CHAPTER-1 and lengthy periods of darkness during winter. The

      CHAPTER-1              GENERAL INTRODUCTION

 

1.1. Introduction

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Innumerable ecosystems had been made during the Earth’s evolution and are diverse by large variation in physio, chemical and biological factors creating our environment (Pikuta & Hoover, 2007). Generally, extremes environment referred to pH, salinity and physical extremes known for temperature, pressure and radiation (Van den Burg, 2003). Life exist in all probable places and conditions on the Earth intermingling with the environment and cross species relations. In general, most ecosystems contain the evolutionarily attuned and functionally related functional communities (consortia and populations). The environments with extreme physico-chemical and climatic parameters are inhabited by a wide spectrum of different microorganisms called extremophiles (Pikuta & Hoover, 2007). Extremophiles are organisms which permanently experience environmental conditions which may be considered as extreme in comparison to the physico- chemical characteristics of the normal environment. Microbial extremophiles are the dominant life forms of the extreme environments. They are able to survive in the extreme environments and have developed mechanisms that allow them to cope with a variety of stressors and have evolved several structural and chemical adaptations, which allow them to survive and grow in extreme environments including rocks, geysers, deserts, glaciers, poles and deep sea. These organisms may survive in freezing temperatures and repeated freeze-thaw cycles, desiccation, high or low levels of salinity or pH, and lengthy periods of darkness during winter.  The limits of growth and reproduction of microbes varies from –12°C to more than +100°C, pH 0 to 12, hydrostatic pressures up to 1400 atm and salt concentrations of saturated brines.  Such extreme factors may vary from one site to another. One of the utmost influencing factors for fungal growth is temperature (Adan 1994; Carlile et al. 2001) and the understanding of fungal growth affected by temperature of the environment is an important part of fungal physiology (Li et al., 2008). Some of microorganisms have potential to grow at very low temperature; (-15 to 10°C) these are called as psychrophiles. The environments they inhabit are ubiquitous on earth, as a large fraction of our planetary surface experiences temperature lower than 15°C. Temperature as low as -15 °C are found in pockets of very salty water (brine) surrounded by sea ice. The first discussion of the term “psychrophile” was ended by Schmidt-Nielsen in 1902 by giving the description of bacteria capable of growth at 0°C (Morita, 1975), but Arctic diatoms had already been studied more than one hundred years ago without defining this term (Van Heurck 1909). Later, the term was also used to refer to a number of species of eukaryotic organisms (diatoms, yeasts, algae, lichens, mosses, insects and also for fishes). The term psychrotroph (also termed psychrotolerant), was retained to denote organisms that have the ability to grow at low temperature, but have their optimal and maximal growth temperature above 15°C and 20°C, respectively. The above identification is a useful one because it has relevance in terms of their respective ecological distributions as psychrophiles are limited to permanently cold environments. A psychrotolerant microorganism may have high metabolism and capable to grow with an extended lag phase at freezing and low temperature, and unlike the psychrophiles they do not die at room temperature and have an optimum growth in the range of mesophilic microrganisms (Pikuta & Hoover, 2007).  Usually Fungi are able to live in a relatively large range of temperatures, but their growth rate and metabolism are different at different temperatures even when other conditions, e.g. nutrient and water activity are constant. Normally, the temperature at which a mold has the highest biomass increase rate is accepted as the optimum temperature level of that mold (Carlile et al. 2001). However, it is not well known whether this is also the temperature at which the fungi is growing most efficiently and under least stress.

