing and was found to be Staphylococcus epidermidis. The optimization of various parameters, namely temperature, pH, carbon, and nitrogen sources on growth, and biosurfactant production was studied. The highest biosurfactant production was obtained when MSS media contains sucrose (carbon source) and urea (nitrogen source) at pH 10 and temperature 55°C. The Fourier transform-infrared (FT-IR) analysis of purified biosurfactant indicated the presence of lipopeptide biosurfactant when compared with reference FT-IR spectra.
The document summarizes the process of producing monoclonal antibodies (mAbs) through hybridoma technology. It involves immunizing an animal, usually a mouse, to elicit an immune response. B cells from the animal's spleen are then fused with myeloma cells to generate immortal hybridoma cells. These hybridoma cells are screened and selected in HAT medium to identify clones that produce the desired mAb. The selected clones are then subjected to further characterization and mass production methods.
BT2252 - ETBT - UNIT 3 - Enzyme Immobilization.pdfpkaviya
This document discusses enzyme immobilization. It begins by outlining some common applications of immobilized enzymes in industries like food, chemicals, pharmaceuticals, cosmetics and medicine. It then compares the characteristics of free enzymes and immobilized enzymes. The main techniques for immobilizing enzymes are described in detail: adsorption, covalent binding, matrix entrapment, encapsulation and cross-linking. Factors affecting enzyme kinetics after immobilization and types of diffusion effects are also summarized. The document concludes by stating that enzyme immobilization is a promising technique for industrial biocatalysis but current limitations need to be addressed.
Site-directed mutagenesis is a molecular biology technique used to make specific changes to DNA sequences. It involves using a primer containing the desired mutation in a PCR reaction to introduce the mutation into the gene of interest. There are different approaches for site-directed mutagenesis using PCR, including using a mutated primer in normal PCR or a primer extension method. The technique is used for applications like protein engineering to study the impact of sequence changes or insert restriction sites. However, it can be difficult to replicate the mutated DNA and screening mutations requires sequencing.
The document discusses nitrogenase enzymes, which are produced by certain bacteria and are responsible for reducing nitrogen gas to ammonia. It notes that nitrogen fixation is essential for life. The summary describes that nitrogenase consists of two components - the MoFe protein that catalyzes nitrogen reduction and the Fe protein that supplies electrons. The mechanism involves electrons flowing from ATP to the Fe-S cluster and then to the FeMo cofactor where nitrogen is reduced to ammonia. Key bacteria that synthesize nitrogenase include rhizobia, cyanobacteria, and azotobacter.
This document discusses biological databases and nucleic acid sequence databases. It describes the three primary nucleotide sequence databases: GenBank, EMBL, and DDBJ. GenBank is hosted by the National Center for Biotechnology Information and contains over 286 million bases and 352,000 sequences. EMBL is hosted by the European Molecular Biology Laboratory and mirrors data daily with GenBank and DDBJ. DDBJ is the DNA Data Bank of Japan and also mirrors data daily with the other two databases. Biological databases are important tools for scientists to understand biology at multiple levels.
This document discusses the biodegradation of starch by microorganisms. It begins by defining biodegradation and starch. Starch is made of amylose and amylopectin and can be degraded aerobically or anaerobically. Many bacteria and fungi produce amylase enzymes that break down starch into simpler sugars like maltose and glucose. The document then covers the industrial applications of starch degradation in food processing, brewing, textiles, fuel production, detergents, and more. Key microbes used include Bacillus species, Aspergillus, and Saccharomyces.
1. There are three main types of gene transfer between bacteria: conjugation, transformation, and transduction.
2. Conjugation involves the direct transfer of genetic material between bacterial cells via cell-to-cell contact through a conjugation tube or pilus. Transformation occurs through the uptake of naked DNA from the environment. Transduction is the transfer of DNA from one bacterium to another via bacteriophages.
3. The mechanisms of conjugation, transformation, and transduction are described, including the roles of F factors, Hfr strains, competence factors, and specialized vs. generalized transduction. Key experiments in the discovery of these processes are also summarized.
The document summarizes the process of producing monoclonal antibodies (mAbs) through hybridoma technology. It involves immunizing an animal, usually a mouse, to elicit an immune response. B cells from the animal's spleen are then fused with myeloma cells to generate immortal hybridoma cells. These hybridoma cells are screened and selected in HAT medium to identify clones that produce the desired mAb. The selected clones are then subjected to further characterization and mass production methods.
BT2252 - ETBT - UNIT 3 - Enzyme Immobilization.pdfpkaviya
This document discusses enzyme immobilization. It begins by outlining some common applications of immobilized enzymes in industries like food, chemicals, pharmaceuticals, cosmetics and medicine. It then compares the characteristics of free enzymes and immobilized enzymes. The main techniques for immobilizing enzymes are described in detail: adsorption, covalent binding, matrix entrapment, encapsulation and cross-linking. Factors affecting enzyme kinetics after immobilization and types of diffusion effects are also summarized. The document concludes by stating that enzyme immobilization is a promising technique for industrial biocatalysis but current limitations need to be addressed.
Site-directed mutagenesis is a molecular biology technique used to make specific changes to DNA sequences. It involves using a primer containing the desired mutation in a PCR reaction to introduce the mutation into the gene of interest. There are different approaches for site-directed mutagenesis using PCR, including using a mutated primer in normal PCR or a primer extension method. The technique is used for applications like protein engineering to study the impact of sequence changes or insert restriction sites. However, it can be difficult to replicate the mutated DNA and screening mutations requires sequencing.
The document discusses nitrogenase enzymes, which are produced by certain bacteria and are responsible for reducing nitrogen gas to ammonia. It notes that nitrogen fixation is essential for life. The summary describes that nitrogenase consists of two components - the MoFe protein that catalyzes nitrogen reduction and the Fe protein that supplies electrons. The mechanism involves electrons flowing from ATP to the Fe-S cluster and then to the FeMo cofactor where nitrogen is reduced to ammonia. Key bacteria that synthesize nitrogenase include rhizobia, cyanobacteria, and azotobacter.
This document discusses biological databases and nucleic acid sequence databases. It describes the three primary nucleotide sequence databases: GenBank, EMBL, and DDBJ. GenBank is hosted by the National Center for Biotechnology Information and contains over 286 million bases and 352,000 sequences. EMBL is hosted by the European Molecular Biology Laboratory and mirrors data daily with GenBank and DDBJ. DDBJ is the DNA Data Bank of Japan and also mirrors data daily with the other two databases. Biological databases are important tools for scientists to understand biology at multiple levels.
This document discusses the biodegradation of starch by microorganisms. It begins by defining biodegradation and starch. Starch is made of amylose and amylopectin and can be degraded aerobically or anaerobically. Many bacteria and fungi produce amylase enzymes that break down starch into simpler sugars like maltose and glucose. The document then covers the industrial applications of starch degradation in food processing, brewing, textiles, fuel production, detergents, and more. Key microbes used include Bacillus species, Aspergillus, and Saccharomyces.
1. There are three main types of gene transfer between bacteria: conjugation, transformation, and transduction.
2. Conjugation involves the direct transfer of genetic material between bacterial cells via cell-to-cell contact through a conjugation tube or pilus. Transformation occurs through the uptake of naked DNA from the environment. Transduction is the transfer of DNA from one bacterium to another via bacteriophages.
3. The mechanisms of conjugation, transformation, and transduction are described, including the roles of F factors, Hfr strains, competence factors, and specialized vs. generalized transduction. Key experiments in the discovery of these processes are also summarized.
Antisense RNA is a single-stranded RNA that is complementary to messenger RNA (mRNA) and inhibits its translation. Historically, antisense RNA effects were confused with RNA interference, which involves small interfering RNAs targeting mRNA for degradation. Antisense RNA works by forming duplexes with mRNA, blocking the ribosome from accessing nucleotides or leading to degradation. Examples include transgenic Flavr Savr tomatoes with reduced ethylene production using antisense RNA against an ethylene-producing enzyme, and natural antisense RNA in mice and humans that blocks insulin-like growth factor 2 receptor mRNA. While promising, antisense RNA drugs still face challenges in design, activity, and delivery that have limited
This document discusses nucleic acid therapeutics, which use nucleic acids like DNA and RNA to treat diseases. It describes different types of nucleic acid therapeutics including DNA-based therapies using plasmids, oligonucleotides, aptamers, and DNAzymes, as well as RNA-based therapies like RNA aptamers, antisense RNA, ribozymes, and microRNAs. The document also discusses various methods for delivering these nucleic acid therapeutics, including mechanical methods, electrical methods, viral vectors, polymeric systems, and liposomal systems.
