Altered production of organic acid and pigments by microbes under influence of microwave radiation

Microwave Mutagenesis

Scientific Study , 2014 , 75 Pages , Grade: A

Biology - Micro- and Molecular Biology

This study attempted at microwave mutagenesis of: (i) Lactobacillus plantarum for lactic acid overproduction, and (ii) Streptococcus mutans for reduced lactic acid production. Lactic acid is among the microbiological products with high market potential. Lactic acid is also an important virulence factor in formation of dental caries by S. mutans, as the acid produced by the bacteria leads to demineralisation of the teeth. Two of the mutants obtained (one from each organism) were able to maintain the altered lactic acid production till 10 generations. However the magnitude of alteration in lactic acid producing ability of the mutants went on decreasing over generations. The microwave effects observed in this study seem largely to be athermal in nature. Investigation of the mutants obtained at molecular level may result in identification of novel mutations responsible for altered lactic acid production. These mutations can then be introduced into a suitable organism either for better industrial production of lactic acid, or for constructing new probiotic strain(s) for possible application in maintenance of oral health.

Effect of low power (90 W) microwave (2,450 MHz) radiation on bacterial growth and pigment production was studied in three different bacteria. Microwave exposure of 2-6 min duration was able to alter growth and pigment production (prodigiosin production by Serratia marcescens, violacein production by Chormobacterium violaceum, and staphyloxanthin production by Staphylococcus aureus) in the test organisms significantly. In this study, pigment production was estimated in the cell population originated from microwave treated inoculum, and not directly in the MW treated cells. Thus the alterations in pigment production and/or secretion might have been transferred from the originally MW treated cells to their daughter cells (who did not receive direct MW exposure), indicating the mutagenic influence of microwave radiation. Heavy prodigiosin overproduction observed in one of the test tubes inoculated with microwave treated S. marcescens could not be sustained by daughter populations corresponding to that tube, indicating the reversible nature of microwave induced mutation(s). The microwave effects observed in this study largely seem to be of athermal nature, as the thermal effect was minimized by use of ice during the microwave treatment.

INHALTSVERZEICHNIS

1. Prologue
1.1. Preamble
1.2. The Importance of the Study
1.3. Statement of the Problem
1.4. Rationale of the Research Work
1.5. Objectives:

2. Literature Review
2.1 Interactions of MW with Biological Materials
2.2. Athermal (Non-thermal) Mechanisms of Interaction
2.4 Biological effects of MW radiation
2.5 Microwave Mutagenesis
2.6 MW and cell phones
2.7 Effect of MW on growth and enzyme activity
2.8 Lactic acid
2.9 Microbial pigment production
2.9.1. Pigment produced by Microorganisms

3. Microwave mutagenesis of Streptococcus mutans for reduced lactic acid production
3.1 Materials and Methods
3.1.1. Culture maintenance
3.1.2 Culture activation
3.1.3 Inoculum preparation
3.1.4 MW oven and its maintenance
3.1.5 MW treatment
3.1.6 Growth measurement:
3.1.7 Expermental condition:
3.1.8 Lactic acid estimation:
3.1.8 Experimental outline
3.2 Results and Discussion

4. Microwave mutagenesis of Lactobacillus plantarum for lactic acid overproduction
4.1 Materials and Methods
4.1.1. Culture maintenance
4.1.2 Culture activation
4.1.3 Inoculum preparation
4.1.4 MW oven and its maintenance
4.1.5 MW treatment
4.2.6 Growth measurement:
4.1.6 Experimental condition:
4.1.7 Lactic acid estimation:
4.1.8 Experimental outline:
4.2 Results and Discussion

5. Effect of low power microwave radiation on pigment production of selected bacteria
5.1 Materials and Methods
5.1.1 Culture maintenance
5.1.2. Culture activation
5.1.3. Inoculum preparation
5.1.4. MW oven and its maintenance
5.1.5. MW treatment
5.1.6. MW treatment to inoculum
5.1.7 Growth measurement:
5.1.8. Experimental condition:
5.1.9 Pigment extraction methods:
5.1.10 Calculation for pigment extraction:
5.1.11 Statistical analysis
5.2. Results and Discussion
5.2.1 Effect of low power MW on prodigiosin production
5.2.2. Effect of low power MW treatment on violacein production
5.2.3 Effect of low power MW treatment on staphyloxanthin production:

6. Microwave mutagenesis for the overproduction of prodigiosin from S. marcescens
6.1. Materials and Methods:
6.1.1 Culture maintenance
6.1.2 Culture activation
6.1.3 Inoculum preparation
6.1.4. MW oven and its maintenance
6.1.5 MW treatment
6.1.6 MW treatment to inoculum
6.1.7 Growth measurement:
6.1.8 Experimental condition and prodigiosin extraction method was using described in chapter 5 (5.1.8 and 5.1.9).
6.1.9 Experimental outline:
6.2 Results and Discussion