1.2. Mechanisms of Adaptation to low temperatures

According to previously reported reviews described by Pikuta & Hoover (2007), the cold adaptation mechanisms to cold temperatures could be associated with the changes in proteins (more flexible structural and conformation changes), increasing the fluidity of membranes by the changing of the unsaturation degree of fatty acids, alterations in ante-iso-/iso- branching patterns, and by shortening in the fatty acid chain length. Likewise, the synthesis of antifreeze glycoproteins and peptides can promote the decrease in freezing point of cellular water. Decline in temperature commonly leads the change in Unsaturation of fatty acid chains, that promotes the increase in the fluidity of the membrane because unsaturated fatty acid groups produce further instabilities to the membrane than saturated chains and it is done by desaturases located in the membrane itself and thus are able to respond faster. Similarly, the average fatty acid chain length may be shortened, which would have the effect of increasing the fluidity of the cell membrane. After a reduction in temperature, an increase in the amount or kind of branched fatty acids also reported. Sometimes, there may be a reduction in the portion of cyclic fatty acids and thus rise in mono-unsaturated straight chain fatty acids may occur. Sometimes, sudden decline in temperature may initiate specific alterations in gene expression of cold shock proteins. Additional adaptive approaches developed by psychrophilic microorganisms comprise the regulation of ion channel permeability, seasonal dormancy and microtubule polymerization (Detrich et al. 2000). The key adaptive strategy of   psychrophiles is the modification of enzyme kinetics, allowing the emergence of metabolic rates compatible to life at low temperatures. The psychrophiles enzymes could be extremely active at low temperatures and could be 10 fold higher than that of their mesophilic homologues.

Fig 1. A typical structure of psychrophilic and psychrotrophic fungi and their adaptability mechanisms in low temperature environments, *Poly-unsaturated fatty acids (source: Hassan et al., 2016).

 

1.3. Secondary metabolites

Secondary metabolites are organic molecules that are not involved in the normal growth and development of an organism. The producer organism can grow in the absence of their synthesis, suggesting that secondary metabolism is not essential, at least for short term survival. A second view proposes that the genes involved in secondary metabolism provide a ”genetic playing field” that allows mutation and natural selection to fix new beneficial traits via evolution. A third view characterizes secondary metabolism as an integral part of cellular metabolism and biology; it relies on primary metabolism to supply the required enzymes, energy, substrates and cellular machinery and contributes to the long term survival of the producer. Fungal secondary metabolites are chemical compounds produced by a limited number of species in a genus, an order, or even phylum, and has a high differentiation power. It consists of compounds produced on one or more media and includes toxins, antibiotics and other outward –directed compounds (Frisvad et al., 2007).  A simple classification of secondary metabolites includes three main groups: terpenes (such as plant volatiles, cardiac glycosides, carotenoids and sterols), phenolics (such as phenolic acids, coumarins, lignans, stilbenes, flavonoids, tannins and lignin) and nitrogen containing compounds (such as alkaloids and glucosinolates). These compounds are an extremely diverse group of natural products synthesized by plants, fungi, bacteria, algae, and animals. Different classes of these compounds are often associated to a narrow set of species within a phylogenetic group and constitute the bioactive compound in several medicinal, aromatic, colorant, and spice plants or functional foods.

Many of the secondary metabolites may be termed ‘stress metabolites’, allowing adaptation to physical and chemical stresses and, in the case of antibiotics, permitting competition against rival soil microorganisms (Dayson, 2009). The presence of psychrophilic and psychrotrophic fungi in cold environments, including; permafrost, off-shore polar waters glaciers, ice sheets and shelves, freshwater ice, sea ice, icebergs, have been widely studied.  To combat harsh conditions, such as very low temperature conditions and others, fungi have adapted special features that are still not fully understood. Although, several cold adaptive mechanisms of psychrophilic fungi have been reported, it is assumed that a combination of strategies including production of secondary metabolites, cold-active enzymes, antifreeze proteins, compatible solutes (glycerol), trehalose and polyols (acyclic sugar alcohols) are employed by psychrophiles for their survival. Psychrophilic fungi exist in some of the coldest environments throughout the world because of their great efficiency of adaptation to cold environment. The natural products obtainable from psychrophilic fungi such as proteins, enzymes (cold enzymes), secondary metabolites and compatible solutes are of great interest due to their potential biotechnological applications (eg. Pharmaceuticals). The likely potential has been increasing exponentially with the isolation of new psychrophilic fungal strains, the identification of novel compounds and pathways, and the molecular and biochemical characterization of cellular components. There are several examples of extremophilic fungi which show the diversity of microbes in the environment and their unique metabolism towards extreme conditions. A profile of secondary metabolites is reported by the mycologists and is based on fungal extracts. Secondary metabolite production by psychrophilic fungi is also useful in their taxonomical classification and identification. Fungal taxonomy, chemotaxonomy based on secondary metabolites has been reported in large ascomycetes, and basidiomycetes genera.  Fungal secondary metabolites are low-molecular weight natural products with restricted taxonomic distribution, often synthesized by non-ribosomal peptide synthetase and the polyketides aflatoxin and sterigmatocystin, which are synthesized through a polyketide pathway, are among the best studied fungal secondary metabolites and their pathways have become paradigms (eg. Penicillin). Because of these bioactive properties, many fungal secondary metabolites have been adopted by humans for use as pharmaceuticals such as antibiotics, anticancer, cholesterol-lowering agents, tumor inhibitors and immune-suppressants for transplant operations. However, the production of secondary metabolites by the fungus may depend upon the nutrient availability, physical and environmental conditions and the production of secondary metabolites in extreme environmental conditions could be an area of interest and new approach as the production of natural products may get modified and enhanced under extreme environmental conditions.