Composite: It compile and filter sequence data from primary database.
Specialized : database—allows targeted searching on one or more specific subject areas
Monoclonal antibodies are identical antibodies produced by one type of immune cell that are clones of a single parent cell. They are produced using hybridoma technology which involves fusing antibody producing B cells from an immunized animal with myeloma tumor cells to create a hybridoma cell line. This hybridoma cell line is capable of indefinite division in culture while producing the same monoclonal antibody. The monoclonal antibodies are then purified from the culture supernatant and have various diagnostic and therapeutic applications such as cancer treatment.
This document discusses global and local sequence alignment. Global alignment aims to align the entire sequences, treating gaps equally across the sequences. It is useful for closely related sequences of similar length. Local alignment finds locally similar regions, allowing gaps to be treated differently. It is useful for more distantly related sequences that may contain similar subsequences. Both use dynamic programming, with global alignment using Needleman-Wunsch and local using Smith-Waterman. Dynamic programming breaks the problem into subproblems by filling a matrix to find the highest scoring alignment.
The document describes the procedure for performing a Dot ELISA assay. It contains 6 sections - Introduction, Principle, Kit Components & Storage, Procedure, Flow Chart, and Result & Interpretation. The Principle section explains that in Dot ELISA, antigen is coated on a nitrocellulose membrane and detected using an enzyme-labeled secondary antibody, appearing as brown dots. The Procedure section provides instructions for preparing reagents and carrying out the assay, including blocking, antibody incubation, washing, and developing with substrate. The Flow Chart further illustrates the step-by-step process. Positive results appear as brown dots, while negative controls have no color.
Estimation of dna by diphenylamine methodjeevithaseyan
This document describes a method to estimate the concentration of DNA using diphenylamine. DNA reacts with diphenylamine under acidic conditions to form a blue-green complex. Various concentrations of a DNA standard are reacted with diphenylamine reagent and measured at 595nm to generate a standard curve. An unknown DNA sample is then reacted, measured and its concentration determined using the standard curve. Reagents, equipment, procedure and calculations are outlined to perform the diphenylamine method of DNA concentration estimation.
This document discusses microbial communities and biofilms. It begins by explaining that microbes thrive in diverse ecosystems under a range of conditions. Microbial communities are heterogeneous mixtures that interact. Biofilms provide advantages like nutrient sharing and protection. The document then discusses techniques to analyze microbial communities, including genetic methods. It covers positive and negative impacts of biofilms in areas like infections, food production, and wastewater treatment. Stress can impact microbial diversity by selecting certain organisms. Modern techniques allow direct analysis of constituent populations in communities.
Conjugation is the transfer of genetic material between bacteria through direct cell-to-cell contact. Joshua Lederberg and Edward Tatum discovered conjugation in 1946 while experimenting with two auxotrophic E. coli strains - strain A could produce certain amino acids while strain B was deficient in those amino acids. When mixed together, prototrophic bacteria emerged that could produce all amino acids. Further experiments showed conjugation requires physical contact between cells. During conjugation, the F plasmid containing the genetic material is replicated in the donor cell and transferred to the recipient cell through a sex pilus. The recipient cell then incorporates the new genetic material into its genome. This allows for the exchange of genetic information between bacterial cells.
This document provides an introduction and overview of data mining. It discusses how data mining extracts knowledge from large amounts of data to discover hidden patterns and predict future trends. It notes that for effective data mining, data sets need to be extremely large. The document outlines some key techniques of data mining including associative learning, artificial neural networks, clustering, genetic algorithms, and hidden Markov models. It also discusses applications of data mining in bioinformatics such as gene finding, protein function prediction, and disease diagnosis. Finally, it acknowledges that while bioinformatics data is rich, developing comprehensive theories remains challenging but creates opportunities for novel knowledge discovery methods.
An immobilized enzyme is an enzyme that has had its movement restricted by attaching it to a solid support. There are several reasons to immobilize enzymes, including protection from degradation, ability to reuse the enzyme for multiple reactions at a lower cost, and easy separation of products from enzymes. Common methods for immobilization include adsorption, entrapment, covalent binding, and cross-linking. Each method has advantages and disadvantages related to stability, activity loss, and reusability of the immobilized enzyme.
The document discusses various computational methods for predicting the three-dimensional structure of proteins from their amino acid sequences. It describes homology modeling, which predicts structures based on known protein structural templates that share sequence homology. It also covers threading/fold recognition and ab initio modeling, which predict structures without templates by using physicochemical principles or energy minimization approaches. Key steps and programs used in each method are outlined.
This document discusses monoclonal antibodies, including their preparation and applications. It begins with an introduction to antibodies and monoclonal antibodies. It then describes the preparation process, which involves immunizing an animal, isolating B cells, fusing them with myeloma cells to form hybridomas, screening clones, and growing selected clones to produce monoclonal antibodies. The document outlines major applications of monoclonal antibodies in diagnosis using techniques like immunoassays and imaging, as well as their use as therapeutic agents and in protein purification.
The document discusses environmental biotechnology and its applications. It provides details about (1) using microorganisms to treat hazardous wastes and pollution, including bioremediation of contaminated soil and water, (2) the treatment process at a common effluent treatment plant (CETP) that cleans waste water from textile industries, and (3) two case studies on bioremediation of oil-contaminated soil and waste water treatment at a CETP.
This document discusses yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs). YACs are engineered chromosomes derived from yeast DNA that can clone very large DNA sequences in yeast cells of up to 1 megabase. BACs are cloning vectors derived from bacterial DNA that can clone DNA fragments of up to 300 kilobases in E. coli. Both systems allow cloning and propagation of large DNA fragments, but YACs can hold more DNA while BACs are more stable and better for functional analysis in mammalian cells.
IntroductionDefinitionPescidesType of pesticidesFate of pesticides in environmentBiodegradation of pesticides in soil Criteria for biodegradation
Strategies for biodegradationDifferent approaches of biodegradationChemical reaction leading to biodegradationChanging the spectrum of toxicityExample of biodegradationAdvantageDisadvantage
This document discusses enzyme immobilization techniques. It defines immobilized enzymes as enzymes that are confined or restricted to a solid support. There are five main methods of immobilization: adsorption, entrapment, encapsulation, covalent binding, and cross-linking. Adsorption involves weak bonds to a carrier surface, while covalent binding uses strong chemical bonds. Entrapment encloses enzymes in a semi-permeable matrix. Immobilization offers benefits like reusability, stability, and easy product separation, but can also reduce enzyme activity. The ideal carrier is inert, strong, inexpensive, and regenerable.
Isoelectric focusing is a technique that separates proteins and other amphoteric molecules based on their isoelectric point, which is the pH at which they have no net charge. During isoelectric focusing, proteins are placed in an immobilized pH gradient formed by carrier ampholytes and an electric field is applied, causing the proteins to migrate through the pH gradient until they reach the pH that matches their isoelectric point and stop migrating. It is useful for analyzing protein heterogeneity and separating isoforms that have small charge differences but similar molecular weights.
