Minh Nhat & Bui Viet Cuong (2012) studied the extraction of glucose oxidase enzyme from Aspergillus niger in freeze-dried form with specific activity of 7000 IU/g.
2.2. IMPROVING MICROBIOLOGICAL STRAINS AND ENZYME PRODUCTION FERMENTATION
2.2.1. The need to improve microbial strains
In recent decades, with the exponential increase in the application of enzymes in various fields, there has been an urgent need to expand and enhance both quality and quantity improvements through strain improvement, media optimization, and the search for efficient fermentation processes to increase enzyme yields.
Increased enzyme production can be achieved by optimizing culture media and growth conditions but this approach is limited to the organism's ability to synthesize the product. Strain improvement is carried out to reduce costs by increasing productivity or reducing production costs and therefore it plays an important role in the fermentation industry. Strain improvement is the process of improving and manipulating microbial strains to enhance their metabolic capabilities for biotechnological applications (Gonzalez et al., 2003). Due to their inherent control systems, microorganisms usually produce commercial metabolites at very low concentrations that are just sufficient for their own benefit, therefore enhanced production of metabolites is rare and although productivity can be increased by optimizing environmental conditions, the final productivity is still controlled by the organism's genome (Pathak et al., 2015). Strain improvement mainly focuses on increasing fermentation efficiency, reducing costs and increasing economic benefits and may also have some other desirable characteristics (Prabakaran et al., 2009; Singh et al., 2011).
Genetic engineering research over the years has contributed greatly to understanding the stability and activity mechanisms for the improvement of industrial enzymes. Most of the wild strains that have potential for use in industrial fermentation require modification to make fermentation economically viable (Mishra et al., 2014).
2.2.2. Method of improving microbial strains to increase enzyme production
Enhancement of production of microbial products can be achieved through mutagenesis, selection/screening and application of recombinant DNA techniques. Improvement of industrially important microbial strains is achieved by various methods such as mutagenesis, protoplast fusion, recombinant technology
DNA and gene cloning. Random mutagenesis and protoplast fusion are simpler and are commonly used as tools of protein engineering to obtain strains with high enzyme biosynthesis efficiency or desired traits (Singh et al., 2011; Pathak et al., 2015).
Mutation is a commonly used tool for strain improvement. It is an effective method for improving industrial microorganisms and must be carried out repeatedly by mutagenesis agents, selecting and screening suitable survivors. Mutation has been used by many scientists as a tool of protein engineering to obtain strains with high enzyme efficiency or desired characteristics. Screening of mutants or high enzyme-producing strains is very important in improving the efficiency and economy of industrial production (Zhao et al., 2014). Mutations can occur spontaneously (natural mutations) or after infection (artificial mutations, induced mutations) with mutagens.
a. Natural mutation
The rate of spontaneous mutation is usually low but can be increased by the use of mutagens. The rate of spontaneous mutation depends on the growth conditions of the organism and the mutation frequency (the rate of mutation in a population) can be increased significantly by the use of mutagens.
b. Artificial mutation
Agents that increase the frequency of mutations above the natural level are called mutagens, including physical and chemical agents. Mutagens of this type are called induced mutations or artificial mutations. For microorganisms, the main mutagens are ultraviolet rays and some chemicals.
Physical agents such as radiation, X-rays, ultraviolet rays. Many chemicals are mutagens such as homologues of nitric bases, HNO 2 (nitro acid), chain alkylating agents...
Physical mutagen
Physical mutagens include UV rays, gamma rays, and X-rays. Among them, UV rays are the most widely used in industry because they are very effective and do not require complicated machinery or equipment (Pathak & cs., 2015).