1.4. Psychrophilic Penicillium sp.

In 1809, the Penicillium term (Latin word ”Penicillus”, a pencil like structure) was given by Johann Heinrich Friedrich Link, to a group of fungi which bear brush like conidiophores (asexual fruiting structure) and included the detailed description of three Penicillium species of the genus viz., P. expansum, P. glaucum and P. candidum (Raper and Thom, 1949; Houbraken and Samson, 2011). Later, it has been known as a huge genus, comprising 67 % of the total fungal biomass in the soil. Many species of the Penicillium genus have been recognized for their ecological and biotechnological applications (Leitao 2009; Visagie et al. 2009; Khan et al. 2010; Gawas-Sakhalkar et al. 2012). John I. Pitt (1973) divided the genus Penicillium in four subgenera (Aspergilloides, Biverticillium, Furcatum and Penicillium) on the basis of their morphological, micro-morphological and growth characteristics (Raper and Thom, 1949). Other example of Penicillium species belongs temperate regions and differs in the morphology of its conidia and production of secondary metabolites such as penicillin is Penicillium svalbardense (Sonjak et al., 2007b).

 Microfungi of the genus Penicillium is one of the most promising sources of physiologically active compounds, including alkaloids, antibiotics, hormones, mycotoxins etc. Production of these compounds is currently intensely searched for among strain of fungi isolated from little studied and practically uninvestigated habitats. Fungi producers, which are representatives of the genus Penicillium, differ by growth characteristics and the biosynthesis of secondary metabolites. The transport and excretion of alkaloids metabolites are also confined to certain peculiarities typical of fungi of this genus.

Fungi of the genus Penicillium is considered as difficult entity for identifying species using conventional microbiological approaches. The general identification of Penicillium fungi by micro and macromorphological features often gives no definite results. The reliability of Penicillium fungus classifications to certain species seems possible due to species specific production of various biologically active compounds called secondary metabolites. Fungi of the genus Penicillium are known to produce secondary metabolites of various classes of chemical compounds i.e.- ergot, alkaloids, diketopiperazines, quinolines, quinozotrines, azatidine and polyketides.

Aim of the work

In the present study, the psychrophilic and mesophilic fungi has been isolated from Temperate region of Leh Ladakh (J&K., India) and B.B.A.U. Campus, Lucknow (U.P., India) respectively. The growth pattern and characteristics of Penicillium strains under extreme environmental conditions has been studied. The biological activity of produced secondary metabolites by Penicillium strains has been examined.  An additional objective is to isolate and identify the volatile and non-volatile secondary metabolites produced by Penicillium strains has also been done. The information of the category of metabolites produced would help to understand the biological pathways of the fungus and define their role in the growth of the disease symptoms. The differences in the types of chemical structures of the compounds produced by both Penicillium strains also tried to   investigate. Secondary metabolites extracted from crude extracts of fungal culture on artificial media were screened and identified using high-performance liquid chromatography and biological assays. Other modern current separation and identification techniques, such as HPLC-UV, GC-MS/MS and LC-MS, had been used for the purification of the compounds and their identification in ethyl acetate crude extracts.

 Followings are the objectives of the present thesis.

Screening, Identification and Optimization of growth conditions of mesophilic and psychrophilic Penicillium strains and their characterization.
Study on the adaptational features of both the Penicillum strains under different environmental conditions.
Screening of both mesophilic and psychrophilic Penicillium strains in terms of secondary metabolite production under different environmental conditions.
Extraction and characterization of biomolecules present in the secondary metabolite of both the Penicillium strains
Study on the antimicrobial and anticancer attributes of the biomolecules extracted from the secondary metabolite of each Penicillium strain.