Screening of Biosurfactant Bioemulsifier Producing Bacteria from Petroleum Co...ijtsrd
The release of impurities in the environment, containing petroleum and petroleum cogitated products, is engenders of global being taint. It is also a hazardous for human and animal health, since many of these impurities have evidenced to be toxic and oncogenic. Hydrocarbon particles that are secreted into the environment are hard to get rid of, since they change state to surfaces and are captured by surface tension in a water immiscible stage. Bioremediation has tested to be an alternate to lessen the effects caused to impureness of soil and water, applying the metabolic abilities of microorganisms that can apply hydrocarbons as source of carbon and energy, or that can alter them by co metabolism. The proficiency of removal is directly related to the compound’s chemical structure, to its bioavailability deliberation, harmfulness, flexibility and approach and to the physicochemical situation present in the atmosphere. Perwez Qureshi | Dr. Reshma Jaweria "Screening of Biosurfactant/Bioemulsifier Producing Bacteria from Petroleum Contaminated Soil" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-5 , August 2021, URL: https://www.ijtsrd.com/papers/ijtsrd46276.pdf Paper URL: https://www.ijtsrd.com/biological-science/microbiology/46276/screening-of-biosurfactantbioemulsifier-producing-bacteria-from-petroleum-contaminated-soil/perwez-qureshi
Effect of Glucose on Biosurfactant Production using Bacterial Isolates from O...ijtsrd
The demand for biosurfactants is gradually increasing and are thus substituting their chemically synthesized counterparts 14 . The production of biosurfactants commercially requires high expenses. For the production of biosurfactant proper optimization of the physico-chemical parameters is very important. Hence the research was conducted to study the effect of glucose as a carbon source for production of biosurfactant using bacterial isolates from oil contaminated sites in MSM medium. Anjali Sharma "Effect of Glucose on Biosurfactant Production using Bacterial Isolates from Oil Contaminated Sites" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-1 , December 2018, URL: http://www.ijtsrd.com/papers/ijtsrd19033.pdf Direct URL: http://www.ijtsrd.com/biological-science/microbiology/19033/effect-of-glucose-on-biosurfactant-production-using-bacterial-isolates-from-oil-contaminated-sites/anjali-sharma
Antisense RNA is a single-stranded RNA that is complementary to messenger RNA (mRNA) and inhibits its translation. Historically, antisense RNA effects were confused with RNA interference, which involves small interfering RNAs targeting mRNA for degradation. Antisense RNA works by forming duplexes with mRNA, blocking the ribosome from accessing nucleotides or leading to degradation. Examples include transgenic Flavr Savr tomatoes with reduced ethylene production using antisense RNA against an ethylene-producing enzyme, and natural antisense RNA in mice and humans that blocks insulin-like growth factor 2 receptor mRNA. While promising, antisense RNA drugs still face challenges in design, activity, and delivery that have limited
This document discusses nucleic acid therapeutics, which use nucleic acids like DNA and RNA to treat diseases. It describes different types of nucleic acid therapeutics including DNA-based therapies using plasmids, oligonucleotides, aptamers, and DNAzymes, as well as RNA-based therapies like RNA aptamers, antisense RNA, ribozymes, and microRNAs. The document also discusses various methods for delivering these nucleic acid therapeutics, including mechanical methods, electrical methods, viral vectors, polymeric systems, and liposomal systems.
Composite: It compile and filter sequence data from primary database.
Specialized : database—allows targeted searching on one or more specific subject areas
Monoclonal antibodies are identical antibodies produced by one type of immune cell that are clones of a single parent cell. They are produced using hybridoma technology which involves fusing antibody producing B cells from an immunized animal with myeloma tumor cells to create a hybridoma cell line. This hybridoma cell line is capable of indefinite division in culture while producing the same monoclonal antibody. The monoclonal antibodies are then purified from the culture supernatant and have various diagnostic and therapeutic applications such as cancer treatment.
This document discusses global and local sequence alignment. Global alignment aims to align the entire sequences, treating gaps equally across the sequences. It is useful for closely related sequences of similar length. Local alignment finds locally similar regions, allowing gaps to be treated differently. It is useful for more distantly related sequences that may contain similar subsequences. Both use dynamic programming, with global alignment using Needleman-Wunsch and local using Smith-Waterman. Dynamic programming breaks the problem into subproblems by filling a matrix to find the highest scoring alignment.
The document describes the procedure for performing a Dot ELISA assay. It contains 6 sections - Introduction, Principle, Kit Components & Storage, Procedure, Flow Chart, and Result & Interpretation. The Principle section explains that in Dot ELISA, antigen is coated on a nitrocellulose membrane and detected using an enzyme-labeled secondary antibody, appearing as brown dots. The Procedure section provides instructions for preparing reagents and carrying out the assay, including blocking, antibody incubation, washing, and developing with substrate. The Flow Chart further illustrates the step-by-step process. Positive results appear as brown dots, while negative controls have no color.
Estimation of dna by diphenylamine methodjeevithaseyan
This document describes a method to estimate the concentration of DNA using diphenylamine. DNA reacts with diphenylamine under acidic conditions to form a blue-green complex. Various concentrations of a DNA standard are reacted with diphenylamine reagent and measured at 595nm to generate a standard curve. An unknown DNA sample is then reacted, measured and its concentration determined using the standard curve. Reagents, equipment, procedure and calculations are outlined to perform the diphenylamine method of DNA concentration estimation.
This document discusses microbial communities and biofilms. It begins by explaining that microbes thrive in diverse ecosystems under a range of conditions. Microbial communities are heterogeneous mixtures that interact. Biofilms provide advantages like nutrient sharing and protection. The document then discusses techniques to analyze microbial communities, including genetic methods. It covers positive and negative impacts of biofilms in areas like infections, food production, and wastewater treatment. Stress can impact microbial diversity by selecting certain organisms. Modern techniques allow direct analysis of constituent populations in communities.
Conjugation is the transfer of genetic material between bacteria through direct cell-to-cell contact. Joshua Lederberg and Edward Tatum discovered conjugation in 1946 while experimenting with two auxotrophic E. coli strains - strain A could produce certain amino acids while strain B was deficient in those amino acids. When mixed together, prototrophic bacteria emerged that could produce all amino acids. Further experiments showed conjugation requires physical contact between cells. During conjugation, the F plasmid containing the genetic material is replicated in the donor cell and transferred to the recipient cell through a sex pilus. The recipient cell then incorporates the new genetic material into its genome. This allows for the exchange of genetic information between bacterial cells.
This document provides an introduction and overview of data mining. It discusses how data mining extracts knowledge from large amounts of data to discover hidden patterns and predict future trends. It notes that for effective data mining, data sets need to be extremely large. The document outlines some key techniques of data mining including associative learning, artificial neural networks, clustering, genetic algorithms, and hidden Markov models. It also discusses applications of data mining in bioinformatics such as gene finding, protein function prediction, and disease diagnosis. Finally, it acknowledges that while bioinformatics data is rich, developing comprehensive theories remains challenging but creates opportunities for novel knowledge discovery methods.
An immobilized enzyme is an enzyme that has had its movement restricted by attaching it to a solid support. There are several reasons to immobilize enzymes, including protection from degradation, ability to reuse the enzyme for multiple reactions at a lower cost, and easy separation of products from enzymes. Common methods for immobilization include adsorption, entrapment, covalent binding, and cross-linking. Each method has advantages and disadvantages related to stability, activity loss, and reusability of the immobilized enzyme.
The document discusses various computational methods for predicting the three-dimensional structure of proteins from their amino acid sequences. It describes homology modeling, which predicts structures based on known protein structural templates that share sequence homology. It also covers threading/fold recognition and ab initio modeling, which predict structures without templates by using physicochemical principles or energy minimization approaches. Key steps and programs used in each method are outlined.
This document discusses monoclonal antibodies, including their preparation and applications. It begins with an introduction to antibodies and monoclonal antibodies. It then describes the preparation process, which involves immunizing an animal, isolating B cells, fusing them with myeloma cells to form hybridomas, screening clones, and growing selected clones to produce monoclonal antibodies. The document outlines major applications of monoclonal antibodies in diagnosis using techniques like immunoassays and imaging, as well as their use as therapeutic agents and in protein purification.
The document discusses environmental biotechnology and its applications. It provides details about (1) using microorganisms to treat hazardous wastes and pollution, including bioremediation of contaminated soil and water, (2) the treatment process at a common effluent treatment plant (CETP) that cleans waste water from textile industries, and (3) two case studies on bioremediation of oil-contaminated soil and waste water treatment at a CETP.
This document discusses yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs). YACs are engineered chromosomes derived from yeast DNA that can clone very large DNA sequences in yeast cells of up to 1 megabase. BACs are cloning vectors derived from bacterial DNA that can clone DNA fragments of up to 300 kilobases in E. coli. Both systems allow cloning and propagation of large DNA fragments, but YACs can hold more DNA while BACs are more stable and better for functional analysis in mammalian cells.