Epilogue

REFERENCES

1. Prologue

1.1. Preamble

Since many years, scientists are interested in studying the interaction of electromagnetic fields (EMFs) and various bio-system and its bioprocesses. All biological systems are electrochemical in nature so EMF may influence them. Attention has been focused on different frequency range waves, of which microwave (MW) is an important part. Microwaves (MW) are non-ionizing electromagnetic waves in 1mm to 1m wavelength range, with a wide frequency band between 300 MHz and 300 GHz [Banik et al., 2003]. They are a very important component of the electromagnetic spectrum as demonstrated by the increasing scope of applications. They have relatively short wavelengths and high frequencies compared to the extremely low frequency fields. They therefore have a greater energy which is sufficient to cause heating in conductive materials (Figure 1.1). Unlike the X-rays and gamma rays, which are ionizing, MW interaction with matter does not result in removal of orbital electrons. Rather, such interactions are known to cause effects like atomic excitations, increased atomic and molecular vibrations, rotations, and heat production [Davis and VanZandt, 1988]. Because of the nature of MW, its interaction mechanisms and the biological effects have previously not been associated with production of free radicals, oxidative modification of cell membranes, lipid peroxidation. These mechanisms are associated with ionizing radiations. Interest in the study of MW interaction with biological systems has been sustained for several decades. The first person to explore the bio effects of MW fields was Antonin Gosset in 1924, when he and his co-workers used short waves to destroy tumours in plants with no damage to the plant itself [Bren, 1996]. During the 1930s, physicists, engineers, and biologists studied the effects of low frequency electromagnetic waves on biological materials. Studies of the effects of microwaves on bacteria, viruses and DNA were performed in the 1960s and included research on heating, biocidal effects, dielectric dispersion, mutagenic effects, etc. [Yaghmaee and Durance, 2005]. In recent year, the industrial MW applications have grown considerably, apart from the usage of domestic MW ovens. Some of the MW applications in industries are tampering of frozen product, thawing, blanching, baking, drying/dehydration/vacuum drying/freeze-drying, pasteurization, sterilization, cooking, etc. In modern world, MW radiations are used in FM radio, television, RADAR (Radio Detection and Ranging), and satellite-to-earth communications. Apart from its use in domestic ovens, many other applications of MW in different areas have been identified.

Applications of MW that are based primarily on its thermal effects include those in food processing industry [Decareau and Kenyon, 1970], disinfection and sterilization [Bhattacharjee et al., 2009; Kothari et al., 2011], moisture removal [Ozbek and Dadali, 2007], waste treatment [Beszedes et al., 2010], etc.

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Figure 1.1 Position of microwave radiation in electromagnetic spectrum

(http://www.chem.ucalgary.ca/courses/350/Carey5th/Ch13/ch13-1.html)

MW irradiation is also used to disinfect household products such as sponges, kitchen utensils, syringes and medicinal devices where bacterial viability is reduced by MW treatment. Microwave assisted extraction (MAE) has emerged as a promising method for preparation of bioactive plant extracts. It is considered suitable for fast extraction of phenolic compounds, and also for extraction of heat-labile phytoconstituents [Mandal et al., 2007;Gupta et al. 2012; Kothari et al., 2012]. Research on non-thermal effects can open the door for new applications which may be based either solely on non-thermal effect, or both thermal and non-thermal effects. Either low power or high power (for very short duration, and with a provision of cooling water circulation to avoid heating) MW can be used for mutagenesis in plants [Jangid et al., 2010] and microorganisms [Li et al., 2009;Lin et al., 2012], protein unfolding [George et al., 2008], tumor detection based on electrical properties of tissues [Stuchly and Stuchly, 1983], rate enhancement of biochemical reactions [Bose et al., 2002; Sun et al., 2011], enzyme immobilization [Wang et al., 2011], and reduction of pathogenic population [Barnabas et al., 2010].

1.2. The Importance of the Study

Sub- lethal MW-microbes interaction may change level of:

Growth rate [Rebrova et al., 1992; Golant et al., 1994; Shub et al., 1995]

Metabolite activity [Dreyfuss et al., 1980]

Plasmid amplification [Otludil et al., 2005]

Rapid Strain identification [Spencer et al., 1985]

Transformation efficiency [Fregel et al., 2008]

Porosity of the cell membrane that allow uptake of drugs into MW-treated cell [Shamis et al., 2011]

Secondary metabolites such as antibiotics [Himabindu et al., 2007]

Lose of virulence of virulent strain [Moore et al., 1979; Tsuji and Yokoigawa 2011]

Mutation [Li et al., 2009;Lin et al., 2012]

1.3. Statement of the Problem

Increasing applications of MW radiation has led to concerns globally due to the suspected bio effects associated with its exposure. Effect of MW, thermal and/or athermal, is inconclusive, complex, and controversial in literature. Thermal effect causes thermogenic effect while athermal effects are other than heat and such effects reported as somatic effect and/or genetic effect.

