This document summarizes improved cultivation and metagenomic methods for bioprospecting in cold environments. Only a small percentage of microorganisms can typically be cultured using standard techniques, leaving much potential undiscovered. Improved cultivation methods aim to better mimic the natural environment, such as using diffusion chambers, hollow-fiber membrane chambers, and gel microdroplets to allow nutrient exchange while separating microbes. Metagenomic methods involve direct sequencing or functional screening of environmental DNA without cultivation. Both approaches can be combined, such as using DNA from enrichment cultures. Bioprospecting in cold environments faces additional challenges like low biomass and restricted access, requiring adaptation of methods.
A study of antibiotic resistance of Extended-Spectrum Beta-Lactamases produci...Premier Publishers
This study investigated antibiotic resistance of Extended-Spectrum Beta-Lactamases (ESBLs) producing Enterobacteriaceae isolated at the University Hospital of Befelatanana in Madagascar. The study found 73 ESBL producing isolates over 6 months, representing 6.3% of total bacteria cultured. The most common ESBL species were Klebsiella spp (34.2%), E. coli (32.9%), and Enterobacter spp (30.1%). All isolates were resistant to amoxicillin, amoxicillin-clavulanic acid, and 3rd generation cephalosporins. Resistance to other antibiotics ranged from 0% to over 75%. Patients under
Science and technology of manipulating and improving microbial strains, in order to enhance their metabolic capacities for biotechnological applications, are referred to as strain improvement.
This document summarizes a presentation on using bioassays to evaluate the genotoxicity of hospital wastewater in Jaipur, India at different concentrations. It describes how hospital wastewater contains various hazardous materials like drugs, chemicals, and pathogens. The Allium cepa (onion) test was used to analyze wastewater samples at concentrations of 100% and 75%. Various parameters like pH, acidity, and alkalinity were measured. Onion root tips exposed to wastewater samples showed increased mitotic abnormalities and chromosomal aberrations compared to controls. The study concludes that direct disposal of untreated hospital wastewater poses genotoxic and carcinogenic risks, so proper treatment is needed before disposal to protect
1) Castanea sativa (European chestnut) leaf extracts containing ursene and oleanene derivatives were found to block Staphylococcus aureus virulence and pathogenesis without inducing resistance.
2) The extracts inhibited all four S. aureus accessory gene regulator (agr) alleles which control virulence factor production, in a concentration-dependent manner without impacting bacterial growth.
3) In vivo testing in a mouse skin infection model showed the extracts attenuated dermatopathology caused by MRSA when administered as a single dose, demonstrating potential as a non-antibiotic therapy.
Removal of Ciprofloxacin (CIP) by bacteria isolated from hospital effluent wa...AI Publications
Most antibiotics are metabolized incompletely by patients after administration and enter the municipal sewage with the patients’ excretion. Therefore, studies on the biodegradability of some clinically important drugs can be taken as a very first step of an environmental risk assessment. The present study reports the biodegradation of CIP by Lactobacillus gesseri, Enterobacter sp., Bacillus sp., Bacillus subtilius and Micrococcus luteus which were isolated as CIP resistance, non pathogenic bacteria. The presence of antibiotic-resistant bacteria was identified using the 16s rRNA sequencing. A 0.5ml of overnight starved bacterial suspensions was introduced into medium containing CIP at 5 ppm. Triplicate samples were incubated at 280C with shaking at 100ppm. A 0.5 ml of subsamples was removed at 2 days interval for a period of 14 days. Samples were subjected to High Performance Liquid Chromatography (HPLC) analysis. Fourier Transform Infrared Spectroscopy (FTIR) analyses were carried out for each sample at the end of the 14 days to find structures of by-products. Complete degradation of CIP by L. gasserri was detected at the end of 14 days of incubation with average degradation rate of 0.182 ±0.15µg /day. Descending degradation rates were followed by Enterobacter sp. (0.75 ±0.03 d-1) and Bacillus sp. (0.41±0.02d-1) at 8 and 6 days respectively. However, clear cut degradation of CIP was not detected for B.subtilis and Micrococcus luteus respectively. Further, FTIR spectrum revealed that incubation of L. gesseri, Enterobacter sp. and Bacillus sp., changed the piperazine ring and quinolone part in the CIP structure while degradation occurred.
This document summarizes a study that evaluated the antimicrobial activity of crude culture filtrate and methanol extract of the mushroom Stereum ostrea against bacteria. Key findings:
- Crude culture filtrate of S. ostrea showed the highest inhibitory activity against both gram-positive and gram-negative bacteria based on zone of inhibition tests, with the strongest effect against Bacillus subtilis. Methanol extract also inhibited bacterial growth but to a lesser degree.
- The minimum inhibitory concentration of both crude extract and methanol extract was 20μl for B. subtilis and 30μl for other tested bacteria.
- Results indicate S. ostrea contains metabolites with potential broad-spectrum antimicrobial properties that
(2016) potential sequencing for detection of pathogenic bacteriaClaudiaTere1
1. The study developed and evaluated a culture-independent method using high-throughput sequencing to detect pathogenic bacteria in spices and herbs.
2. Model samples of paprika and oregano were contaminated with mixtures of Salmonella, E. coli, and S. aureus at levels from 101 to 107 CFU/g.
3. For spices like paprika, the method had detection limits in the range of 104-105 CFU/g and provided semi-quantitative results. For herbs like oregano, sensitivity was lower with detection limits of 107 CFU/g or greater.
- Compounds 2, 12, and 16 were found to enhance biofilm formation in commensal bacteria (Streptococcus cristatus, Streptococcus gordonii, and Streptococcus sanguinis) without enhancing biofilm in pathogenic bacteria.
- These compounds are structurally similar to other known biofilm/quorum sensing inhibitors.
- Using a combination of these structurally related compounds may selectively reduce pathogenic biofilm while retaining commensal bacteria.
A study of antibiotic resistance of Extended-Spectrum Beta-Lactamases produci...Premier Publishers
This study investigated antibiotic resistance of Extended-Spectrum Beta-Lactamases (ESBLs) producing Enterobacteriaceae isolated at the University Hospital of Befelatanana in Madagascar. The study found 73 ESBL producing isolates over 6 months, representing 6.3% of total bacteria cultured. The most common ESBL species were Klebsiella spp (34.2%), E. coli (32.9%), and Enterobacter spp (30.1%). All isolates were resistant to amoxicillin, amoxicillin-clavulanic acid, and 3rd generation cephalosporins. Resistance to other antibiotics ranged from 0% to over 75%. Patients under
Science and technology of manipulating and improving microbial strains, in order to enhance their metabolic capacities for biotechnological applications, are referred to as strain improvement.
This document summarizes a presentation on using bioassays to evaluate the genotoxicity of hospital wastewater in Jaipur, India at different concentrations. It describes how hospital wastewater contains various hazardous materials like drugs, chemicals, and pathogens. The Allium cepa (onion) test was used to analyze wastewater samples at concentrations of 100% and 75%. Various parameters like pH, acidity, and alkalinity were measured. Onion root tips exposed to wastewater samples showed increased mitotic abnormalities and chromosomal aberrations compared to controls. The study concludes that direct disposal of untreated hospital wastewater poses genotoxic and carcinogenic risks, so proper treatment is needed before disposal to protect
1) Castanea sativa (European chestnut) leaf extracts containing ursene and oleanene derivatives were found to block Staphylococcus aureus virulence and pathogenesis without inducing resistance.
2) The extracts inhibited all four S. aureus accessory gene regulator (agr) alleles which control virulence factor production, in a concentration-dependent manner without impacting bacterial growth.
3) In vivo testing in a mouse skin infection model showed the extracts attenuated dermatopathology caused by MRSA when administered as a single dose, demonstrating potential as a non-antibiotic therapy.
Removal of Ciprofloxacin (CIP) by bacteria isolated from hospital effluent wa...AI Publications
Most antibiotics are metabolized incompletely by patients after administration and enter the municipal sewage with the patients’ excretion. Therefore, studies on the biodegradability of some clinically important drugs can be taken as a very first step of an environmental risk assessment. The present study reports the biodegradation of CIP by Lactobacillus gesseri, Enterobacter sp., Bacillus sp., Bacillus subtilius and Micrococcus luteus which were isolated as CIP resistance, non pathogenic bacteria. The presence of antibiotic-resistant bacteria was identified using the 16s rRNA sequencing. A 0.5ml of overnight starved bacterial suspensions was introduced into medium containing CIP at 5 ppm. Triplicate samples were incubated at 280C with shaking at 100ppm. A 0.5 ml of subsamples was removed at 2 days interval for a period of 14 days. Samples were subjected to High Performance Liquid Chromatography (HPLC) analysis. Fourier Transform Infrared Spectroscopy (FTIR) analyses were carried out for each sample at the end of the 14 days to find structures of by-products. Complete degradation of CIP by L. gasserri was detected at the end of 14 days of incubation with average degradation rate of 0.182 ±0.15µg /day. Descending degradation rates were followed by Enterobacter sp. (0.75 ±0.03 d-1) and Bacillus sp. (0.41±0.02d-1) at 8 and 6 days respectively. However, clear cut degradation of CIP was not detected for B.subtilis and Micrococcus luteus respectively. Further, FTIR spectrum revealed that incubation of L. gesseri, Enterobacter sp. and Bacillus sp., changed the piperazine ring and quinolone part in the CIP structure while degradation occurred.
This document summarizes a study that evaluated the antimicrobial activity of crude culture filtrate and methanol extract of the mushroom Stereum ostrea against bacteria. Key findings:
- Crude culture filtrate of S. ostrea showed the highest inhibitory activity against both gram-positive and gram-negative bacteria based on zone of inhibition tests, with the strongest effect against Bacillus subtilis. Methanol extract also inhibited bacterial growth but to a lesser degree.