IntroductionDefinitionPescidesType of pesticidesFate of pesticides in environmentBiodegradation of pesticides in soil Criteria for biodegradation
Strategies for biodegradationDifferent approaches of biodegradationChemical reaction leading to biodegradationChanging the spectrum of toxicityExample of biodegradationAdvantageDisadvantage
This document discusses enzyme immobilization techniques. It defines immobilized enzymes as enzymes that are confined or restricted to a solid support. There are five main methods of immobilization: adsorption, entrapment, encapsulation, covalent binding, and cross-linking. Adsorption involves weak bonds to a carrier surface, while covalent binding uses strong chemical bonds. Entrapment encloses enzymes in a semi-permeable matrix. Immobilization offers benefits like reusability, stability, and easy product separation, but can also reduce enzyme activity. The ideal carrier is inert, strong, inexpensive, and regenerable.
Isoelectric focusing is a technique that separates proteins and other amphoteric molecules based on their isoelectric point, which is the pH at which they have no net charge. During isoelectric focusing, proteins are placed in an immobilized pH gradient formed by carrier ampholytes and an electric field is applied, causing the proteins to migrate through the pH gradient until they reach the pH that matches their isoelectric point and stop migrating. It is useful for analyzing protein heterogeneity and separating isoforms that have small charge differences but similar molecular weights.
Similar to Isolation, Screening, and Characterization of Biosurfactant-Producing Microorganisms from Petroleum-Contaminated Soil and Further Optimization of Parameters for Biosurfactant Production
Screening of Biosurfactant Bioemulsifier Producing Bacteria from Petroleum Co...ijtsrd
The release of impurities in the environment, containing petroleum and petroleum cogitated products, is engenders of global being taint. It is also a hazardous for human and animal health, since many of these impurities have evidenced to be toxic and oncogenic. Hydrocarbon particles that are secreted into the environment are hard to get rid of, since they change state to surfaces and are captured by surface tension in a water immiscible stage. Bioremediation has tested to be an alternate to lessen the effects caused to impureness of soil and water, applying the metabolic abilities of microorganisms that can apply hydrocarbons as source of carbon and energy, or that can alter them by co metabolism. The proficiency of removal is directly related to the compound’s chemical structure, to its bioavailability deliberation, harmfulness, flexibility and approach and to the physicochemical situation present in the atmosphere. Perwez Qureshi | Dr. Reshma Jaweria "Screening of Biosurfactant/Bioemulsifier Producing Bacteria from Petroleum Contaminated Soil" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-5 , August 2021, URL: https://www.ijtsrd.com/papers/ijtsrd46276.pdf Paper URL: https://www.ijtsrd.com/biological-science/microbiology/46276/screening-of-biosurfactantbioemulsifier-producing-bacteria-from-petroleum-contaminated-soil/perwez-qureshi
Effect of Glucose on Biosurfactant Production using Bacterial Isolates from O...ijtsrd
The demand for biosurfactants is gradually increasing and are thus substituting their chemically synthesized counterparts 14 . The production of biosurfactants commercially requires high expenses. For the production of biosurfactant proper optimization of the physico-chemical parameters is very important. Hence the research was conducted to study the effect of glucose as a carbon source for production of biosurfactant using bacterial isolates from oil contaminated sites in MSM medium. Anjali Sharma "Effect of Glucose on Biosurfactant Production using Bacterial Isolates from Oil Contaminated Sites" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-1 , December 2018, URL: http://www.ijtsrd.com/papers/ijtsrd19033.pdf Direct URL: http://www.ijtsrd.com/biological-science/microbiology/19033/effect-of-glucose-on-biosurfactant-production-using-bacterial-isolates-from-oil-contaminated-sites/anjali-sharma
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Phycoremediation is a green technology that supports the direct use of living green microalgae for in situ, or in place removal, degradation, of contaminants in soils, sludge, sediments, surface water and ground waters by the mechanisms of bio-transformation, bio-accumulation, bio-concentration, bio-sparging.
It can be said by the current study that microalgae has a great potential for the treatment of industrial and municipal wastewaters as compared to the chemical treatments available commercially. Biological systems are much more efficient in cleaning the excess nutrients from the waste water followed by generation of valuable biomass which can be applied in the food, fertilizer, energy production as use of inorganic chemicals like lime and ferrous sulphate generates huge amount of sludge in textile industries, but on the other hand static anaerobic treatment using acclimatized MLSS gives better colour reduction with zero sludge generation. Microalgal cells can be used in free form to treat waste waters containing high C.O.D., high ammonical nitrogen and high TDS. It not only provides a better reduction of chemicals from wastewaters but it also helps to reduce the operational cost of ETP. Microalgaes not only helps to remediate industrial waste waters but also to treat sweage water and to restore natural water bodies like lakes and ponds. As they are active in remediating the chemicals but also it shows an antagonistic effect against some pathogenic germs like total coliforms and fecal coliforms.
These microalgal cells can also be combined with bacterial biomass of activated sludge process to develop an Algal-Bacterial consortium (ALBA) for better enhancement in the reduction of chemicals from the wastewaters as this symbiotic relation of algae and bacteria provides high satiability of the microalgae along with MLSS and faceable in terms of price and economy for instance the bacterial biomass provides carbon dioxide to algal cells for photosynthesis and in return the bacteria acquires oxygen from algae. The harvested biomass from the ETP’s can be used as bio-fertilizers as it consists of appropriate ratio of vital macro and micro nutrients like N,P,K etc. which enhance the growth of plantlets. It can also be used as aqua feeds for shrimps, fishes and molluscs. Furthermore these microlgal cells are non-toxic in the environment as it becomes a part of food chain and do not cause eutrophication. Therefore, micro-algal based treatment is most suitable for the treating the waste waters and restoring the natural water bodies as compared to other chemical treatments.
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This document summarizes a study on treating dairy industry wastewater using a hybrid upflow anaerobic sludge blanket (UASB) reactor. The dairy wastewater has high levels of biochemical oxygen demand (BOD), chemical oxygen demand (COD), and total solids that require treatment before disposal. The study constructed a laboratory-scale UASB reactor and analyzed parameters like BOD, COD, pH, and total solids at different hydraulic retention times. Results showed reductions in BOD and COD and increases in pH and total solids, with optimal treatment achieved at a 36 hour retention time. The UASB reactor was found to effectively treat dairy wastewater through simple biological processes.
A REVIEW ON APPLICATIONS OF BIOSURFACTANTS PRODUCED FROM UNCONVENTIONAL INEXP...SUS GROUP OF INSTITUTIONS
Biosurfactants can serve as green alternative in different areas due to
their ecological acceptance as they are biodegradable and nontoxic.
Nowadays biosurfactants are predominantly used in pharmaceutical,
oil industry, and for the bioremediation of pollutants. Apart from these,
biosurfactants also show potential applications in many sectors of food
industry and agriculture. Allied with emulsion forming and breaking,
antiadhesive, functional ingredient, are some properties that can be
exploited in agro-food biotechnology. Potential role of biosurfactants
in food and agricultural sectors as well as present concern of lowering
the production cost of biosurfactants by using the unconventional
wastes as substrate is discussed in this article.
This document discusses bioremediation, which uses microorganisms to remove environmental pollutants or prevent pollution. It describes various types of bioremediation including biostimulation and bioaugmentation. Key organisms used in bioremediation are discussed, such as Pseudomonas bacteria, white rot fungi, and plants. Methods like phytoremediation, biosurfactants, and bioremediation of sites, soils, wastes, and hydrocarbons are summarized. Advantages include being natural and enabling complete destruction of contaminants, while disadvantages are limitations to biodegradable compounds and length of time needed.
This document provides an overview of bioremediation. Some key points:
- Bioremediation uses microorganisms like bacteria and fungi to remove or break down pollutants in the environment. It can be used to treat contamination in soil, water, and solid waste.
- There are different types of bioremediation including biostimulation, bioaugmentation, and intrinsic bioremediation. Genetically engineered microbes are also used.
- The microbes degrade pollutants through redox reactions and metabolic pathways. Bioremediation can be done on-site (in situ) or by removing contaminated material to another location (ex situ).