      CHAPTER-1              GENERAL INTRODUCTION

 

1.1. Introduction

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Innumerable ecosystems had been made during the Earth’s evolution and are diverse by large variation in physio, chemical and biological factors creating our environment (Pikuta & Hoover, 2007). Generally, extremes environment referred to pH, salinity and physical extremes known for temperature, pressure and radiation (Van den Burg, 2003). Life exist in all probable places and conditions on the Earth intermingling with the environment and cross species relations. In general, most ecosystems contain the evolutionarily attuned and functionally related functional communities (consortia and populations). The environments with extreme physico-chemical and climatic parameters are inhabited by a wide spectrum of different microorganisms called extremophiles (Pikuta & Hoover, 2007). Extremophiles are organisms which permanently experience environmental conditions which may be considered as extreme in comparison to the physico- chemical characteristics of the normal environment. Microbial extremophiles are the dominant life forms of the extreme environments. They are able to survive in the extreme environments and have developed mechanisms that allow them to cope with a variety of stressors and have evolved several structural and chemical adaptations, which allow them to survive and grow in extreme environments including rocks, geysers, deserts, glaciers, poles and deep sea. These organisms may survive in freezing temperatures and repeated freeze-thaw cycles, desiccation, high or low levels of salinity or pH, and lengthy periods of darkness during winter.  The limits of growth and reproduction of microbes varies from –12°C to more than +100°C, pH 0 to 12, hydrostatic pressures up to 1400 atm and salt concentrations of saturated brines.  Such extreme factors may vary from one site to another. One of the utmost influencing factors for fungal growth is temperature (Adan 1994; Carlile et al. 2001) and the understanding of fungal growth affected by temperature of the environment is an important part of fungal physiology (Li et al., 2008). Some of microorganisms have potential to grow at very low temperature; (-15 to 10°C) these are called as psychrophiles. The environments they inhabit are ubiquitous on earth, as a large fraction of our planetary surface experiences temperature lower than 15°C. Temperature as low as -15 °C are found in pockets of very salty water (brine) surrounded by sea ice. The first discussion of the term “psychrophile” was ended by Schmidt-Nielsen in 1902 by giving the description of bacteria capable of growth at 0°C (Morita, 1975), but Arctic diatoms had already been studied more than one hundred years ago without defining this term (Van Heurck 1909). Later, the term was also used to refer to a number of species of eukaryotic organisms (diatoms, yeasts, algae, lichens, mosses, insects and also for fishes). The term psychrotroph (also termed psychrotolerant), was retained to denote organisms that have the ability to grow at low temperature, but have their optimal and maximal growth temperature above 15°C and 20°C, respectively. The above identification is a useful one because it has relevance in terms of their respective ecological distributions as psychrophiles are limited to permanently cold environments. A psychrotolerant microorganism may have high metabolism and capable to grow with an extended lag phase at freezing and low temperature, and unlike the psychrophiles they do not die at room temperature and have an optimum growth in the range of mesophilic microrganisms (Pikuta & Hoover, 2007).  Usually Fungi are able to live in a relatively large range of temperatures, but their growth rate and metabolism are different at different temperatures even when other conditions, e.g. nutrient and water activity are constant. Normally, the temperature at which a mold has the highest biomass increase rate is accepted as the optimum temperature level of that mold (Carlile et al. 2001). However, it is not well known whether this is also the temperature at which the fungi is growing most efficiently and under least stress.