- The minimum inhibitory concentration of both crude extract and methanol extract was 20μl for B. subtilis and 30μl for other tested bacteria.
- Results indicate S. ostrea contains metabolites with potential broad-spectrum antimicrobial properties that
(2016) potential sequencing for detection of pathogenic bacteriaClaudiaTere1
1. The study developed and evaluated a culture-independent method using high-throughput sequencing to detect pathogenic bacteria in spices and herbs.
2. Model samples of paprika and oregano were contaminated with mixtures of Salmonella, E. coli, and S. aureus at levels from 101 to 107 CFU/g.
3. For spices like paprika, the method had detection limits in the range of 104-105 CFU/g and provided semi-quantitative results. For herbs like oregano, sensitivity was lower with detection limits of 107 CFU/g or greater.
- Compounds 2, 12, and 16 were found to enhance biofilm formation in commensal bacteria (Streptococcus cristatus, Streptococcus gordonii, and Streptococcus sanguinis) without enhancing biofilm in pathogenic bacteria.
- These compounds are structurally similar to other known biofilm/quorum sensing inhibitors.
- Using a combination of these structurally related compounds may selectively reduce pathogenic biofilm while retaining commensal bacteria.
Strain development techniques of industrially important microorganismsMicrobiology
Strain improvement and development involves manipulating microbial strains to enhance their metabolic capacities for biotechnology applications. Targets of improvement include rapid growth, genetic stability, non-toxicity, large cell size, ability to use cheaper substrates, increased productivity, and reduced cultivation costs. Methods for optimization include modifying environmental conditions, nutrition, mutagenesis, transduction, conjugation, transformation, and genetic engineering. Common industrial microorganisms are bacteria such as Bacillus subtilis and yeasts such as Saccharomyces cerevisiae.
This document outlines a proposed seminar that will conduct a comparative microbiological and physicochemical analysis of bottled water and water from treatment plants in Ondo State, Nigeria. The objectives are to determine the microbiological and mineral quality of the samples, compare their levels of purity, and assess antibiotic sensitivity of any isolates. Samples will be collected and tested for bacteria, fungi, coliforms, physicochemical properties. Results will provide information on the quality of bottled water and treated water distributed in Ondo State.
1) The study evaluated the antimicrobial activity of crude culture filtrate and methanol extract of the mushroom Stereum ostrea against both Gram-positive and Gram-negative bacteria.
2) Crude culture filtrate showed higher inhibitory activity compared to methanol extract, as evidenced by larger inhibition zones. The crude filtrate was most effective against Bacillus subtilis and least effective against Klebsiella pneumoniae.
3) The minimal inhibitory concentration of both crude and methanol extracts was 20 μl for Bacillus subtilis and 30 μl for the other bacteria tested, indicating Stereum ostrea was most potent against Bacillus subtilis.
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
The papers for publication in The International Journal of Engineering& Science are selected through rigorous peer reviews to ensure originality, timeliness, relevance, and readability.
This document summarizes the development and testing of water dispersible powder (WDP) formulations containing a novel mosquitocidal bacteria, Bacillus thuringiensis subsp. israelensis/tochigiensis (VCRC B-474), and different carrier materials for mosquito larval control. The bacterial strain was characterized and formulated into WDPs with chalk, bentonite, or talc as carriers. The formulations were tested for mosquito larvicidal activity against Culex quinquefasciatus larvae over 12 months. The chalk formulation showed the highest activity with LC50 values ranging from 0.274-0.523 μg/ml and maintained activity for up to 6 months of storage at room
In vitro experiments of prokaryotic and eukaryotic antimicrobial peptide cyto...AI Publications
These proteinaceous molecules, called antimicrobial peptides (AMPs), are a varied collection of antimicrobial peptides. The ability of AMPs to combat gut infections necessitates further study of the AMP-GI tract interaction. These peptides need to be tested in vitro for cytotoxicity before they may be considered for use in clinical infections. Using the MTT conversion assay, neutral red dye absorption assay, and a comparison to vancomycin, researchers examined the cytotoxicity of gallidermin, nisin A, natural magainin peptides, and melittin in two gastrointestinal cell types (HT29 and Caco-2). Sheep erythrocyte hemolytic activity was also studied, and the influence of AMPs on paracellular permeability was assessed using transepithelial resistance (TEER) and TEM. Gallidermin, nisin A, magainin I, magainin II, and melittin were the least cytotoxic AMPs. To our knowledge, only Melittin and NIS caused considerable hemolysis. There are two distinct ways that melittin and nisin differ in their ability to kill bacteria. It was the only AMP that had an effect on the permeability of the paracellular space. Intestinal tight junctions and cell–cell adhesion were destroyed by long-term melittin therapy, as were microvilli, cell debris, and cell–cell adhesion. Antimicrobial activity and low cytotoxicity make Gallidermin a promising therapeutic drug. The antibacterial properties of Melittin are limited, but its ability to transport poorly bioavailable medicines may be useful.
Strain development techniques of industrially important microorganismsMicrobiology
This document discusses techniques for strain improvement of industrially important microorganisms. The goals of strain improvement include increasing productivity, growth rate, and substrate utilization while decreasing toxicity and costs. Methods include physical and chemical mutagenesis to generate genetic diversity, as well as optimization of environmental and nutritional conditions. Specific techniques covered are mutagenesis, transduction, transformation, conjugation, and genetic engineering. Commonly used microorganisms in industry that are generally recognized as safe include various bacteria and yeast species.
Ppt.strain improvement by ghalia nawalGhalia Nawal
Microbial strain improvement involves enhancing the biosynthetic capabilities of microbes to produce desired products in higher quantities. It can be achieved through various approaches like mutagenesis, genetic recombination, and cloning/genetic engineering. Mutagenesis uses chemical or physical agents to induce mutations in microbes. Genetic recombination combines DNA from two strains to generate superior hybrids. Cloning and genetic engineering allow assembling new DNA combinations to create novel microbes. Precision engineering evaluates relationships between growth, substrate use, and production to optimize industrial microbe performance. Strain improvement has various biotechnological applications such as producing medicines and improving microbe productivity and characteristics.
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
An assessment of medicinal cocus nucifera plant extracts as natural antibioti...Alexander Decker
1. The study assessed the antimicrobial properties of Cocus nucifera plant extracts against five bacterial strains using the agar well diffusion method.
2. Ethanol extracts of C. nucifera roots showed the highest antimicrobial activity, with zones of inhibition ranging from 5.55mm to 13.08mm. The highest activity was seen against Escherichia coli.
3. Phytochemical analysis of C. nucifera root extracts revealed the presence of compounds such as saponins, tannins and flavonoids that are associated with antimicrobial effects and support the traditional use of this plant to treat infectious diseases.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
The document summarizes a study conducted over six months to identify common bacterial contaminants in a microbiology laboratory. Samples were collected from surfaces, air, hands, and clothing of laboratory personnel using culture methods and identified using biochemical and molecular tests. The most common contaminants isolated were Micrococcus (52.94% of cultures) and Bacillus subtilis (23.52% of cultures), primarily from surfaces and air, respectively. The study concludes that proper disinfection, sterilization, and personal hygiene are needed to reduce laboratory contaminants and prevent false positive culture reports and laboratory-acquired infections.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
This document provides a detailed literature review for a research proposal on isolating and characterizing antibiotic-producing microorganisms from soil samples near industrial areas. The review covers soil microbial communities and interactions, common types of antibiotic-producing microbes found in soil like Actinomycetes and Bacillus species, methods for isolating pure cultures of microbes, and techniques for identifying isolated microbes and determining their antibiotic activity. The proposed research aims to screen soil samples for new antibiotics that could have applications in industry and medicine.
This document discusses strain improvement and preservation in biotechnology. It defines a strain as a group of species with distinguishing characteristics. The main approaches to strain improvement discussed are mutant selection, recombination, and recombinant DNA technology. Mutant selection involves applying mutagens to induce beneficial mutations for traits like increased productivity. Recombination generates new combinations of genes between strains. Recombinant DNA technology transfers genes to modify metabolic activities or products. Proper strain preservation methods are also outlined, including freezing, lyophilization, and storage in glycerol or liquid nitrogen. Applications include production of vaccines, enzymes, and other industrial biomolecules.
Strain improvement technique (exam point of view)Sijo A
The development of industrial strains, that can tolerate cultural environment and produces the desired metabolite in large amount from wild type strain is called strain improvement.
The rate of production is controlled by genome of an organism.
Hence the rate of production can be increased by inducing necessory changes in genome of the organism. Hence it is also called genetic improvement of microbial strain.
Metabolomics Analysis on Antifungal Activities Produced by Penicillium oxalic...Agriculture Journal IJOEAR
—In-vitro antagonist tests such as disc diffusion and minimum inhibition concentration (MIC) were conducted against C. gloeosporioides. 1 H-NMR coupled with multivariate statistical analysis was carried out to identify possible compounds produced. Glucose crude extract exhibited the highest percent inhibition of radial growth (PIRG) with 75% and the lowest MIC value with 78 µg mL-1. For metabolomics, different metabolites produced were clustered according to the carbon sources used and gave a representative impression of the metabolites produced by P. oxalicum T3.3. The study has shown the potential of using a combination of 1 H-NMR spectroscopy and multivariate statistical analysis and their correlation with MIC in differentiating the effect of carbon sources used based on the identification of possible metabolites contributing to their differences. Findings from this work may potentially provide the basis for further studies on both antimicrobial activities against plant pathogen and elucidation of the metabolite compounds produced by P. oxalicum T3.3.
Strain improvement through genetic engineeringSulov Saha
Strain improvement through genetic engineering involves manipulating microbial strains to enhance their metabolic capacities for biotechnological applications. The objectives are to get multiple copies of specific genes, produce high amounts of specific proteins or products, and integrate genes of interest between organisms. Strategies for strain improvement include using sources of DNA, vectors, host cells, and metabolic engineering. Genetic engineering has applications in food technology, agriculture, microbiology, and industry such as improving industrial strains.