This document provides an overview of research on treating vegetable waste. It characterizes the physical, chemical, and biological properties of various vegetable wastes. It then describes several treatment methods for vegetable waste, including anaerobic digestion to produce biogas, fluidized bed combustion, biodiesel production, and composting. The document also discusses identifying compounds in artichoke waste and comparing different waste treatment methods.
IRJET- Bioremediation of Waste Water from Natural Rubber Processing PlantIRJET Journal
This document summarizes a study on using bioremediation to treat wastewater from a natural rubber processing plant. Three bacterial species (Pseudomonas sp., Bacillus sp., and Lactobacillus sp.) were isolated from the plant's effluent and used individually and together in a consortium to degrade various pollutants in the wastewater over 15 days. Testing showed that the bacterial consortium was most effective at reducing levels of BOD, COD, total solids, and ammonia compared to using the individual bacteria species. Pseudomonas sp. performed best as an individual species, while Lactobacillus sp. showed the lowest efficiency. The consortium improved wastewater pH
This document summarizes a review article about the potential applications of biosurfactants in the food industry. It discusses how biosurfactants have properties like emulsion formation and stabilization, as well as antiadhesive and antimicrobial activities, that could be useful in food processing. Biosurfactants are naturally derived and biodegradable alternatives to chemical surfactants. They are generally non-toxic and tolerant of various environmental conditions like temperature, pH, and salt concentrations. The document outlines several classes of biosurfactants and their producing microorganisms. It also discusses emulsification abilities and how biosurfactants could potentially be used as emulsifiers in foods.
Isolation and Screening of Hydrogen Producing Bacterial Strain from Sugarcane...Editor IJCATR
The aim of this study is to isolate a highly competent bacterium with potent cellulose degrading capability and a better
hydrogen producer. Soil sample from sugarcane bagasse yard was isolated, serially diluted and plated on cellulose specific nutrient
agar plate. Four colonies have been isolated in which a single colony has potent cellulose degrading ability and the highest hydrogen
productivity of 275.13 mL H2 L-1. The newly isolated bacterium was morphologically and biochemically characterized. The
molecular characterization of the bacterium was carried out using 16S rDNA sequencing and the organism was identified as
Bacilllus subtilis AuChE413. Proteomic analysis such as MALDI-TOF was carried out to differentiate the isolated Bacillus subtilis
from Bacillus thuringiensis and Bacillus amyloliquefaciens. Phylogenetic tree was constructed to analyze the evolutionary
relationship among different genus and species with the newly isolated strain.
FEASIBILITY STUDY OF TREATMENT OF EFFLUENT FROM A BULK DRUG MANUFACTURING IND...Journal For Research
A study has been carried out on aerobic biological treatment of a bulk drug industrial effluent which is highly acidic in nature and shows high value of BOD5 (≈ 36000 mg/l), COD (≈ 84000 mg/l). Chemical treatment conducted for neutralizing the pH followed by biological treatment using a lab-scale reactor with acclimatized bacterial consortia isolated from natural soil has confirmed its feasibility for biological treatment. About 99% removal of COD from starting value of around 8000 mg/l has been achieved. The COD value in different hydraulic retention time (HRT) has been brought down to less than 100 mg/l in treated effluent, showing high removal of dissolved organics by aerobic biological treatment.
POTENTIAL BIOMEDICAL AND PHARMACEUTICAL APPLICATIONS OF MICROBIAL SURFACTANTSSUS GROUP OF INSTITUTIONS
Many microorganisms are able to produce a wide range of amphipathic
compounds, with both hydrophilic and hydrophobic moieties present
within the same molecule which allow them to exhibit surface
activities at interfaces and are generally called biosurfactants.
Biosurfactants are versatile, structurally diverse group of surface-active
substances produced by microorganisms and have variety of
applications in the sectors including bioremediation, food industry,
agriculture and pharmaceuticals. Interest in biosurfactant production
has markedly increased during the past decade, although large-scale
production has not been possible because of low production yields and
high total costs. At present, biosurfactants have gained importance in environmental
applications, while new applications in the pharmaceutical, biomedical, cosmetic and food
industry, with a high added value, are still developing. Recently, the potential applications of
biosurfactants in the biomedical field have increased. Their antibacterial, antifungal and
antiviral activities make them relevant molecules for applications in combating many
diseases and as therapeutic agents. In addition, their role as anti-adhesive agents against
several pathogens indicates their utility as suitable anti-adhesive coating agents for medical
insertional materials leading to a reduction in a large number of hospital infections without
the use of synthetic drugs and chemicals. This article emphasizes the medicinal and
therapeutic perspective of biosurfactants. With these specialized and cost-effective
applications, biosurfactants can be considered as an interesting option for the near future.
Cashew nut processing industry waste water treatmentVishnu Raj
This document discusses the treatment of waste water from the cashew nut processing industry in India. It provides background on the industry and production levels in India. It then describes the sources and characteristics of the wastewater, which contains toxic cashew nut shell liquid (CNSL) that is difficult to treat using conventional biological methods. The document reviews various treatment alternatives that have been studied including aerobic reactors with fungal inoculums, bioaugmentation with specific microorganisms, and enzymatic bioremediation approaches. It concludes that biological remediation technologies show promise for treating this type of wastewater but further optimization is needed given the recalcitrant nature of some CNSL components.
isolation and characterization of pgpr from paper mill effluent infested areaijtsrd
Paper and pulp industry are considered as 17th most polluting industry due to its highly coloured and toxic wastewater discharge in the environment. A number of heavy metals get mixed within the agricultural soil through the paper mill discharge. These heavy metals are potent phytotoxic and they have huge negative impact on plant health. To minimize these impacts and to improve crop health deliberate application of chemical fertilizers is very common in present days but these chemical fertilizers are destroying the soil and plant health tremendously. In this situation rhizosphere researches suggest the concept of the application of PGPR. The group of root colonizing bacteria which enhance plant growth and development are called Plant Growth Promoting Rhizobacteria PGPR . This group of bacteria improve plant’s growth by direct as well as indirect mechanisms. These mechanisms involve IAA production, siderophore production, soil structure formation, decomposition of organic matter, solubilization of minerals, degrading organic pollutants, biocontrol of seed borne pathogens etc. In present study we have focused on isolation and characterization of PGPRs from paper mill effluent infested soil. Ananya Roy Chowdhury "Isolation and Characterization of PGPR from Paper Mill Effluent Infested Area" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-4 | Issue-5 , August 2020, URL: https://www.ijtsrd.com/papers/ijtsrd32930.pdf Paper Url :https://www.ijtsrd.com/biological-science/botany/32930/isolation-and-characterization-of-pgpr-from-paper-mill-effluent-infested-area/ananya-roy-chowdhury
Microbial biotransformation uses microorganisms like bacteria, fungi, and actinomycetes to modify organic compounds through enzymatic reactions. Key reactions include oxidation, reduction, hydrolysis, and others. These transformations are used commercially to produce pharmaceuticals, vitamins, antibiotics, and other chemicals. For example, microbes can hydroxylate steroids through oxidation or reduce ketones and aldehydes. Biotransformation offers advantages like selectivity and mild reaction conditions compared to chemical synthesis.
Isolation and antimicrobial activity of rhamnolipid (biosurfactant) from oil ...eSAT Publishing House
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology
DOI: 10.21276/ijlssr.2016.2.4.4
ABSTRACT- Microorganisms are the important factors in the degradation of the toxic substances in our environment.
Petrol and diesel oil is one of the complex mixtures which cannot be easily degraded. The Bacillus cereus was involved in
the degradation of oil during which the complex toxic substances were detoxified by the production of biosurfactants. In
our study we have identified that the biosurfactant producing Bacillus cereus have a high potential for hydrocarbon
degradation. The Bacillus cereus was isolated from hydrocarbon contaminated soil and identified based on morphology
and biochemical test according to the Bergey’s manual of systematic bacteriology. The maximum hydrocarbon degrading
biosurfactant producing Bacillus cereus was obtained by qualitative and quantitative methods. In optimization studies, the
best results observed for Bacillus cereus were, Olive oil as the suitable carbon source, Sodium nitrate as the best Nitrogen
source and Optimum pH is 7 and Optimum temperature is 37°C. The ability of these isolates to degrade hydrocarbons and
survive in the oil contaminated soil is attributed to the development of resistance by mutation on the plasmid. It is also
clearly evident that the specific gene was responsible for the production of biosurfactant and the degradation process.