Table 2.2. Mutagenic agents for strain development
Mutagen
Mutations arise | Impact on DNA | Effect | |
Radiation Ionizing radiation | |||
1. X-rays, gamma rays | Destroy single strand or double stranded DNA | Loss, structural change | High |
Short wavelength | |||
2. Ultraviolet rays (UV) | Pyridimine dimer formation and cross-linking in DNA molecule | Move, lose, repeat, replace from GC -> AT | Medium |
Chemistry | |||
Base analogs | |||
3. 5-Chlorouracil, 5-Bromouracil | wrong pairing | Transfer AT->GC, GC->AT | Short |
4. 2-Aminopurine deaminating agents | Error in copying DNA | Short | |
5.Hydroxylamine (NH 2 OH) | Cytosine amino acid | GC -> AT transfer | Short |
6. Nitrous acid (HNO 2 ) | The deamine of A, C and G | Bi-directional traslation, loss, AT ->GC and/or GC- transfer >AT | Medium |
Alkylation factor | |||
7. N-methyl-N'-nitro N-Nitrosoguanidine (NTG) | Methylation, high pH | GC -> AT transfer | High |
8.Ethyl methanesulfonate | Alkylation of C and A | GC -> AT transfer | High |
9. Mustards di-(2- chloroethyl)-sulfide | Alkylation of C and A | GC -> AT transfer | High |
Intervening factors | |||
10. Ethidium bromide, acridine | Insert between 2 pairs of bp | Frame translation | Short |
Biological factors | |||
11. Phages, plasmids, DNA transposing | Replace, destroy the base | Lost, added, replaced | Short |
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Source: Pathak & cs. (2015)
UV rays have a wavelength of 200-300nm, DNA has the ability to absorb a maximum wavelength of 254nm, this is the wavelength that increases the frequency of mutations in microorganisms. UV rays excite electrons in the molecule leading to the formation of bonds between neighboring pyrimidine molecules, resulting in the creation of dimers (thymine-thymine, thymine-cytosine and cytosine-cytosine) or between pyrimidines of complementary chains leading to the formation of crosslinks. UV rays mainly cause mutations of transfer, deletion, duplication, substitution of nitrogenous bases (Pathak & cs., 2015). UV rays are known to be harmful, but they have the ability to induce mutations to improve performance due to better adaptation to the living environment (Pathak & cs., 2015). Agrawal & cs. (1999) have shown that UV rays are a strong mutagen. Most industrial enzyme production is increased mainly by UV treatment.
Chemical mutagen
There are many chemicals that can cause genetic mutations. To date, chemicals that have been found to be more effective than radiation have been found. Chemical mutagens include: Inhibitors of basic nitrogen synthesis in the DNA structure such as caffeine, ethyl urethane, etc.; Substances similar to basic nitrogen such as caffeine, 5-bromuracil, and substances similar to basic nitrogen, so they cause DNA to attach incorrectly during synthesis; Alkylating agents that break DNA strands such as ethyl methane sulfonate (EMS), methyl methasulfonate (MMS), ethylene imine (EI), nitrosoguanidine (NG), etc.; Other substances such as oxidizing and reducing groups. In contrast to replication errors, mutagens such as nitrous acid and nitrogen mustard can cause direct changes in DNA. Substances that insert into DNA (proflavine, acridine, etc.).
Direct point mutation
Recently, many studies have shown that the mutation rate of specific genes is increased by direct mutagenesis. This is done to obtain the maximum frequency of desired mutations and requires extensive knowledge of the gene controlling the target product and the genetic map of the organism. Recently in vitro mutagenesis is used by recombination along with genetic engineering to modify the isolated gene or part of the gene. It involves changing the base sequence of DNA and changing the codon in the gene coding for amino acids. It can be done by protein engineering. The desired improvement can be increased thermotolerance, change in substrate range, reduction in negative feedback inhibition, change in pH range (Pathak et al., 2015).
2.2.3. Fermentation enzyme production
In the process of enzyme production, there are many fermentation methods accepted by manufacturers, of which the two main methods are submerged fermentation and aerated fermentation.
(Submerged fermentation - SmF) and solid state fermentation (Solid state fermentation
- SSF). In recent decades, researchers and manufacturers have tended to use solid surface fermentation (SSF) technique to produce many enzymes from microorganisms (Singh & cs., 2011).