Mutagenesis; A conventional tool for strain improvement in industry Zohaib HUSSAIN
The strain improvement is the process of improvement and manipulation of microbial strains for the icreasment of metabolic level for industrial applications. The yield of microbial enzymes can be increased by using microbe specific medium for fermentation, improving the fermentation process and strain improvement for higher yield of product.
All these things lead to decrease in cost production. Microbe produce product according to its need therefore there is great need for overproduction. There is tremendous contribution of conventional Mutagenesis for strain improvement. Mutagenesis is important tool for the production of mutants which are capable to produce large product i.e. hyperactive.
Microbial strain selection techniques are used to genetically modify microorganisms for improved industrial applications. Strains can be improved through environmental and nutritional optimization as well as genetic manipulation methods like mutagenesis, transduction, transformation, conjugation and protoplast fusion. The goals of strain improvement are to increase productivity, growth rate, substrate utilization and product yield while reducing costs. Improved microbial strains have various applications in medicine, agriculture and industry for the production of enzymes, antibiotics, amino acids and biofuels.
strain improvement to increase yeild of selected molecules.pptxberciyalgolda1
This document discusses strain improvement to increase the yield of selected molecules. Strain improvement involves manipulating microbial strains to enhance their metabolic capacities for biotechnological applications. Targets of strain improvement include rapid growth, genetic stability, non-toxicity, large cell size, ability to use cheaper substrates, increased productivity, and reduction of cultivation costs. Methods for strain improvement discussed include mutagenesis, optimization of environmental and nutritional conditions, transduction, transformation, conjugation, protoplast fusion, genetic engineering, metabolic engineering, and genome shuffling. The goal is to develop strains with desirable industrial applications.
Strain development techniques of industrially important microorganismsMicrobiology
Strain improvement and development involves manipulating microbial strains to enhance their metabolic capacities for biotechnology applications. Targets of improvement include rapid growth, genetic stability, non-toxicity, large cell size, ability to use cheaper substrates, increased productivity, and reduced cultivation costs. Methods for optimization include modifying environmental conditions, nutrition, mutagenesis, transduction, conjugation, transformation, and genetic engineering. Common industrial microorganisms are bacteria such as Bacillus subtilis and yeasts such as Saccharomyces cerevisiae.
This document outlines a proposed seminar that will conduct a comparative microbiological and physicochemical analysis of bottled water and water from treatment plants in Ondo State, Nigeria. The objectives are to determine the microbiological and mineral quality of the samples, compare their levels of purity, and assess antibiotic sensitivity of any isolates. Samples will be collected and tested for bacteria, fungi, coliforms, physicochemical properties. Results will provide information on the quality of bottled water and treated water distributed in Ondo State.
1) The study evaluated the antimicrobial activity of crude culture filtrate and methanol extract of the mushroom Stereum ostrea against both Gram-positive and Gram-negative bacteria.
2) Crude culture filtrate showed higher inhibitory activity compared to methanol extract, as evidenced by larger inhibition zones. The crude filtrate was most effective against Bacillus subtilis and least effective against Klebsiella pneumoniae.
3) The minimal inhibitory concentration of both crude and methanol extracts was 20 μl for Bacillus subtilis and 30 μl for the other bacteria tested, indicating Stereum ostrea was most potent against Bacillus subtilis.
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
The papers for publication in The International Journal of Engineering& Science are selected through rigorous peer reviews to ensure originality, timeliness, relevance, and readability.
This document summarizes the development and testing of water dispersible powder (WDP) formulations containing a novel mosquitocidal bacteria, Bacillus thuringiensis subsp. israelensis/tochigiensis (VCRC B-474), and different carrier materials for mosquito larval control. The bacterial strain was characterized and formulated into WDPs with chalk, bentonite, or talc as carriers. The formulations were tested for mosquito larvicidal activity against Culex quinquefasciatus larvae over 12 months. The chalk formulation showed the highest activity with LC50 values ranging from 0.274-0.523 μg/ml and maintained activity for up to 6 months of storage at room
In vitro experiments of prokaryotic and eukaryotic antimicrobial peptide cyto...AI Publications
These proteinaceous molecules, called antimicrobial peptides (AMPs), are a varied collection of antimicrobial peptides. The ability of AMPs to combat gut infections necessitates further study of the AMP-GI tract interaction. These peptides need to be tested in vitro for cytotoxicity before they may be considered for use in clinical infections. Using the MTT conversion assay, neutral red dye absorption assay, and a comparison to vancomycin, researchers examined the cytotoxicity of gallidermin, nisin A, natural magainin peptides, and melittin in two gastrointestinal cell types (HT29 and Caco-2). Sheep erythrocyte hemolytic activity was also studied, and the influence of AMPs on paracellular permeability was assessed using transepithelial resistance (TEER) and TEM. Gallidermin, nisin A, magainin I, magainin II, and melittin were the least cytotoxic AMPs. To our knowledge, only Melittin and NIS caused considerable hemolysis. There are two distinct ways that melittin and nisin differ in their ability to kill bacteria. It was the only AMP that had an effect on the permeability of the paracellular space. Intestinal tight junctions and cell–cell adhesion were destroyed by long-term melittin therapy, as were microvilli, cell debris, and cell–cell adhesion. Antimicrobial activity and low cytotoxicity make Gallidermin a promising therapeutic drug. The antibacterial properties of Melittin are limited, but its ability to transport poorly bioavailable medicines may be useful.
Strain development techniques of industrially important microorganismsMicrobiology
This document discusses techniques for strain improvement of industrially important microorganisms. The goals of strain improvement include increasing productivity, growth rate, and substrate utilization while decreasing toxicity and costs. Methods include physical and chemical mutagenesis to generate genetic diversity, as well as optimization of environmental and nutritional conditions. Specific techniques covered are mutagenesis, transduction, transformation, conjugation, and genetic engineering. Commonly used microorganisms in industry that are generally recognized as safe include various bacteria and yeast species.
Ppt.strain improvement by ghalia nawalGhalia Nawal
Microbial strain improvement involves enhancing the biosynthetic capabilities of microbes to produce desired products in higher quantities. It can be achieved through various approaches like mutagenesis, genetic recombination, and cloning/genetic engineering. Mutagenesis uses chemical or physical agents to induce mutations in microbes. Genetic recombination combines DNA from two strains to generate superior hybrids. Cloning and genetic engineering allow assembling new DNA combinations to create novel microbes. Precision engineering evaluates relationships between growth, substrate use, and production to optimize industrial microbe performance. Strain improvement has various biotechnological applications such as producing medicines and improving microbe productivity and characteristics.
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
An assessment of medicinal cocus nucifera plant extracts as natural antibioti...Alexander Decker
1. The study assessed the antimicrobial properties of Cocus nucifera plant extracts against five bacterial strains using the agar well diffusion method.
2. Ethanol extracts of C. nucifera roots showed the highest antimicrobial activity, with zones of inhibition ranging from 5.55mm to 13.08mm. The highest activity was seen against Escherichia coli.
3. Phytochemical analysis of C. nucifera root extracts revealed the presence of compounds such as saponins, tannins and flavonoids that are associated with antimicrobial effects and support the traditional use of this plant to treat infectious diseases.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
The document summarizes a study conducted over six months to identify common bacterial contaminants in a microbiology laboratory. Samples were collected from surfaces, air, hands, and clothing of laboratory personnel using culture methods and identified using biochemical and molecular tests. The most common contaminants isolated were Micrococcus (52.94% of cultures) and Bacillus subtilis (23.52% of cultures), primarily from surfaces and air, respectively. The study concludes that proper disinfection, sterilization, and personal hygiene are needed to reduce laboratory contaminants and prevent false positive culture reports and laboratory-acquired infections.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
This document provides a detailed literature review for a research proposal on isolating and characterizing antibiotic-producing microorganisms from soil samples near industrial areas. The review covers soil microbial communities and interactions, common types of antibiotic-producing microbes found in soil like Actinomycetes and Bacillus species, methods for isolating pure cultures of microbes, and techniques for identifying isolated microbes and determining their antibiotic activity. The proposed research aims to screen soil samples for new antibiotics that could have applications in industry and medicine.
This document discusses strain improvement and preservation in biotechnology. It defines a strain as a group of species with distinguishing characteristics. The main approaches to strain improvement discussed are mutant selection, recombination, and recombinant DNA technology. Mutant selection involves applying mutagens to induce beneficial mutations for traits like increased productivity. Recombination generates new combinations of genes between strains. Recombinant DNA technology transfers genes to modify metabolic activities or products. Proper strain preservation methods are also outlined, including freezing, lyophilization, and storage in glycerol or liquid nitrogen. Applications include production of vaccines, enzymes, and other industrial biomolecules.
Strain improvement technique (exam point of view)Sijo A
The development of industrial strains, that can tolerate cultural environment and produces the desired metabolite in large amount from wild type strain is called strain improvement.
The rate of production is controlled by genome of an organism.
Hence the rate of production can be increased by inducing necessory changes in genome of the organism. Hence it is also called genetic improvement of microbial strain.
Metabolomics Analysis on Antifungal Activities Produced by Penicillium oxalic...Agriculture Journal IJOEAR
—In-vitro antagonist tests such as disc diffusion and minimum inhibition concentration (MIC) were conducted against C. gloeosporioides. 1 H-NMR coupled with multivariate statistical analysis was carried out to identify possible compounds produced. Glucose crude extract exhibited the highest percent inhibition of radial growth (PIRG) with 75% and the lowest MIC value with 78 µg mL-1. For metabolomics, different metabolites produced were clustered according to the carbon sources used and gave a representative impression of the metabolites produced by P. oxalicum T3.3. The study has shown the potential of using a combination of 1 H-NMR spectroscopy and multivariate statistical analysis and their correlation with MIC in differentiating the effect of carbon sources used based on the identification of possible metabolites contributing to their differences. Findings from this work may potentially provide the basis for further studies on both antimicrobial activities against plant pathogen and elucidation of the metabolite compounds produced by P. oxalicum T3.3.