According to the results from the present study the Bacillus cereus has high potential for hydrocarbon degradation and can
be used especially for Microbial Enhanced Oil Recovery and bioremediation of hydrocarbons in near future.
Key-words- Bacillus cereus, Biosurfactant, Hydrocarbon, Biodegradation, Plasmid DNA
IRJET-Biogas Generation from Combination of Food Waste and Water HyacinthIRJET Journal
1. The document discusses biogas generation from anaerobic digestion of various ratios of food waste and water hyacinth using cow dung as a seed material. Ratios studied included 80:20, 70:30, 60:40, and 0:100 of food waste to water hyacinth.
2. For the 80:20 ratio, biogas generation was 450 mL and reductions in parameters like BOD, COD, and TS were 58%, 68%, and 19% respectively. Nutrient levels like phosphates and nitrates increased.
3. For the 70:30 ratio, biogas generation was highest at 480 mL. Reductions in BOD, COD, and TS were
Similar to Isolation, Screening, and Characterization of Biosurfactant-Producing Microorganisms from Petroleum-Contaminated Soil and Further Optimization of Parameters for Biosurfactant Production (20)
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A Review on Polyherbal Formulations and Herbal Medicine for Management of Ul...BRNSS Publication Hub
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Evaluation of Cordia Dichotoma gum as A Potent Excipient for the Formulation ...BRNSS Publication Hub
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The document discusses Goldbach's problems and their solutions. It summarizes that the ternary Goldbach problem, which states that every odd number greater than 7 can be represented as the sum of three odd primes, was solved in 2013. It also discusses Ramare's 1995 proof that any even number can be represented as the sum of no more than 6 primes. The document then provides proofs for theorems related to representing numbers as sums of primes and concludes there are an infinite number of twin primes.
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Chapter wise All Notes of First year Basic Civil Engineering.pptxDenish Jangid
Chapter wise All Notes of First year Basic Civil Engineering
Syllabus
Chapter-1
Introduction to objective, scope and outcome the subject
Chapter 2
Introduction: Scope and Specialization of Civil Engineering, Role of civil Engineer in Society, Impact of infrastructural development on economy of country.
Chapter 3
Surveying: Object Principles & Types of Surveying; Site Plans, Plans & Maps; Scales & Unit of different Measurements.
Linear Measurements: Instruments used. Linear Measurement by Tape, Ranging out Survey Lines and overcoming Obstructions; Measurements on sloping ground; Tape corrections, conventional symbols. Angular Measurements: Instruments used; Introduction to Compass Surveying, Bearings and Longitude & Latitude of a Line, Introduction to total station.
Levelling: Instrument used Object of levelling, Methods of levelling in brief, and Contour maps.
Chapter 4
Buildings: Selection of site for Buildings, Layout of Building Plan, Types of buildings, Plinth area, carpet area, floor space index, Introduction to building byelaws, concept of sun light & ventilation. Components of Buildings & their functions, Basic concept of R.C.C., Introduction to types of foundation
Chapter 5
Transportation: Introduction to Transportation Engineering; Traffic and Road Safety: Types and Characteristics of Various Modes of Transportation; Various Road Traffic Signs, Causes of Accidents and Road Safety Measures.
Chapter 6
Environmental Engineering: Environmental Pollution, Environmental Acts and Regulations, Functional Concepts of Ecology, Basics of Species, Biodiversity, Ecosystem, Hydrological Cycle; Chemical Cycles: Carbon, Nitrogen & Phosphorus; Energy Flow in Ecosystems.
Water Pollution: Water Quality standards, Introduction to Treatment & Disposal of Waste Water. Reuse and Saving of Water, Rain Water Harvesting. Solid Waste Management: Classification of Solid Waste, Collection, Transportation and Disposal of Solid. Recycling of Solid Waste: Energy Recovery, Sanitary Landfill, On-Site Sanitation. Air & Noise Pollution: Primary and Secondary air pollutants, Harmful effects of Air Pollution, Control of Air Pollution. . Noise Pollution Harmful Effects of noise pollution, control of noise pollution, Global warming & Climate Change, Ozone depletion, Greenhouse effect
Text Books:
1. Palancharmy, Basic Civil Engineering, McGraw Hill publishers.
2. Satheesh Gopi, Basic Civil Engineering, Pearson Publishers.
3. Ketki Rangwala Dalal, Essentials of Civil Engineering, Charotar Publishing House.
4. BCP, Surveying volume 1
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Complications of wound healing like infection, hyperpigmentation of scar, contractures, and keloid formation.
Isolation, Screening, and Characterization of Biosurfactant-Producing Microorganisms from Petroleum-Contaminated Soil and Further Optimization of Parameters for Biosurfactant Production
2. Deo and Rana: Isolation, screening, and characterization of biosurfactant-producing microorganisms from petroleum-
contaminated soil and further optimization of parameters for biosurfactant production
IJPBA/Jul-Sep-2018(Suppl)/Vol 9/Issue 3 54
and particulate biosurfactant. Among the
various classes of biosurfactants
-
rhamnolipids
and surfactins are best-studied biosurfactants.
Rhamnolipid is one of the types of glycolipids, in
which one or two molecules of rhamnose are linked
to one or two molecules of β-hydroxydecanoic
acid while the
-OH group of one of the acids
is involved in glycosidic linkage with the
reducing end of the rhamnose disaccharide, in
ester formation. Rhamnolipids are produced
by Pseudomonas aeruginosa, Gram-negative,
motile, and non-spore-forming bacteria. Surfactin
is a cyclic lipopeptide commonly used as an
antibiotic. In the various course of studies of its
properties, surfactin was found to exhibit effective
characteristics such as antibacterial, antiviral,
antifungal, antimycoplasma, and hemolytic
activities. Surfactin is produced by Bacillus
subtilis, Gram-positive, motile, and spore-forming
bacteria. Surface activity in most hydrocarbon-
degrading microorganisms is attributed to several
cell surface constituents, which includes structures
such as M protein and lipoteichoic acid in Group A
Streptococci, Protein A in Staphylococcus aureus,
Layer A in Aeromonas salmonicida, prodigiosin
in Serratia spp., gramicidins in Bacillus brevis
spores, and thin fimbriae in A. calcoaceticus.[5]
The oil and gas industry is one of the most
important sectors in India. Unfortunately, oil
spills and industrial discharges from petroleum-
related industries have been identified as
the two major sources of pollution. The
biodegradation of petroleum pollutant is limited
by its poor availability to the microorganisms,
its hydrophobicity, and low aqueous solubility, [6]
The applications of biosurfactants are not limited
to bioremediation purposes, but they can be
used in petroleum engineering to reduce heavy
oil viscosity, clean oil storage tanks, increase
petroleum transport in pipelines, and stabilize
fuel water-oil emulsions.[7]
The features that make
biosurfactants as an alternative to commercially
synthesized surfactants are its low toxicity, higher
biodegradabilityand,hence,greaterenvironmental
compatibility, better foaming properties (useful in
mineral processing), and stable activity at extreme
pH, temperature, and salinity.[8]
Biosurfactants
also found to be important in therapeutic and
biomedical field as they possess antimicrobial,
antiviral, and antifungal properties also they
inhibit fibrin clot formation and have antiadhesive
properties. These biomolecules can replace the
harsh surfactants, presently being used in million
dollar pesticide industries.[9]
To reduce the production costs, different routes
could be investigated such as the increase of
yields and product accumulation, the development
of economical engineering processes, and the
use of cost-free or cost-credit feedstock for
microorganism growth and surfactant production.
The choice of low cost or waste substrates is
important to overall economy of the process, and
they account for 30–50% of final product cost and
also minimize the expenses cost of waste treatment.