2.2.3.1. Solid state fermentation (SSF)
Solid surface fermentation is defined as a fermentation process involving solid substrates and carried out in the absence or near absence of free water; however, the substrate must have sufficient moisture to support the growth and metabolism of microorganisms (Singhania et al., 2009). The potential of SSF lies in the introduction of microorganisms in an environment with the highest substrate concentration for fermentation, the SSF system closely resembles the natural habitat of microorganisms and is therefore a suitable choice for growth and production of valuable products (Singhania et al., 2009). SSF fermentation has great potential for enzyme production. In addition to the conventional applications in the food and fermentation industries, microbial enzymes also play an important role in the biotransformation of organic solvents, mainly to bioactive compounds (Pandey et al., 1999).
a. Advantages and disadvantages of solid surface fermentation
Solid surface fermentation has received much attention from researchers for the production of many types of industrial enzymes due to its advantages as follows: Solid surface fermentation is easy to perform, the technological process is often not complicated, the requirements for fermentation equipment are simple, inexpensive, thus minimizing costs in the production process. The culture medium in solid surface fermentation is simple. Some substrates can be used directly as solid medium or supplemented with some nutrients. Solid surface fermentation gives higher productivity in a shorter period of time, the amount of enzyme produced from surface fermentation is often much higher than that of submerged fermentation. This is a very important superiority in explaining why the surface culture method is currently developing strongly again. The product recovered after fermentation has a small amount of solvent, limiting wastewater discharged into the environment. On the other hand, the product after collection is easy to dry, easy to preserve and easy to purify. Low moisture content, thus limiting contamination by other microorganisms. Easy to handle when contaminated during fermentation (Singhania & cs., 2009).
Although solid surface fermentation has many advantages, it still has some disadvantages as follows: Microorganisms cultured by the SSF method are limited by barriers.
barriers to the moisture content of the medium. Difficulty in determining fermentation medium parameters such as pH, free oxygen, CO 2 (Singhania & cs., 2009).
b. Microorganisms used for enzyme production by solid surface fermentation
In SSF fermentation, most groups of microorganisms (yeasts, filamentous fungi, bacteria) can grow and develop on the surface of solid media to produce different groups of enzymes. The choice of a particular strain depends on the purpose, requirements and a number of factors, especially the nature of the substrate and environmental conditions. However, filamentous fungi are suitable for SSF fermentation because the substrate and conditions in SSF are similar to the natural living conditions of filamentous fungi (Tengerdy & Szakacs, 2003).
Hydrolytic enzymes such as cellulases, xylanases, pectinases etc. are mostly produced by filamentous fungi, as these enzymes are used in nature for their growth. Trichoderma sp. and Aspergillus niger have been most widely used for these enzymes. Starch-degrading enzymes are also commonly produced by filamentous fungi and the preferred strains belong to the genera Aspergillus and Rhizopus . Although commercial production of amylases is carried out using both fungal and bacterial strains, bacterial α-amylase is commonly used for starch degradation due to its high temperature stability. To achieve high yields at low production costs, it seems that genetically modified strains will play a major role in enzyme production (Pandey et al., 1999).
c. Substrates used in solid surface fermentation for enzyme production
In solid surface fermentation medium, microorganisms will grow on the surface of the medium, receive nutrients from the medium particles and biosynthesize intracellular and extracellular enzymes. Extracellular enzymes will permeate into the medium particles, while intracellular enzymes are located in the microbial biomass.
The choice of substrate for enzyme production by SSF fermentation depends on many factors, mainly related to the cost and convenience of the substrate, which may include the choice of some agricultural wastes. In the SSF process, the substrate not only provides nutrients for microbial growth but also acts as an anchor for the cells. Substrates that provide all the nutrients required for microbial growth are considered ideal substrates. However, some of the nutrients may be present in suboptimal concentrations, or even absent from the substrate. In such cases, it is necessary
Other nutrients must be added externally. Some substrates also need to be pretreated before being used for SSF (chemical or mechanical) such as ligno-cellulose, to make them more usable by microorganisms for their growth (Pandey & cs., 1999).