Strain improvement through genetic engineeringSulov Saha
Strain improvement through genetic engineering involves manipulating microbial strains to enhance their metabolic capacities for biotechnological applications. The objectives are to get multiple copies of specific genes, produce high amounts of specific proteins or products, and integrate genes of interest between organisms. Strategies for strain improvement include using sources of DNA, vectors, host cells, and metabolic engineering. Genetic engineering has applications in food technology, agriculture, microbiology, and industry such as improving industrial strains.
Mutagenesis; A conventional tool for strain improvement in industry Zohaib HUSSAIN
The strain improvement is the process of improvement and manipulation of microbial strains for the icreasment of metabolic level for industrial applications. The yield of microbial enzymes can be increased by using microbe specific medium for fermentation, improving the fermentation process and strain improvement for higher yield of product.
All these things lead to decrease in cost production. Microbe produce product according to its need therefore there is great need for overproduction. There is tremendous contribution of conventional Mutagenesis for strain improvement. Mutagenesis is important tool for the production of mutants which are capable to produce large product i.e. hyperactive.
Microbial strain selection techniques are used to genetically modify microorganisms for improved industrial applications. Strains can be improved through environmental and nutritional optimization as well as genetic manipulation methods like mutagenesis, transduction, transformation, conjugation and protoplast fusion. The goals of strain improvement are to increase productivity, growth rate, substrate utilization and product yield while reducing costs. Improved microbial strains have various applications in medicine, agriculture and industry for the production of enzymes, antibiotics, amino acids and biofuels.
strain improvement to increase yeild of selected molecules.pptxberciyalgolda1
This document discusses strain improvement to increase the yield of selected molecules. Strain improvement involves manipulating microbial strains to enhance their metabolic capacities for biotechnological applications. Targets of strain improvement include rapid growth, genetic stability, non-toxicity, large cell size, ability to use cheaper substrates, increased productivity, and reduction of cultivation costs. Methods for strain improvement discussed include mutagenesis, optimization of environmental and nutritional conditions, transduction, transformation, conjugation, protoplast fusion, genetic engineering, metabolic engineering, and genome shuffling. The goal is to develop strains with desirable industrial applications.
The document describes several methods for developing transgenic plants, including direct gene transfer methods like microinjection and electroporation, and indirect methods using Agrobacterium. It also discusses some achievements of transgenic plants, including improved nutritional quality, insect and disease resistance, and herbicide tolerance. A new study is described that develops a double right border binary vector to more easily produce transgenic plants without selectable marker genes. This allows the marker gene to be separated from the gene of interest to generate "clean" transgenic plants.
Role of biotechnology in enhancing fruit crop production and qualityankit gawri
It was evident that developed biotechnological approaches have the potential to enhance the yield, quality, and shelf-life of fruits and vegetables to meet the demands of the 21st century. However, the developed biotech approaches for fruits and vegetables were more of academic jargon than a commercial reality
This document discusses current trends in plant breeding. It begins by defining plant breeding as the genetic improvement of crops using both traditional and modern techniques to select for desired traits. It then provides background on the history of plant breeding, including the Green Revolution. The document outlines various modern breeding technologies like phenomics, proteomics, transcriptomics, genetic modification, and the role of bioinformatics in data analysis. It discusses using these omics approaches and genome sequencing to enable a second Green Revolution with crops that are higher yielding, more nutritious, and tolerant of environmental stresses. The goal is to produce more food to feed a growing global population in a sustainable way.
Industrial microorganisms are microbes used in industry to produce products like chemicals, food, detergents, textiles, and bioenergy. Important industrial microbes include Saccharomyces cerevisiae and Aspergillus niger. Microorganisms are isolated using techniques like enrichment culture and indicator systems. Isolated strains can be improved through mutation, genetic engineering, and hybridization to develop strains with desired properties like increased yield, stability, and safety. Improved strains are preserved long-term using cryopreservation or freeze drying for future use in industrial fermentation and production.
Basic Knowledge about industrial microorganism. why industry choose microorganism rather than chemical. isolation technique of microorganism. source of microorganisms. Process of using microorganism. Disadvantages of using microorganisms in industry. Process of genetic modification of microorganisms. Storage process of microorganism. preservation methods of microorganism. Reculture methods of microorganism.
Traditional phenotypic methods and newer genotypic methods can both be used to identify bacteria. Phenotypic methods include gram staining, culturing, and analyzing biochemical characteristics and reactions. These methods have limitations as some bacteria cannot be cultured. Genotypic methods like MALDI-TOF, PCR, and microarrays identify bacteria based on their genetic material and can identify bacteria directly from clinical samples faster than phenotypic methods. A variety of biochemical tests are used as part of phenotypic identification to analyze carbohydrate metabolism, production of specific compounds, enzyme activity, and other characteristics.
Presentation from the International Life Sciences Institute, India - "Scientific Workshop on Safety Assessment of GM Foods" held on 14-15 October, 2015 in New Delhi, India
This document discusses plant molecular pharming (PMP), which uses plants as bioreactors for producing recombinant pharmaceutical proteins. It covers the definition, history, strategies, host systems, production of antibiotics/enzymes/vaccines in plants, advantages/disadvantages of plant systems, and issues of transgene pollution. Key points include:
- PMP uses whole plants, plant cells or tissues to produce commercially valuable proteins like vaccines via recombinant DNA.
- Early work in 1986 produced human growth hormone in tobacco and sunflower. Commercial production of various proteins in plants has occurred.
- Strategies include transforming host plants, growing biomass, processing/purifying the product of interest.
- Plants,
The document describes a seminar presentation on using plant tissue culture techniques to induce variation and improve crops. It provides background on tissue culture, discusses types of culture and variation that can occur. It also presents a case study on developing insect-resistant transgenic brinjal (eggplant) and studies on tissue culture-induced variation in tomato and torenia plants. The conclusion states that tissue culture has been valuable for plant biotechnology and improving food security.
This document discusses various methods for improving microbial strains, including selecting naturally occurring variants, manipulating existing genetics, and introducing new genetics. It focuses on mutation and selection techniques like chemical or UV mutagenesis followed by selection on selective media. Genetic engineering techniques are also summarized, including restriction digestion, ligation into vectors, transformation, and screening of recombinants. Common vectors like pBR322, pUC18, phages like M13, and cosmids are described. The overall goal is to outline strategies for isolating industrially useful microbial mutants.
New concepts in maintenance of plant breeding promises and prospectsZuby Gohar Ansari
1. The document discusses new concepts in plant breeding that promise to improve crops, including exploiting genes from wild plants and breaking complex traits into components to select for improved crops.
2. Recent advances in breeding technology such as wide hybridization between crops and wild relatives, molecular marker-assisted selection, and selecting for quantitative trait components are allowing plant breeders to make faster progress in improving yields and stress tolerance.
3. While genetic engineering holds promise, conventional plant breeding continues to advance and ensure contributions to agriculture through increasingly sophisticated methods.
A powerful non-transgenic reverse genetics method that combines chemical mutagenesis with PCR based screening to identify point mutations in regions of interest.
EcoTILLING is a molecular technique that is similar to TILLING, except that its objective is to uncover natural genetic variation as opposed to induced mutations.
This research article summarizes a study investigating potential artifacts in 1H NMR-based metabolomic studies on cell cultures due to contaminants from plastic cell culture dishes. The researchers found that brief rinsing or incubation of culture medium in plastic dishes eluted chemicals that could confound assays of certain metabolites. Extraction of "null samples" using perchloric acid, methanol-chloroform, or acetonitrile also produced artifacts from plastic dishes, though to a lesser extent with methanol and acetonitrile. The best practice is to run extraction of blank dishes with every batch of experiments to identify background contamination and provide a reference spectrum.
This document summarizes advances in seed testing technologies for major crops. It discusses the history and concepts of seed testing, including assessing genetic purity, physical purity, physiological quality, and seed health. Modern methods like molecular markers, image analysis, and spectral imaging provide non-destructive, quick, and highly accurate testing compared to traditional techniques. These advances allow for improved evaluation of seed quality attributes and performance.
Detection of Genetically modified plants and Organic Seed production.NSStudents
The Presentation is prepared by the N.S Institution of science, Markapur.
It consists of a basic introduction related to Detection of Genetically modified plants and Organic Seed production.
Current approaches toward production ofsecondary plant metabolitesshahnam azizi
In this presentation you can familiar with:
Primary metabolite vs secondary metabolite
Importance and function of secondary metabolite
Approaches for increasing secondary metabolite production in plant tissue culture
Similar to improved cultivation and metagenomics as new tools for bioprospecting in cold enviroments (20)
Climate Change All over the World .pptxsairaanwer024
Climate change refers to significant and lasting changes in the average weather patterns over periods ranging from decades to millions of years. It encompasses both global warming driven by human emissions of greenhouse gases and the resulting large-scale shifts in weather patterns. While climate change is a natural phenomenon, human activities, particularly since the Industrial Revolution, have accelerated its pace and intensity
Improving the viability of probiotics by encapsulation methods for developmen...Open Access Research Paper
The popularity of functional foods among scientists and common people has been increasing day by day. Awareness and modernization make the consumer think better regarding food and nutrition. Now a day’s individual knows very well about the relation between food consumption and disease prevalence. Humans have a diversity of microbes in the gut that together form the gut microflora. Probiotics are the health-promoting live microbial cells improve host health through gut and brain connection and fighting against harmful bacteria. Bifidobacterium and Lactobacillus are the two bacterial genera which are considered to be probiotic. These good bacteria are facing challenges of viability. There are so many factors such as sensitivity to heat, pH, acidity, osmotic effect, mechanical shear, chemical components, freezing and storage time as well which affects the viability of probiotics in the dairy food matrix as well as in the gut. Multiple efforts have been done in the past and ongoing in present for these beneficial microbial population stability until their destination in the gut. One of a useful technique known as microencapsulation makes the probiotic effective in the diversified conditions and maintain these microbe’s community to the optimum level for achieving targeted benefits. Dairy products are found to be an ideal vehicle for probiotic incorporation. It has been seen that the encapsulated microbial cells show higher viability than the free cells in different processing and storage conditions as well as against bile salts in the gut. They make the food functional when incorporated, without affecting the product sensory characteristics.