[10-12]
Hydrocarbons such as crude oil and diesel and
various carbohydrates such as glucose, sucrose, and
glycerol have been commonly used as substrates
for the production of biosurfactants. Since the
biological function of biosurfactant is related to
hydrocarbon uptake, a spontaneous release occurs
with these substrates,
[13]
It is well known that
combination of different environmental factors
such as temperature, pH, salinity, and hydrocarbon
toxicity would affect growth of microorganisms,
hence, the amount of their bioproducts. The
combination of these constraints can be expected
to limit the number of suitable organisms that
would grow and produce bioproducts.[14]
The
objective of this study is, therefore, to screen
hydrocarbon-degrading bacteria from petroleum-
contaminated soil and to optimize the physical and
nutrient parameters to enhance the production of
biosurfactants.
MATERIALS AND METHODS
Isolation of organisms from soil
Soil samples were collected from different petrol
pumps in Pune, India. All the samples were
collected using polyethylene containers with
utmost care to avoid contamination. 1 g of soil
sample was taken and serially diluted in 0.85%
sterile saline. Flasks were maintained in a shaker
with 150 rpm at room temperature for 4 days.
Screening was performed using serial dilutions of
the sample and plating on minimal salt medium
(MSM) with palm oil (1% w/v) as carbon
source for the isolation of bacteria. The MSM
of following composition was used (g/L) - 0.8 g
dipotassium hydrogen phosphate (K2
HPO4
), 0.2 g
potassium dihydrogen potassium (KH2
PO4
),
3. IJPBA/Jul-Sep-2018(Suppl)/Vol 9/Issue 3 55
Deo and Rana: Isolation, screening, and characterization of biosurfactant-producing microorganisms from petroleum-
contaminated soil and further optimization of parameters for biosurfactant production
0.05 g calcium chloride (CaCl2
), 0.5 g magnesium
chloride (MgCl2
), 0.01 g ferric chloride (FeCl2
),
1 g diammonium sulfate (NH4
)2
SO4
), and 5 g
sodium chloride (NaCl). Plates were incubated at
37°C for 72 h. Different bacterial colonies were
selected based on the colony morphology and
then streaked on nutrient agar. Morphologically,
distinct colonies were reisolated by transferring
them to fresh palm oil containing agar plates at
least 3 times to obtain pure cultures. The colonies
were then screened for biosurfactant production.
Screening for biosurfactant activity
Hemolytic activity
Pure culture of bacterial isolates was streaked on
the freshly prepared blood agar plates containing
5% sheep blood and incubated at 37°C for 48 h.
Results were recorded based on the type of zone of
clearance observed, i.e. α-hemolysis when colony
was surrounded by greenish zone, β-hemolysis
when colony was surrounded by clear white zone,
and γ-hemolysis when there was no change in
the medium surrounding the colony.[15]
This is
preliminary method for screening biosurfactant
activity.
Emulsification index (%EI 24
)
Colonies of pure culture were suspended in test
tube containing 2 ml of MSM and incubated at
37°C for 24 h. 2 ml hydrocarbon (oil) was added
to each tube. Then, the mixture was vortexed at
high speed for 2 min and allowed to stand for 24 h
at room temperature and emulsification index was
calculated. The %EI24
was calculated by dividing
the height of emulsified layer (mm) by total height
of the liquid in tube (mm) and then multiplying
by100.[16]
Emulsification
index E24
Height of emulsion layer
Total h
( )=
e
eight
×100
Drop-collapse test
The cultures that showed hemolytic activity
and emulsification index were grown in MSM
supplemented with 1% palm oil for 48 h at room
temperature for drop-collapse test. Screening of
biosurfactant production was performed using
the qualitative drop-collapse test described by
Bordour and Maier.[17]
2 µl of palm oil was added
to each well of 96-well microtiter plate lid and
these were left to equilibrate for 24
h. 5 µl of
cultural supernatant was added to the surface of
oil and drop size was observed after 1 min. The
result was considered positive for biosurfactant
production and flat rounded drops indicated lack
of biosurfactant production.[18]
Oil displacement method
20 µl of palm oil was placed on surface of distilled
water (20 ml) in a Petri dish, then 20 µl of culture
supernatant was gently put on center of oil film. If
the biosurfactant is present in the supernatant, the
oil is displaced and diameter of the clear hallow
area visualized under visible light was measured
and calculated after 30 s.[19]
Identification of biosurfactant-producing
bacteria
Based on the screening test results, the positive
isolates were inoculated into the mineral salt
medium for the biosurfactant production. Based
on the quantification of biosurfactant produced,
the best strain was selected and then identified
by its microscopic appearance and biochemical
tests based on Bergey’s Manual of Determinative
Bacteriology. In addition to morphological
and biochemical tests, the phylogeny of strain
is determined using sequence analysis of
polymerase chain reaction (PCR) amplified
16s rRNA gene using the primer pair RPP2
(CCAAGCTTCTAGA CGGITACCTTGTTA
CGACTT) and FDD2 (CCGGATCCGTCGACA
GAGTTTGATCI TGGCTCAG). RPP2 and
FDD2 are universal primers for 1.5 kb fragment
amplification for eubacteria. The following PCR
conditions were used: 95°C for 5 min, followed
by 25 cycles of 95°C for 1 min, 50°C for 30 s, and
72°C for 5 min, followed by 72°C for 5 min. After
completion of PCR amplification, the samples
were purified and loaded on the sequencer - Avant
3100 Gene Analyzer. The sequencing output was
analyzed using the accompanying DNA Sequence
Analyzer computer software. The sequence was
compared with National Center for Biotechnology
Information GenBank entries using BLAST
algorithm.
4. Deo and Rana: Isolation, screening, and characterization of biosurfactant-producing microorganisms from petroleum-
contaminated soil and further optimization of parameters for biosurfactant production
IJPBA/Jul-Sep-2018(Suppl)/Vol 9/Issue 3 56
Effect of temperature, pH, carbon, and
nitrogen sources on growth and biosurfactant
production
Optimization study was conducted to find out the
optimum conditions for the large-scale production
ofbiosurfactant.Theselectedisolatewasinoculated
in 100 ml of minimal salt media in 250 ml flask,
and the pH of the media was adjusted to 4, 6, 8,
10, and 12, and incubated for 48 h. The cells were
removed by centrifugation, and supernatant was
used for the estimation of biosurfactant production
by measuring OD at 420 nm. Similarly, a range
of different temperatures was used (15°C, 25°C,
35°C, 45°C, and 55°C) to find the optimum
temperature.
Growth and biosurfactant production by the isolate
was evaluated using mineral salt media (MSS)
with different carbon and nitrogen sources. The
MSS consisted of gl−1
:NaCl 10.0; Na2
HPO4
5.0;
KH2
PO4
2.0; and MgSO4
7H2
O 0.2. The carbon
sources used were sodium acetate, paraffin,
sucrose, and starch using ammonium nitrate as the
nitrogen source (2/g). The different carbon sources
were added to MSS at a concentration of 10/gl. The
different nitrogen sources used were - ammonium
sulfate, ammonium nitrate, sodium nitrate, and urea
while sucrose is used as carbon source (10/gl).The
nitrogen sources added to MSS at a concentration
of 2/gl.All media were adjusted to a pH of 7.Assays
were performed in 100 ml flasks containing 50 ml
of different media. Each flask was inoculated with
1% of preculture grown in same medium for 24 h.
Cultures were incubated at 40°C without shaking
for 120 h. After every 24 h, samples were taken,
centrifuged at 10,000, 20 min, 20°C, and cell-free
supernatants were used to measure biosurfactant
production using various parameters.
Extraction and purification of biosurfactant
Biosurfactant for chemical composition analysis
was extracted from cell-free supernatant using
Folch extraction method that is commonly used
to extract lipids from biomolecules.[20]
Briefly, a
chloroform/methanol mixture (2:1) was added
to supernatant sample to a final chloroform/
methanol/water ratio of 8:4:3. The mixture was
centrifuged (9000 g, 5 min), the organic layer was
collected, and samples were evaporated to dryness
under N2
at 37°C for 30 min.
Fourier transform-infrared (FTIR)
Spectroscopy
FTIR is most useful for identifying types of
chemical bonds (functional groups) and therefore
can be used to elucidate some components of
the unknown sample. 10 mg of freeze-dried
pure biosurfacant was grounded with 100 mg
of KBr and pressed with 7500 kg for 30 s to
obtain translucent pellets. The FTIR spectra, with
resolution of 1/cm, were collected from 400 to
4000 wavenumbers (cm−1
) and are an average of
128 scans using a Tensor 27 infrared spectrometer
operating in attenuated total reflection mode.