1.2. Mechanisms of Adaptation to low temperatures

According to previously reported reviews described by Pikuta & Hoover (2007), the cold adaptation mechanisms to cold temperatures could be associated with the changes in proteins (more flexible structural and conformation changes), increasing the fluidity of membranes by the changing of the unsaturation degree of fatty acids, alterations in ante-iso-/iso- branching patterns, and by shortening in the fatty acid chain length. Likewise, the synthesis of antifreeze glycoproteins and peptides can promote the decrease in freezing point of cellular water. Decline in temperature commonly leads the change in Unsaturation of fatty acid chains, that promotes the increase in the fluidity of the membrane because unsaturated fatty acid groups produce further instabilities to the membrane than saturated chains and it is done by desaturases located in the membrane itself and thus are able to respond faster. Similarly, the average fatty acid chain length may be shortened, which would have the effect of increasing the fluidity of the cell membrane. After a reduction in temperature, an increase in the amount or kind of branched fatty acids also reported. Sometimes, there may be a reduction in the portion of cyclic fatty acids and thus rise in mono-unsaturated straight chain fatty acids may occur. Sometimes, sudden decline in temperature may initiate specific alterations in gene expression of cold shock proteins. Additional adaptive approaches developed by psychrophilic microorganisms comprise the regulation of ion channel permeability, seasonal dormancy and microtubule polymerization (Detrich et al. 2000). The key adaptive strategy of   psychrophiles is the modification of enzyme kinetics, allowing the emergence of metabolic rates compatible to life at low temperatures. The psychrophiles enzymes could be extremely active at low temperatures and could be 10 fold higher than that of their mesophilic homologues.

Fig 1. A typical structure of psychrophilic and psychrotrophic fungi and their adaptability mechanisms in low temperature environments, *Poly-unsaturated fatty acids (source: Hassan et al., 2016).

 

1.3. Secondary metabolites

Secondary metabolites are organic molecules that are not involved in the normal growth and development of an organism. The producer organism can grow in the absence of their synthesis, suggesting that secondary metabolism is not essential, at least for short term survival. A second view proposes that the genes involved in secondary metabolism provide a ”genetic playing field” that allows mutation and natural selection to fix new beneficial traits via evolution. A third view characterizes secondary metabolism as an integral part of cellular metabolism and biology; it relies on primary metabolism to supply the required enzymes, energy, substrates and cellular machinery and contributes to the long term survival of the producer. Fungal secondary metabolites are chemical compounds produced by a limited number of species in a genus, an order, or even phylum, and has a high differentiation power. It consists of compounds produced on one or more media and includes toxins, antibiotics and other outward –directed compounds (Frisvad et al., 2007).  A simple classification of secondary metabolites includes three main groups: terpenes (such as plant volatiles, cardiac glycosides, carotenoids and sterols), phenolics (such as phenolic acids, coumarins, lignans, stilbenes, flavonoids, tannins and lignin) and nitrogen containing compounds (such as alkaloids and glucosinolates). These compounds are an extremely diverse group of natural products synthesized by plants, fungi, bacteria, algae, and animals. Different classes of these compounds are often associated to a narrow set of species within a phylogenetic group and constitute the bioactive compound in several medicinal, aromatic, colorant, and spice plants or functional foods.

Many of the secondary metabolites may be termed ‘stress metabolites’, allowing adaptation to physical and chemical stresses and, in the case of antibiotics, permitting competition against rival soil microorganisms (Dayson, 2009). The presence of psychrophilic and psychrotrophic fungi in cold environments, including; permafrost, off-shore polar waters glaciers, ice sheets and shelves, freshwater ice, sea ice, icebergs, have been widely studied.  To combat harsh conditions, such as very low temperature conditions and others, fungi have adapted special features that are still not fully understood. Although, several cold adaptive mechanisms of psychrophilic fungi have been reported, it is assumed that a combination of strategies including production of secondary metabolites, cold-active enzymes, antifreeze proteins, compatible solutes (glycerol), trehalose and polyols (acyclic sugar alcohols) are employed by psychrophiles for their survival. Psychrophilic fungi exist in some of the coldest environments throughout the world because of their great efficiency of adaptation to cold environment. The natural products obtainable from psychrophilic fungi such as proteins, enzymes (cold enzymes), secondary metabolites and compatible solutes are of great interest due to their potential biotechnological applications (eg. Pharmaceuticals). The likely potential has been increasing exponentially with the isolation of new psychrophilic fungal strains, the identification of novel compounds and pathways, and the molecular and biochemical characterization of cellular components. There are several examples of extremophilic fungi which show the diversity of microbes in the environment and their unique metabolism towards extreme conditions. A profile of secondary metabolites is reported by the mycologists and is based on fungal extracts. Secondary metabolite production by psychrophilic fungi is also useful in their taxonomical classification and identification. Fungal taxonomy, chemotaxonomy based on secondary metabolites has been reported in large ascomycetes, and basidiomycetes genera.  Fungal secondary metabolites are low-molecular weight natural products with restricted taxonomic distribution, often synthesized by non-ribosomal peptide synthetase and the polyketides aflatoxin and sterigmatocystin, which are synthesized through a polyketide pathway, are among the best studied fungal secondary metabolites and their pathways have become paradigms (eg. Penicillin). Because of these bioactive properties, many fungal secondary metabolites have been adopted by humans for use as pharmaceuticals such as antibiotics, anticancer, cholesterol-lowering agents, tumor inhibitors and immune-suppressants for transplant operations. However, the production of secondary metabolites by the fungus may depend upon the nutrient availability, physical and environmental conditions and the production of secondary metabolites in extreme environmental conditions could be an area of interest and new approach as the production of natural products may get modified and enhanced under extreme environmental conditions.