Kinetic studies on malachite green dye adsorption from aqueous solutions by A...Open Access Research Paper
Water polluted by dyestuffs compounds is a global threat to health and the environment; accordingly, we prepared a green novel sorbent chemical and Physical system from an algae, chitosan and chitosan nanoparticle and impregnated with algae with chitosan nanocomposite for the sorption of Malachite green dye from water. The algae with chitosan nanocomposite by a simple method and used as a recyclable and effective adsorbent for the removal of malachite green dye from aqueous solutions. Algae, chitosan, chitosan nanoparticle and algae with chitosan nanocomposite were characterized using different physicochemical methods. The functional groups and chemical compounds found in algae, chitosan, chitosan algae, chitosan nanoparticle, and chitosan nanoparticle with algae were identified using FTIR, SEM, and TGADTA/DTG techniques. The optimal adsorption conditions, different dosages, pH and Temperature the amount of algae with chitosan nanocomposite were determined. At optimized conditions and the batch equilibrium studies more than 99% of the dye was removed. The adsorption process data matched well kinetics showed that the reaction order for dye varied with pseudo-first order and pseudo-second order. Furthermore, the maximum adsorption capacity of the algae with chitosan nanocomposite toward malachite green dye reached as high as 15.5mg/g, respectively. Finally, multiple times reusing of algae with chitosan nanocomposite and removing dye from a real wastewater has made it a promising and attractive option for further practical applications.
Epcon is One of the World's leading Manufacturing Companies.EpconLP
Epcon is One of the World's leading Manufacturing Companies. With over 4000 installations worldwide, EPCON has been pioneering new techniques since 1977 that have become industry standards now. Founded in 1977, Epcon has grown from a one-man operation to a global leader in developing and manufacturing innovative air pollution control technology and industrial heating equipment.
Optimizing Post Remediation Groundwater Performance with Enhanced Microbiolog...Joshua Orris
Results of geophysics and pneumatic injection pilot tests during 2003 – 2007 yielded significant positive results for injection delivery design and contaminant mass treatment, resulting in permanent shut-down of an existing groundwater Pump & Treat system.
Accessible source areas were subsequently removed (2011) by soil excavation and treated with the placement of Emulsified Vegetable Oil EVO and zero-valent iron ZVI to accelerate treatment of impacted groundwater in overburden and weathered fractured bedrock. Post pilot test and post remediation groundwater monitoring has included analyses of CVOCs, organic fatty acids, dissolved gases and QuantArray® -Chlor to quantify key microorganisms (e.g., Dehalococcoides, Dehalobacter, etc.) and functional genes (e.g., vinyl chloride reductase, methane monooxygenase, etc.) to assess potential for reductive dechlorination and aerobic cometabolism of CVOCs.
In 2022, the first commercial application of MetaArray™ was performed at the site. MetaArray™ utilizes statistical analysis, such as principal component analysis and multivariate analysis to provide evidence that reductive dechlorination is active or even that it is slowing. This creates actionable data allowing users to save money by making important site management decisions earlier.
The results of the MetaArray™ analysis’ support vector machine (SVM) identified groundwater monitoring wells with a 80% confidence that were characterized as either Limited for Reductive Decholorination or had a High Reductive Reduction Dechlorination potential. The results of MetaArray™ will be used to further optimize the site’s post remediation monitoring program for monitored natural attenuation.
Presented by The Global Peatlands Assessment: Mapping, Policy, and Action at GLF Peatlands 2024 - The Global Peatlands Assessment: Mapping, Policy, and Action
ENVIRONMENT~ Renewable Energy Sources and their future prospects.tiwarimanvi3129
This presentation is for us to know that how our Environment need Attention for protection of our natural resources which are depleted day by day that's why we need to take time and shift our attention to renewable energy sources instead of non-renewable sources which are better and Eco-friendly for our environment. these renewable energy sources are so helpful for our planet and for every living organism which depends on environment.
Recycling and Disposal on SWM Raymond Einyu pptxRayLetai1
Increasing urbanization, rural–urban migration, rising standards of living, and rapid development associated with population growth have resulted in increased solid waste generation by industrial, domestic and other activities in Nairobi City. It has been noted in other contexts too that increasing population, changing consumption patterns, economic development, changing income, urbanization and industrialization all contribute to the increased generation of waste.
With the increasing urban population in Kenya, which is estimated to be growing at a rate higher than that of the country’s general population, waste generation and management is already a major challenge. The industrialization and urbanization process in the country, dominated by one major city – Nairobi, which has around four times the population of the next largest urban centre (Mombasa) – has witnessed an exponential increase in the generation of solid waste. It is projected that by 2030, about 50 per cent of the Kenyan population will be urban.
Aim:
A healthy, safe, secure and sustainable solid waste management system fit for a world – class city.
Improve and protect the public health of Nairobi residents and visitors.
Ecological health, diversity and productivity and maximize resource recovery through the participatory approach.
Goals:
Build awareness and capacity for source separation as essential components of sustainable waste management.
Build new environmentally sound infrastructure and systems for safe disposal of residual waste and replacing current dumpsites which should be commissioned.
Current solid waste management situation:
The status.
Solid waste generation rate is at 2240 tones / day
collection efficiently is at about 50%.
Actors i.e. city authorities, CBO’s , private firms and self-disposal
Current SWM Situation in Nairobi City:
Solid waste generation – collection – dumping
Good Practices:
• Separation – recycling – marketing.
• Open dumpsite dandora dump site through public education on source separation of waste, of which the situation can be reversed.
• Nairobi is one of the C40 cities in this respect , various actors in the solid waste management space have adopted a variety of technologies to reduce short lived climate pollutants including source separation , recycling , marketing of the recycled products.
• Through the network, it should expect to benefit from expertise of the different actors in the network in terms of applicable technologies and practices in reducing the short-lived climate pollutants.
Good practices:
Despite the dismal collection of solid waste in Nairobi city, there are practices and activities of informal actors (CBOs, CBO-SACCOs and yard shop operators) and other formal industrial actors on solid waste collection, recycling and waste reduction.
Practices and activities of these actor groups are viewed as innovations with the potential to change the way solid waste is handled.
CHALLENGES:
• Resource Allocation.
Microbial characterisation and identification, and potability of River Kuywa ...Open Access Research Paper
Water contamination is one of the major causes of water borne diseases worldwide. In Kenya, approximately 43% of people lack access to potable water due to human contamination. River Kuywa water is currently experiencing contamination due to human activities. Its water is widely used for domestic, agricultural, industrial and recreational purposes. This study aimed at characterizing bacteria and fungi in river Kuywa water. Water samples were randomly collected from four sites of the river: site A (Matisi), site B (Ngwelo), site C (Nzoia water pump) and site D (Chalicha), during the dry season (January-March 2018) and wet season (April-July 2018) and were transported to Maseno University Microbiology and plant pathology laboratory for analysis. The characterization and identification of bacteria and fungi were carried out using standard microbiological techniques. Nine bacterial genera and three fungi were identified from Kuywa river water. Clostridium spp., Staphylococcus spp., Enterobacter spp., Streptococcus spp., E. coli, Klebsiella spp., Shigella spp., Proteus spp. and Salmonella spp. Fungi were Fusarium oxysporum, Aspergillus flavus complex and Penicillium species. Wet season recorded highest bacterial and fungal counts (6.61-7.66 and 3.83-6.75cfu/ml) respectively. The results indicated that the river Kuywa water is polluted and therefore unsafe for human consumption before treatment. It is therefore recommended that the communities to ensure that they boil water especially for drinking.
Evolving Lifecycles with High Resolution Site Characterization (HRSC) and 3-D...Joshua Orris
The incorporation of a 3DCSM and completion of HRSC provided a tool for enhanced, data-driven, decisions to support a change in remediation closure strategies. Currently, an approved pilot study has been obtained to shut-down the remediation systems (ISCO, P&T) and conduct a hydraulic study under non-pumping conditions. A separate micro-biological bench scale treatability study was competed that yielded positive results for an emerging innovative technology. As a result, a field pilot study has commenced with results expected in nine-twelve months. With the results of the hydraulic study, field pilot studies and an updated risk assessment leading site monitoring optimization cost lifecycle savings upwards of $15MM towards an alternatively evolved best available technology remediation closure strategy.
2. 18 Extremophiles (2015) 19:17–29
1 3
can be due to many factors, including lack of specific nutri-
ents, oxygen level, temperature, pH and osmotic condi-
tions as well as missing growth factors, possibly produced
by other organisms in the community (Vartoukian et al.
2010). Culturing will always favor the organisms that are
best at adapting to the conditions applied in the laboratory,
and these organisms are not necessarily the most dominant
or ecologically important organisms in the environment.
Insights into the uncultured fraction have been fueled by
sequencing of the 16S rRNA genes of bacteria from envi-
ronmental samples, and this in turn has resulted in the iden-
tification of numerous new bacterial phyla, of which very
few are represented by cultivated strains (Achtman and
Wagner 2008; Stewart 2012).