RESULTS AND DISCUSSION
Biosurfactants play an important role in
food industry, bioremediation, food industry,
pharmaceuticals, oil industry, petrochemistry,
paper, and pulp industry.[21]
Furthermore, due to
diversity of microorganisms and different niches,
they inhabit that there is need of efficient isolation,
screening, and optimization of biosurfactant-
producing bacteria from the natural environment.
In the present study, the contaminated soil was
collected from various petrol pumps. A
total of
29 strains were isolated from the soil sample
and screened for biosurfactant production using
various screening tests, and it was found that only
17 colonies were positive in the primary screening
through hemolytic activity in blood agar plate. The
hemolytic activity was first studied by Bernheimer
andAvigad[22]
in B. subtilis. Carrillo et al.[15]
found an
associationbetweenhemolyticactivityandsurfactant
production, and they recommended the use of blood
agar lysis as a primary screening method. All the 17
strains were further screened by drop-collapse test
to confirm biosurfactant production, and the results
revealed only 10 colonies were potent biosurfactant
producers. Bodour et al.[16]
used drop-collapse
assay and found that only biosurfactant-producing
organisms formed microemulsion in this assay
[Table 1]. For screening of biosurfactant-producing
isolates,theyemulsifiedthecoatedoilinculturebroth
and made microemulsion, because of the formation
of microemulsion, the culture drops collapsed in
coated oil. The presence of organisms present in
the soil depends on the nature and nutrient content
of the soil. These colonies were further screened to
select the most potent biosurfactant producer. For the
5. IJPBA/Jul-Sep-2018(Suppl)/Vol 9/Issue 3 57
Deo and Rana: Isolation, screening, and characterization of biosurfactant-producing microorganisms from petroleum-
contaminated soil and further optimization of parameters for biosurfactant production
detection of potential strain, E24 value of the isolates
was compared. It was found that strain C8 showed
77.77% emulsification, which was significantly high
compared to otherstrains[Table 1].From10selected
colonies, therefore, only one colony was selected
as a highly potent biosurfactant producer based on
emulsification (77.77%), oil spread (1.7 cm), drop
collapse, and hemolytic activity (α-hemolysis).
The strains showed α, γ, or no hemolysis [Table 1].
The potent strain was characterized by standard
bacteriological procedure through biochemical
tests, and the results were interpreted with Bergey’s
ManualofDeterminativeBiologyandwereidentified
to be Staphylococcus spp. [Table 2]. For further
identification and phylogenetic relation analysis,
16s rRNA sequencing was performed which shows
that isolated biosurfactant-producing bacteria was
Staphylococcus epidermidis [Figure 1].
Effect of pH and temperature on biosurfactant
production
The applications of biosurfactants in various fields
depend on their stability at different ranges of
temperature and pH. The stability of biosurfactant
was tested over a wide range of temperature
ranging from 15°C to 55°C and pH 4–12. The pH
of the medium was important characteristic for cell
growth of organism and production of secondary
metabolites. At pH 5, the biosurfactant production
was severely decreased, and the cell growth
was significantly retarded. This low pH created
unfavorable growth conditions for the bacterial
population. When the initial pH was set to 8, the
emulsification activity increased (E24 = 45.45%).
In case of S. epidermidis, the emulsification
activity was 51.25% for pH 10 [Figure 2a]. The
emulsification index decreased with any further
increase in pH.Therefore, it was concluded that any
change to both lower and higher pH values caused
an appreciable drop in biosurfactant production.
Temperature is one of the critical parameters
that greatly affected the culture growth and the
biosurfactant production. The results in the present
study revealed that the biosurfactant activity
reached the highest for the isolate S. epidermidis
grown at 45°C (E24 = 55%) followed by E24 =
Table 1: Comparison of emulsification, oil spreading, drop collapse, and hemolytic activity of the 10 isolated strains from
petroleum‑contaminated soils
Colony number E24
% Oil spreading (cm) Drop collapse Hemolysis
C1 24.24 0.25 + α‑hemolysis
C2 37.83 1.9 + No hemolysis
C3 2.57 0 ‑ No hemolysis
C4 57.14 2 ++ α‑hemolysis
C5 34.28 1.2 + No hemolysis
C6 21.21 1.65 ‑ No hemolysis
C7 43.75 0.3 + No hemolysis
C8 77.77 1.7 +++ α‑hemolysis
C9 37.14 1.5 + No hemolysis
C10 45.71 1.9 ++ γ‑hemolysis
Table 2: Biochemical characterization of the selected
strain (C8)
Characteristics/test Result
Shape Circular
Size 0.1 cm
Margin Smooth
Elevation Flat
Color Pale yellow
Opacity Translucent
Consistency Soft
Gram stain +
Catalase +
Oxidase -
Motility -
Figure 1: Unrooted phylogenetic tree based on 16s rRNA
gene comparison of the biosurfactant-producing bacteria
isolated from petroleum-contaminated soil and the nearest
relative in Genbank, only values 50% are given. NCBI
accession numbers are given
6. Deo and Rana: Isolation, screening, and characterization of biosurfactant-producing microorganisms from petroleum-
contaminated soil and further optimization of parameters for biosurfactant production
IJPBA/Jul-Sep-2018(Suppl)/Vol 9/Issue 3 58
50% at 25°C, and this clearly indicates moderately
thermostable nature of biosurfactant [Figure 2b].
When the incubation temperature increased
to 55°C, bacterial growth and biosurfactant
production were totally inhibited, indicating that
the biosurfactant production by S. epidermidis is
greatly reduced at high temperature.
Effect of carbon and nitrogen sources on
biosurfactant production
Biosurfactants can only act as substitutes of
synthetic surfactants if the cost of the raw material
isminimal.Theuseofvariousalternativesubstrates
is one of the attractive strategies for economical
biosurfactant production. The production of
biosurfactant was found to be dependent on the
composition of the medium. The MSS media
optimization was carried out using various carbon
sources, namely sodium acetate, paraffin, sucrose,
and starch using ammonium nitrate as nitrogen
source. The highest biosurfactant production
was achieved using sucrose (10% w/v) being the
sole source of the carbon [Figure 3a]. Various
nitrogen sources selected were ammonium
sulfate, ammonium nitrate, sodium nitrate, and
urea using sucrose as carbon source, and highest
biosurfactant production was observed with
urea as nitrogen source [Figure 3b]. The highest
biosurfactant production obtained when MSS
media contains sucrose (carbon source) and urea
(nitrogen source) at pH 10 and temperature 55°C.
FTIR analysis
Purification of biosurfactant was carried out
using Folch extraction method and characterized
using FT-IR analysis. It was observed that
characteristic absorbance bands of peptides at
3435–3404.47/cm (NH stretching mode); C-O
stretching mode (aromatic) at 1259.56/cm; and
C=C stretching mode was observed at 1614–
1642/cm. The presence of vinyl chains (HC=CH2
)
was confirmed at 2975/cm [Figure 4].
The FT-IR analysis indicates the presence of
aromatic hydrocarbons combined with a peptide
moiety that is characteristic of lipopeptide
Figure 2: (a) Effect of pH on biosurfactant production as
measured by emulsification index (E24%). (b) Effect of
temperature on biosurfactant production as measured by
emulsification index (E24%)
Figure 3: (a) Effect of different carbon sources on
biosurfactant production as measured by emulsification
index (E24%). (b) Effect of different nitrogen sources on
biosurfactant production as measured by emulsification
index (E24%)
Figure 4: Fourier transform infrared spectra of crude biosurfactant extracted from Staphylococcus epidermidis
a b
a b
7. IJPBA/Jul-Sep-2018(Suppl)/Vol 9/Issue 3 59
Deo and Rana: Isolation, screening, and characterization of biosurfactant-producing microorganisms from petroleum-
contaminated soil and further optimization of parameters for biosurfactant production
biosurfactant when compared with reference FT-
IR spectra.[23]
ACKNOWLEDGMENT
The authors wish to thank Modern College ofArts,
Science and Commerce, Ganeshkhind, Pune, for
providing necessary facilities for carrying out the
research work.
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