Among the factors required for microbial growth and enzyme production using specific substrates, particle size and moisture/water activity are the most important (Pandey et al., 1999). Microorganisms grow not only on the surface of the medium, which separates the solid phase (medium) and the gaseous phase (air), but also on the surface of the medium particles located entirely within the medium. The culture medium should be both highly porous and have adequate moisture content. Smaller substrate particles provide a larger surface area for microbial attack, which is a desirable factor. However, substrate particles that are too small cause the substrate to clump together, which can hinder the respiration of aerobic microorganisms, resulting in poor growth. In contrast, larger particles will facilitate better respiration (due to increased inter-particle space), but provide limited surface area for microbial attack. Therefore, the size of the particles must be considered and selected to suit each specific process (Pandey & cs., 1999).
Porous fermentation differs from liquid fermentation, as microbial growth and product production occur on or near the surface of low-moisture solid substrate particles. Therefore, it is important to provide an optimum water content and control the water activity of the substrate for fermentation, as the presence of water at lower or higher concentrations will adversely affect microbial activity. Furthermore, water has a major impact on the physicochemical properties of the solids and this affects the productivity of the fermentation (Pandey et al., 1999).
Agricultural waste is considered the best substrate source for the SSF process. In recent years, many agro-industrial by-products have been used as substrates for SSF such as: bagasse, wheat bran, rice bran, corn bran, bean bran, straw, rice husk, soybean pulp, coconut fiber, cassava pulp, palm oil waste, cassava flour, wheat flour, corn flour, paper pulp, beet pulp, peanut flour, etc. Among them, cassava pulp and sugarcane pulp have more advantages than other substrates such as rice straw, because of low ash content and high water retention capacity. Compared to sugarcane pulp, cassava pulp has the advantage that it does not require pretreatment and can be decomposed by most microorganisms for different purposes. Cassava pulp is widely used in the production of citric acid, flavorings, and metabolites.
other (Pandey & cs., 1999).
d. Factors affecting enzyme production in solid surface fermentation systems
The main factors affecting microbial enzyme synthesis in porous fermentation systems include: selection of suitable substrate and microorganisms; substrate pretreatment; particle size (interparticle space and surface area) of the substrate; water content and water activity of the substrate; relative humidity; type and amount of inoculum; temperature control of the fermenting material/heat removal of metabolism; maintaining uniformity in the environment of the porous fermentation system and the surrounding air, such as oxygen uptake rate and CO 2 release (Pandey & cs., 1999).
2.2.3.2. Submerged Fermentation/Liquid Fermentation (LF)
Submerged fermentation is the process of culturing microorganisms in a liquid medium, all nutrients in the medium for the growth of microorganisms during the fermentation process must be submerged. Factors that need to be controlled in submerged fermentation are: temperature, pH, stirring speed, oxygen concentration... Bioactive compounds are secreted in the fermentation medium. The substrate in the fermentation process is used quite quickly, so it needs to be replaced or nutrients added continuously (Subramaniyam & cs., 2012). Submerged fermentation is a method commonly used in industrial fermentation processes because it can control all parameters of the fermentation process, so it can ferment large volumes with volumes ranging from thousands to hundreds of thousands of liters.
a. Advantages and disadvantages of submerged fermentation
Compared with solid surface fermentation method, submerged fermentation has many advantages: It takes up little space, microorganisms only need to be cultured in a large volume device, small surface area, so sterility is well controlled, the entire process is easy to control, easy to mechanize and automate during monitoring, less manpower is needed and product uniformity is ensured (because they are all collected from the same device).
However, the submerged fermentation method has some disadvantages as follows: Requires a lot of investment in equipment. In submerged fermentation, it is necessary to stir and aerate continuously because microorganisms can only use dissolved oxygen in the environment. The gas is compressed through a filter system to clean impurities, this system is relatively complicated and can easily contaminate the culture environment. If a batch of fermentation for some reason