Several methods that allow exploitation of the vast
majority of microorganisms that are difficult to cultivate in
the laboratory have been described. The methods can basi-
cally be divided into (1) improved cultivation techniques or
(2) metagenomic methods. The improved cultivation tech-
niques can be used to establish pure cultures or complex
communities available for culture-dependent screenings or
metagenomic approaches. The metagenomic methods are
either based on sequencing of metagenomes and bioinfor-
matic analysis or functional expression of metagenomic
libraries to identify genes or gene clusters of interest.
Bioprospecting in extreme environments with non-
standard conditions, such as low temperature, is met with
additional challenges. Conventional cultivation may be dif-
ficult because of insufficient knowledge on media require-
ments or very prolonged incubation time, enrichment in
situ may be problematic if the environment is located in
remote areas, metagenomic analyses may be hampered
by low amounts of environmental DNA, and heterologous
expression of enzymes and other bioactive molecules may
be challenging since most host organisms are not devel-
oped for extreme conditions. This review will present the
methods developed for improved cultivation as well as the
metagenomic approaches for bioprospecting with focus on
the challenges faced by bioprospecting in cold environ-
ments. The methods that have been developed to deal with
these problems will be summarized and possible routes to
future improvements will be highlighted.
Cultivation‑based bioprospecting
A number of techniques have been developed for improved
cultivation of natural microorganisms in the laboratory or
in situ in the environment. Although only a few of these
methods have been used specifically to bring cold-adapted
microorganisms into culture, they all have the potential
to increase the output of cultivation-based bioprospecting
projects in cold environments. It is important, however, to
keep in mind that cold-adapted microorganisms are faced
with several challenges imposed by the low temperature.
These include increased water viscosity, decreased diffu-
sion rates, and not least reduced biochemical reaction rates
(Feller 2013). For most biological systems, the activity of
a mesophilic enzyme will be 16–80 times lower at 0 °C
than at 37 °C (Georlette et al. 2004). In general, cold-active
enzymes maintain a high biochemical reaction rate at low
temperature by having a more flexible structure, result-
ing in a lower substrate affinity and increased heat lability
(Feller and Gerday 2003; Feller 2013). Therefore, when
designing experiments, care should be taken not to expose
enzymes or organisms to elevated temperatures, as this
might lead to irreversible inactivation of enzymes.
If communities or pure cultures can be established
under laboratory settings, they will in many cases be suit-
able for bioprospecting efforts. Most techniques have been
developed for traditional laboratory conditions, e.g., 20 °C,
1 atm., pH 7, etc., and are often not directly applicable to
organisms originating from extreme environments. How-
ever, since most laboratories are equipped with facilities
that allow incubation at low temperatures, screening of cul-
tivated cold-adapted organisms can be carried out simply
using existing assays at low temperature. A classic exam-
ple is plate-based screening using chromogenic substrates
that develop detectable colors upon activity (Fathallh et al.
2012), and by combining a mixture of different insoluble
chromogenic substrates, multiple activities can be detected
by various color combinations in single plate experiments
(Ten et al. 2004, 2005). Subsequent identification and
cloning of genes of interest have been aided by the emer-
gence of genomics and access to affordable whole genome
sequencing of bacterial isolates.
The cultivation-dependent method of bioprospecting
can involve several steps, ranging from traditional cultur-
ing to the various versions of optimized culturing condi-
tions discussed below (Fig. 1). The subsequent isolation,
screening and purification of microorganisms are typically
done by plating on Petri dishes to obtain pure cultures for
further analysis. Although improved cultivation methods
may increase the proportion of cultivable microorganisms,
many of these organisms will not grow as pure cultures in
the laboratory. In addition, the specific gene or gene cluster
responsible for the trait of interest often has to be identi-
fied to facilitate more efficient expression and characteriza-
tion than possible with the natural isolate. Organisms solely
capable of growing as microcolonies or in mixed commu-
nities can be analyzed by metagenomic approaches, which
may also be applied directly to environmental samples. A
useful coupling of culture-dependent and metagenomic
methods is to use genomic DNA (gDNA) or cDNA from
enrichment cultures or pure natural isolates for metagen-
omic methods (Hobel et al. 2004).
3. 19Extremophiles (2015) 19:17–29
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Methods for improving cultivation
To bring more microorganisms into culture, it is reasonable
to try to mimic the natural environment in the culturing
conditions, e.g., by simulating temperature, pH, oxygen
level or light, and using water or materials taken from the
environment. An alternative taking this one step further is
to bring the natural environment into the laboratory and
Fig. 1 Simplified and general overview of culture-dependent and
metagenomic methods for bioprospecting. In principle any environ-
mental sample can be used. Culture-dependent methods (gray boxes)
include a culturing step ranging from traditional culturing to various
combinations of improved culturing techniques, leading to isola-
tion and screening of natural isolates. Metagenomic methods (blue
boxes) rely on DNA extraction, which can be either direct or indirect.
Extracted DNA can be used for either sequence-based (red) or func-
tional (green) screening approaches, where the latter might require
amplification. Identified activities from natural isolates or recom-
binantly expressed proteins are then characterized. Links between
culture-dependent and metagenomic methods include using DNA
from enrichment cultures or natural isolates (dotted lines). See text
for details. Inspired by Akondi and Lakshmi (2013)
5. 21Extremophiles (2015) 19:17–29
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low nutrient flux to simulate environmental conditions.
The pores in the gel microdroplets allow for exchange of
metabolites between organisms captured in separate drop-
lets. Millions of microdroplets can be present in each
growth column, and each growth column can be placed in
specific media and growth conditions. Gel microdroplets
with microcolonies are then identified by flow cytometry.
The system was tested using seawater samples, and the
highest diversity was obtained when filtered seawater was
used as growth medium compared to seawater with nutri-
ents added, suggesting that most of the diversity is outcom-
peted by relatively few fast growing organisms in a rich
environment (Zengler et al. 2002). A technique combining
the advantageous in situ incubation of diffusion chambers
and hollow-fiber membrane chambers with the small size
of the gel microdroplets was later developed by encapsulat-
ing microorganisms in agar and then further encasing them
in a polysulfonic polymeric membrane, which permits
exchange of chemical components and allows more flex-
ible in situ incubation by being small (Fig. 2c). By inocu-
lating with microorganisms from coral mucus and placing
the encased agar on corals for incubation, approximately
50 % of the cultured organisms represented novel ribotypes
(Ben-Dov et al. 2009). Unfortunately, no attempts were
made to cultivate the microorganisms outside the encased
agar. A similar method, the I-tip, was recently developed
based on standard laboratory equipment (e.g., micropipette
tips). It was used to cultivate bacteria associated with a
sponge and was superior to a standard agar-based cultiva-
tion with respect to cultured diversity (Jung et al. 2014).
Dilution to extinction
In the dilution to extinction technique, natural samples
are diluted to very low cell concentrations and cultured
to establish clonal populations. Connon and Giovannoni
(2002) developed a high-throughput version of this method,
which allows for cell enumeration of small volumes with
low cell densities, making it possible to run the incubations
in microtiter plates. This technique was used to culture
members of the SAR11 clade which, despite its ubiquity,
had previously evaded cultivation (Rappe et al. 2002).
Resuscitation‑promoting factors (Rpf)
Resuscitation-promoting factors (Rpf) are involved in
resuscitation of dormant organisms and thereby in increas-
ing their cultivability. Rpf have been intensively studied
in Mycobacterium tuberculosis (Mukamolova et al. 1998;
Kana et al. 2008) and this knowledge was used to cultivate
the otherwise uncultivable strain Psychrobacter sp. strain
MSC33 on standard media supplemented with a short Rpf
peptide (Nichols et al. 2008).
Gelling agent
Agar is by far the most commonly used gelling agent for
microbiology. However, other gelling agents have been
shown to be superior to agar in supporting growth of
diverse bacteria. Using gellan gum, a water-soluble poly-
saccharide produced by Pseudomonas elodea, instead of
agar, Tamaki et al. (2009) demonstrated that gellan gum
not only supported much faster growth of bacteria from a
freshwater lake sediment compared to agar, but also sup-
ported growth of more novel isolates than agar.
Incubation time
There are several examples of increased cultivability with
increased incubation time. Rarely isolated phylogenetic
groups of soil bacteria have been cultivated by prolonged
incubation (up to 3 months) (Davis et al. 2005; Steven et al.
2007) and a general increase in the diversity of haloarchaeal
groups was observed with increasing incubation time (up
to 12 weeks) (Burns et al. 2004). In another study, the cul-
tured fraction of sea ice bacteria increased from 0.005 to
0.2 % if the incubation time was prolonged from 5 weeks
to 3 months (Lanoil et al. 2009). Extended incubation time
(more than 8 months) was found to significantly increase
the diversity of cultured bacteria from a cold and alkaline
environment, as well as the fraction of unknown genera and
species (Vester et al. 2013). Also, many previously uncul-
tured bacteria were discovered if multiple growth media
were used (Joseph et al. 2003) and an increase in cultured
diversity was observed if solid media were used rather than
liquid (Vester et al. 2013).
Sub‑zero incubation
Cryophiles are extremely cold-adapted organisms capa-
ble of living in brine veins surrounding soil particles in
permafrost soils or in ice brine veins at sub-zero tempera-
tures. Such organisms can be cultured in media with high
salt concentration supplemented with glycerol as shown
for Planococcus halocryophilus, which was able to grow at
temperatures as low as −15 °C (Mykytczuk et al. 2013).