1.4. Psychrophilic Penicillium sp.

In 1809, the Penicillium term (Latin word ”Penicillus”, a pencil like structure) was given by Johann Heinrich Friedrich Link, to a group of fungi which bear brush like conidiophores (asexual fruiting structure) and included the detailed description of three Penicillium species of the genus viz., P. expansum, P. glaucum and P. candidum (Raper and Thom, 1949; Houbraken and Samson, 2011). Later, it has been known as a huge genus, comprising 67 % of the total fungal biomass in the soil. Many species of the Penicillium genus have been recognized for their ecological and biotechnological applications (Leitao 2009; Visagie et al. 2009; Khan et al. 2010; Gawas-Sakhalkar et al. 2012). John I. Pitt (1973) divided the genus Penicillium in four subgenera (Aspergilloides, Biverticillium, Furcatum and Penicillium) on the basis of their morphological, micro-morphological and growth characteristics (Raper and Thom, 1949). Other example of Penicillium species belongs temperate regions and differs in the morphology of its conidia and production of secondary metabolites such as penicillin is Penicillium svalbardense (Sonjak et al., 2007b).

 Microfungi of the genus Penicillium is one of the most promising sources of physiologically active compounds, including alkaloids, antibiotics, hormones, mycotoxins etc. Production of these compounds is currently intensely searched for among strain of fungi isolated from little studied and practically uninvestigated habitats. Fungi producers, which are representatives of the genus Penicillium, differ by growth characteristics and the biosynthesis of secondary metabolites. The transport and excretion of alkaloids metabolites are also confined to certain peculiarities typical of fungi of this genus.

Fungi of the genus Penicillium is considered as difficult entity for identifying species using conventional microbiological approaches. The general identification of Penicillium fungi by micro and macromorphological features often gives no definite results. The reliability of Penicillium fungus classifications to certain species seems possible due to species specific production of various biologically active compounds called secondary metabolites. Fungi of the genus Penicillium are known to produce secondary metabolites of various classes of chemical compounds i.e.- ergot, alkaloids, diketopiperazines, quinolines, quinozotrines, azatidine and polyketides.

Aim of the work

In the present study, the psychrophilic and mesophilic fungi has been isolated from Temperate region of Leh Ladakh (J&K., India) and B.B.A.U. Campus, Lucknow (U.P., India) respectively. The growth pattern and characteristics of Penicillium strains under extreme environmental conditions has been studied. The biological activity of produced secondary metabolites by Penicillium strains has been examined.  An additional objective is to isolate and identify the volatile and non-volatile secondary metabolites produced by Penicillium strains has also been done. The information of the category of metabolites produced would help to understand the biological pathways of the fungus and define their role in the growth of the disease symptoms. The differences in the types of chemical structures of the compounds produced by both Penicillium strains also tried to   investigate. Secondary metabolites extracted from crude extracts of fungal culture on artificial media were screened and identified using high-performance liquid chromatography and biological assays. Other modern current separation and identification techniques, such as HPLC-UV, GC-MS/MS and LC-MS, had been used for the purification of the compounds and their identification in ethyl acetate crude extracts.

 Followings are the objectives of the present thesis.

Screening, Identification and Optimization of growth conditions of mesophilic and psychrophilic Penicillium strains and their characterization.
Study on the adaptational features of both the Penicillum strains under different environmental conditions.
Screening of both mesophilic and psychrophilic Penicillium strains in terms of secondary metabolite production under different environmental conditions.
Extraction and characterization of biomolecules present in the secondary metabolite of both the Penicillium strains
Study on the antimicrobial and anticancer attributes of the biomolecules extracted from the secondary metabolite of each Penicillium strain.

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