Metagenome‑based bioprospecting
Since the culture-dependent methods only catch a very
limited fraction of the total diversity of a given sample,
metagenomic methods for bioprospecting can be highly
valuable. Although these methods do not require cultiva-
tion, they may be restrained by the amount and quality of
DNA obtainable from the environment or community of
interest. The metagenomic approach can either be sequence
6. 22 Extremophiles (2015) 19:17–29
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based, involving high-throughput sequencing and bioin-
formatic analysis, or function based, aimed at functional
expression of metagenomic libraries to identify genes or
gene clusters of interest (Fig. 1).
Sequence‑based metagenomics
The sequence-based approach includes screening for genes
by hybridization with labeled DNA probes or by PCR, both
of which are based on sequences of known genes (Aakvik
et al. 2011; Simon and Daniel 2011; Lee and Lee 2013).
Since the price for direct high-throughput sequencing of
metagenomic DNA has reached a level affordable for many
laboratories (de Pascale et al. 2012; Hunter et al. 2012), this
is now the standard technology for gene discovery (Ekkers
et al. 2012). As outlined in Fig. 1, the sequence-based
approach consists of three main steps: (1) the sequencing
itself (2) bioinformatic analyses including functional anno-
tation of genes, and finally (3) the heterologous expression
of identified genes to document activity. For each of these
steps, different opportunities exist and choices have to be
made.
The various high-throughput sequencing platforms each
have their advantages and disadvantages and the gener-
ated sequences subsequently go through a series of steps
including quality control, filtering, assembly, and func-
tional annotation of assembled sequences. Details of the
sequencing platforms and sequence handling procedures
have recently been reviewed by Loman et al. (2012) and
Kim et al. (2013), respectively. It is important to note
that functional annotation is generally based on sequence
homology (Hunter et al. 2012; Kim et al. 2013) and since
annotations are based on already known and characterized
sequences, completely novel sequences are likely not to be
annotated correctly or simply not annotated at all. To illus-
trate this, of six million proteins identified in a survey of
fully sequenced bacterial genomes, the function of approxi-
mately 40 % was classified as unknown (Blaby-Haas and
de Crecy-Lagard 2011).
The outcome of the sequencing and bioinformatics pipe-
line is typically a long list of genes of interest, which has
to be investigated further to choose the best candidates for
expression. Only 2.8 % of more than 20 million proteins
in the UniProt database have had their existence confirmed
at either the protein or transcript level (Temperton and
Giovannoni 2012), indicating that a significant part of the
annotated genes might not be expressed in the laboratory
or at a too low level in Nature to be detected. Nevertheless,
a prioritized list of expression targets can be made from
analyses of properties of similar proteins, signal peptides,
degree of novelty, etc., and the sequence information can
be used for codon optimization and choice of expression
organism.
Function‑based metagenomics
Functional metagenomics is the study of a metagenome by
expression in a foreign host. This approach involves criti-
cal decisions regarding DNA fragment size, vectors and
expression hosts (Ekkers et al. 2012). As for the sequence-
based approach, the starting point is DNA (or cDNA). This
has to be of good quality in terms of purity and fragment
length. If the concentration of the purified DNA is too low
for cloning, as is often the case for extreme environments,
the DNA can be amplified by multiple displacement ampli-
fication (MDA) (Taupp et al. 2011).
DNA isolation
Both gDNA and cDNA can be used for metagenomics and
the DNA can be obtained from different sources includ-
ing environmental samples (directly or indirectly isolated),
enrichment cultures or pure isolates. Due to the difficulties
in isolating mRNA from environmental samples, studies
using cDNA are rare (Simon and Daniel 2011). For both
enrichment cultures and pure isolates, isolation of mRNA
is more feasible than for complex environmental samples.
Also, if eukaryotic genes are of interest for a functional
bioprospecting approach using a prokaryotic host for
expression, cDNA is necessary to circumvent problems
introduced by introns in gDNA (Aakvik et al. 2011). Direct
extraction of DNA from environmental samples involves
the in situ lysis of cells within the sample matrix and DNA
extraction, whereas indirect sampling involves the isolation
of cells from the sample matrix before ex situ lysis. The
result from direct extraction is typically higher yield but
more fragmented DNA, whereas indirect extraction yields
are lower but with larger DNA fragments. Which method
to choose depends on the sample matrix as well as the
intended uses. Direct extraction can result in co-extraction
of inhibitory substances such as humic acids from soil or
the matrix may itself adsorb the DNA. Unwanted DNA
such as eukaryotic or extracellular DNA can also consti-
tute a significant part of the extracted DNA (Kakirde et al.
2010a; Aakvik et al. 2011; Williamson et al. 2011; Akondi
and Lakshmi 2013; Lee and Lee 2013). These problems
can be circumvented by indirect extraction. This is time
consuming and biased, as it is unlikely that all types of
cells separate from the sample matrix or remain intact dur-
ing separation, with the result that the representativeness
of the DNA will be affected (Ekkers et al. 2012). A study
comparing direct and indirect DNA extraction from soil
communities revealed that although the yield and quality
of DNA were significantly different between the two meth-
ods, functional diversity was similar, probably due to func-
tional redundancy in the community (Delmont et al. 2011).
Bias is also introduced by the choice of lysis and DNA
7. 23Extremophiles (2015) 19:17–29
1 3
extraction method and it is likely that an accurate repre-
sentation of the community will never be achieved. In gen-
eral, it is advisable to apply multiple extraction methods,
and when working with environments with low biomass,
as is the case for many extreme environments, DNA yields
are often low (Aakvik et al. 2011; Vester et al. 2014a, b).
To obtain sufficient amounts of DNA for further analyses,
MDA with the ϕ29 DNA polymerase can be applied (Spits
et al. 2006). MDA is, however, heavily biased and will not
generate quantitative results (Yilmaz et al. 2010; Vester
et al. 2014b) and there is a risk of introducing point muta-
tions (Aakvik et al. 2011).
DNA from more defined sources than environmental
samples can also be used for bioprospecting. Several cold-
active enzymes have been identified by screening genome
libraries from single isolates with desirable properties,
including a β-glucosidase from Paenibacillus xylanilyticus
KJ-03 (Park et al. 2013), a β-d-galactosidase from Paracoc-
cus sp. 32d (Wierzbicka-Wos et al. 2011) and an esterase
from Psychrobacter pacificensis (Wu et al. 2013).
Enrichment cultures can be used to select for microor-
ganisms with specific desirable traits. The relative fraction
of DNA most likely to contain genes of interest is increased
by reducing the overall complexity of the community
(Cowan et al. 2005; Taupp et al. 2011; Ekkers et al. 2012).
Among other studies, this approach has been used to enrich
organisms capable of utilizing chitin as a carbon source at
alkaline pH (Kielak et al. 2013) and identifying cellulo-
lytic organisms (Grant et al. 2004). Although most micro-
organisms cannot be cultivated, more are able to grow in
the mixed community of an enrichment culture than as pure
isolates and using DNA extracted from these cultures will,
therefore, yield different results than those obtained from
culture-dependent screenings from the same enrichment
culture (Entcheva et al. 2001; Voget et al. 2003). An alter-
native approach to enrichment is to use stable isotope prob-
ing (SIP) where a labeled (13
C or 15
N) substrate is added
and utilized by metabolically active organisms that incor-
porate the heavier atoms into their DNA, which can sub-
sequently be separated by density gradient centrifugation
(Kakirde et al. 2010a; Ekkers et al. 2012).
Vectors
The length of the fragments used for cloning depends on
the type of screening library. There are three main types
of functional expression libraries: plasmid (15 kb), fos-
mid (25–35 kb)/cosmid (25–40 kb), and bacterial artificial
chromosomes (BACs, 100–200 kb) (Ekkers et al. 2012).
Plasmids are typically easy to handle and are suitable for
single gene products such as most enzymes, and transfor-
mation efficiencies are high. Due to the small insert size,
they are not useful when whole operons need to be detected
(Taupp et al. 2011) and endogenous promoters might not
be included. Using a plasmid with two promoters flank-
ing the multiple cloning site may facilitate gene expres-
sion independently of insert orientation and endogenous
promoter sequences (Lammle et al. 2007). For larger frag-
ments, cosmids, fosmids, or BACs are used. These larger
fragments and vectors are more difficult to handle, but offer
the possibility of including entire operons and gene clusters
as well as achieving a better sequence coverage with fewer
clones. The commercially available CopyControl kit from
Epicentre Biotechnologies can be used to generate fosmid
libraries with ~40 kb inserts with a high cloning efficiency
and stability in Escherichia coli, and is a frequently used
method for expression libraries of metagenomic DNA
(Table 1) (Lee and Lee 2013; Cheng et al. 2014). Since the
hit rate in functional screening is typically very low (2
out of 10,000 clones screened), high cloning efficiency and
high-throughput screening are crucial for successful iden-
tification of activities (Akondi and Lakshmi 2013; Lee and
Lee 2013). Shuttle vectors carrying multiple origins of rep-
lication can be used to move the library between different
expression hosts, such as Gram-negative and Gram-positive
bacteria. This is particularly useful for libraries made from
environments with mixed communities.
Expression hosts
The choice of expression host will influence the design
and result of functional expression on all levels. E. coli
is by far the most commonly used host due to the sub-
stantial genetic toolbox available (Aakvik et al. 2011),
but methods for using other organisms have been devel-
oped. Functional expression faces the same challenges
associated with heterologous expression: codon usage,
improper promoter recognition, missing initiation fac-
tors, protein misfolding, missing co-factors, breakdown
of product, improper secretion of product, toxicity of
product or intermediates, and formation of inclusion
bodies (Ekkers et al. 2012). Since the origin of metagen-
omic DNA is unknown, it is impossible to predict the
effect of these potential problems, as they may vary
from gene to gene and may depend on the expression
host. Gabor et al. (2004) estimated that 40 % of the
genes from 32 different bacterial and archaeal genomes
contained expression signals that would be recognized in
E. coli. However, the actual fraction of genes that can
be successfully expressed in E. coli is probably signifi-
cantly lower, as missing co-factors, chaperones, secre-
tion, etc., was not considered. Not surprisingly, heterolo-
gous expression in E. coli is more efficient for closely
related organisms (Warren et al. 2008). E. coli has been
engineered to increase the expression of heterologous
genes by improving the recognition of ribosomal binding
9. 25Extremophiles (2015) 19:17–29
1 3
increased by 20–30 % (Liebl et al. 2014). Alternatively,
the psychrophilic Gram-negative bacterium Pseudoalte-
romonas haloplanktis TAC125 has been established as a
system for expression and secretion of recombinant pro-
teins (Cusano et al. 2006). To deal with improper secre-
tion, Li et al. (2007) developed a vector system that ena-
bles forced cell lysis inducible by UV radiation to avoid
the use of costly, time-consuming, and possibly denatur-
ing lysing agents.
If phylogenetic information on the metagenomic DNA
is available, or if the proteins of interest are known to be
highly abundant in a specific type of bacteria, a related
host can be chosen to enhance the chance of successful
expression. Alternatively, shuttle vectors that allow for
library expression in multiple hosts can be used. The pCC-
1FOS vector for fosmid and BAC cloning, which normally
only replicates in E. coli, has been modified so it can be
transferred to other species by conjugation (Aakvik et al.
2009). Another broad-host-range vector is the pGNS-BAC
vector, which features an inducible copy number and has
been successfully used in six different γ-Proteobacteria
(Kakirde et al. 2010b). The broad-host-range cosmid vec-
tor, pJWC, was used to establish a metagenomic library
in six different Proteobacteria, including α-, β-, and
γ-Proteobacteria, with very little overlap in expression
(Craig et al. 2010), confirming the usefulness of multiple
hosts.
The shuttle vectors mentioned above have been used
in organisms from the same phylum as E. coli and may
not be suitable for expression of genes from more dis-
tantly related organisms (e.g., Gram-positive bacteria).
They can, however, be useful for transferring a library to
a host better adapted to the screening conditions. As an
example, E. coli is not an optimal host for screening for
cold- and alkaline-active enzymes since it does not grow
well at low temperatures and not at all at high pH, but
a library can be established in E. coli and subsequently
transferred to a related host adapted to these conditions
to allow for direct assays at low temperature and high
pH (Liebl et al. 2014). To deal with a similar problem,
Angelov et al. (2009) developed a two-host fosmid sys-
tem, which permits transfer of a library from E. coli to
the thermophilic Gram-negative Thermus thermophi-
lus and confirmed that the expression patterns between
these two organisms were very different. BAC shut-
tle vectors that can replicate in both Gram-positive and
Gram-negative bacteria have been developed (Hain et al.
2008; Ouyang et al. 2010), including the E. coli–Bacil-
lus subtilis shuttle vector pHT01 from MoBiTech (Biver
et al. 2013). Additional commercial shuttle vectors with
increased transfer efficiency and flexibility are currently
being developed (David Mead, Lucigen, personal com-
munication), which could prove beneficial for increasing
the expression rate in metagenomic libraries. Also, the
pCC1FOS vector has been modified to allow transfer
to Mycobacterium spp. (Ly et al. 2011), and McMahon
et al. (2012) developed a shuttle vector for E. coli and
Streptomyces lividans and an optimized S. lividans strain
for expression. Recently, Cheng et al. (2014) created
cosmid vectors for transfer of cloned metagenomic DNA
by pentaparental conjugation to Gateway®
destination
vectors that are able to replicate in hosts such as Bacillus
and Saccharomyces.
Screening
The challenges and limitations of the function-based
approach highlight the necessity for a robust and high-
throughput screening setup with low detection thresh-
old to capture the few positive clones (Taupp et al. 2011).
With function-based metagenomics, it is possible to per-
form heterologous complementation screenings where the
metagenomic inserts complement a given trait in the host
organism with the advantage that only positive clones will
be able to grow. This approach was used to screen for cold-
active DNA polymerase activities using an E. coli strain
with a cold-sensitive lethal mutation in DNA polymerase
I (Nagano et al. 1999) as a host for a metagenomic library
from glacial ice (Simon et al. 2009). Clones with resistance
to antibiotics or heavy metals can be selected for by includ-
ing these substances in the growth medium (Kakirde et al.
2010a).
Another approach for functional screening is sub-
strate-induced gene expression screening (SIGEX) where
metagenomic DNA is cloned upstream of a promoter-
less GFP. This allows for detection of promoters induced
by the conditions applied after which, cells can be sorted
using fluorescence-activated cell sorting (FACS). This
method has been used to identify genes induced by aro-
matic-hydrocarbon compounds in a groundwater metage-
nome library in E. coli (Uchiyama et al. 2005). A similar
approach is the product-induced gene expression screening
(PIGEX) where a reporter strain with a product-sensitive
promoter coupled to GFP is co-cultivated with a metagen-
omic library to facilitate detection of product formation by
fluorescence. This has been used to identify amidase activ-
ities in E. coli carrying a metagenome library from acti-
vated sludge of a wastewater treatment facility (Uchiyama
and Miyazaki 2010).
The functional approach has significant benefits, most
importantly that it enables the detection of truly novel
activities as no prior sequence information is needed. Fur-
thermore, targeted activities are often directly available in a
relevant production organism, making the road to optimized
expression less troublesome than for the sequence-based
approach. Among the main disadvantages are the rather
10. 26 Extremophiles (2015) 19:17–29
1 3
labor-intensive setup and the necessity for high-throughput
screening to maintain a reasonable hit rate (Aakvik et al.
2011; Simon and Daniel 2011; Taupp et al. 2011).
Cold‑active enzymes identified by functional
metagenomics
Table 1 lists cold-active enzymes identified by functional
metagenomics. It can be seen that bioprospecting for
cold-active enzymes has been conducted in many differ-
ent environments including sediments from both poles, the
deep-sea, cold deserts, mountain soils, lakes, and glacial
ice. The characteristic low hit rate as well as the preferred
use of fosmid libraries and E. coli as host is also obvi-
ous. Lipases and esterases dominate the enzyme activities
identified, most likely as a result of the easy detection of
these activities. The results clearly demonstrate the need
for further development of alternative hosts and assays to
facilitate easy and efficient screening for additional enzyme
activities, since cold-active enzymes have many useful
applications.
Applications of cold‑active enzymes
Enzymes from psychrophiles are generally cold active and
heat labile, which has three main advantages for biotech-
nological applications: (1) because of the higher specific
activity, less (expensive) enzyme is required; (2) processes
can run at the temperature of tap water or ambient temper-
ature reducing the need for heating; and (3) their activity
can be (selectively) inactivated by a moderate temperature
increase (Feller 2013). Many industrial and biotechnologi-
cal processes make use of cold-active enzymes or could
benefit from their use, as the reduced temperature can be
beneficial in multiple ways. Such processes may save
energy and production costs, improve hygiene, maintain
taste and other organoleptic properties, and reduce the risk
of contamination. Cold-active enzymes are used in fine
chemical synthesis, environmental biotechnology, biofuels
and energy production, and in the food and feed, detergent,
pharmaceutical, medical and textile industries (Cavicchioli
et al. 2011). Specific examples include the use of cold-
active β-galactosidases to hydrolyze lactose to generate
lactose-free milk (Feller 2013), heat-labile α-amylases in
the baking industry (Coronado et al. 2000) as well as heat-
labile phosphatases and nucleases for molecular biology
(Feller 2013). In laundry and dish-washing detergents, the
use of cold-active enzymes (proteases, cellulases, lipases,
and amylases) is especially promising, since they allow for
environment-friendly low temperature washing (Horikoshi
1999; van der Maarel et al. 2002; Fujinami and Fujisawa
2010; Mojallali et al. 2013).
Conclusions and perspectives
Approximately 75 % of the Earth’s biosphere is cold
(5 °C) (Huston 2008), and since microbial life is pre-
sent practically everywhere, this constitutes an enormous
reservoir for bioprospecting for cold-adapted activities of
interest. Culture-dependent bioprospecting is an important
and valuable tool in this regard, but since most microor-
ganisms cannot be easily cultured, if at all, it is limited to
covering only a fraction of the total biodiversity. The large
uncultivable fraction of microorganisms can potentially be
accessed by improved cultivation or through metagenomic
approaches and these technologies each have their advan-
tages and disadvantages. Culture-based methods result in
organisms that are known to produce potentially novel,
active enzymes but the re-discovery rate can be very high
(Aakvik et al. 2011; Vester et al. 2014a, b). Sequence-based
metagenomics often identifies a large number of genes
encoding putative enzyme activities, but there is no guar-
antee that the genes can be expressed as active enzymes
in available heterologous hosts and it also relies on previ-
ous sequence knowledge for identification. Function-based
metagenomics may result in novel, functionally active
enzymes, but the hit rate can be extremely low.
Bioprospecting at low temperature is faced with addi-
tional difficulties including low biomass and DNA avail-
ability, and the lack of cold-adapted expression hosts for
functional metagenomics. Both academia and industry
could benefit from a more diverse collection of host strains
with various extremophilic properties, including adaptation
to low temperature, to increase the likelihood of identify-
ing specific activities of interest. Furthermore, the develop-
ment of more versatile vectors as well as strain engineering
should improve the low hit rate associated with functional
metagenomics. The potential of the uncultured microbial
diversity is enormous, and with the methods presently
available and those currently being developed, this biologi-
cal resource is becoming increasingly accessible.
Acknowledgments This work was supported by a Grant to JKV
from University of Copenhagen.
Conflict of interest The authors declare that they have no conflict
of interest.
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution License which permits any use, distribu-
tion, and reproduction in any medium, provided the original author(s)
and the source are credited.
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