This study basically deals with the athermal effects and is aimed at investigating the hypothesis that the exposure of microbial cells to MW (low power) may cause athermal effect, which affect on growth of microbes, lactic acid production, and production of pigment like prodigiosin, staphyloxanthin and violacein. Furthermore, Our study also gives information that MW athermal effects causes changes at genetic level and can be passed on to next generation.

1.4. Rationale of the Research Work

There are numerous and increasing applications of MW energy and technology in the industries, in homes, in medical, research institutions etc., and there is greater awareness and concern of the public over the suspected potential health hazards associated with such exposures [ICNIRP Guidelines, 1998]. There is therefore, a need for deeper understanding of the bio-effects of exposure to this radiation. Due to the ease of handing them in laboratory, microorganisms can be conveniently used to study the effect of MW on living systems. Besides, employing mutagenic frequencies of MW radiation for microbial strain improvement can be of considerable industrial significance.

1.5. Objectives:

a. Microwave mutagenesis of Streptococcus mutans for reduced lactic acid production
b. Microwave mutagenesis of Lactobacillus plantarum for lactic acid overproduction
c. Effect of low power microwave radiation on pigment production of selected bacteria

2. Literature Review

2.1 Interactions of MW with Biological Materials

The interactions can be considered at various levels of organization of a living organism: atomic, molecular, subcellular, cellular, entire organism. These interactions with biosystem arise because of three processes: (i) penetration by electromagnetic waves and their propagation into the living system, (ii) primary interaction of the waves with biosystem; and (iii) possible secondary effects arising from the primary interaction. One of the first fundamental steps in evaluating the effects of a certain exposure to radiation in a living organism is determination of the induced internal electromagnetic field and its spatial distribution. Further, various possible biophysical mechanisms of interaction can be applied. Any such interactions, which may be considered primary, elicit one or more secondary reactions in the living system. For instance, when MW energy absorption results in a temperature increase within cell (a primary interaction), the activation of the thermoregulatory, compensatory mechanism is a possible secondary interaction [Czerski, 1975] While the primary interactions are becoming better understood, there is still insufficient attention devoted to the interaction mechanisms involving molecular level. Studies on the biological effects of MWs reveal several areas of established effects and mechanisms on the one hand and speculative effects on the other. There are known thermal and athermal interaction mechanisms of MWs with biological systems.

2.2. Athermal (Non-thermal) Mechanisms of Interaction

MW radiation seems to affect system in a manner, which cannot be explained by thermal effects alone [Spencer et al., 1985]. MW has ability to destroy bacterial cells at specific parameters without causing heating of the substrate [Barnabas et al., 2010]. MW plays role in dielectric saturation [Hyland, 1988], formation of oxidative stress [Sokolovic et al., 2008], protein unfolding [George et al., 2008], changing the structures by differentially partitioning the ions [Asadi et al., 2011], others chemical transformation of small molecules such as chemical bond cleavage [Oslen, 1966], vibrational resonance in DNA molecules [Edwards et al., 1985]. The oscillating EMF of MW couples energy into large biomolecules with several oscillations. When a large number of dipoles are present in one molecule (DNA, protein, RNA etc.) and kept under MW, enough energy can be transferred to the molecules, which would be able to break the bond.

Biological effects of MW radiation can be divided into two categories: thermal effects and non-thermal effects. Thermal effect is the one in which the MW energy is converted into heat energy in the living systems. These effects can be macroscopic where whole organisms or major portions of them participate in the heat transfer process or microscopic where cellular component like bound water is vaporized by the selective application of the MW heating [Richmond, 1969]. The dielectric effect of MW on polar molecules has been known for more than a century [Debye, 1922]. Polar molecules are present in the cells in the form of water, DNA, and proteins and they respond to an electromagnetic field by rotating. This rotation creates an angular momentum which results in friction with neighbouring molecules, thereby developing a linear momentum (vibrational energy) [Saifuddin et al., 2009]. In this way, radiation energy is converted into thermal energy. Effect generated from vibrational energy is thermal effect which occurs in a biosystem due to penetration of electromagnetic waves (MW) into biological materials and heating up the intra- and extra- cellular fluids by transfer of vibrational energy [Tahir et al., 2009]. However, MW thermal effect is different from conventional heating effect. Dipolar polarization and rotation of molecules in an attempt to align the dipoles with applied MW field (Fig. 2.1) produces effects which cannot be achieved by conventional heating [Zelentsova et al., 2004].

The non-thermal effect of MW is highly controversial and has been a matter of debate in scientific community. Non-thermal effects are postulated to result from a direct stabilizing interaction of electric field with specific (polar) molecules in reaction medium with no rise in temperature [Herrero et al., 2007]. Thermal effects solely cannot explain the manner in which the MW affects biological systems. Several studies have revealed that MW radiation can kill microbial cells; however it is still not clear if non-thermal effects of MW have any contribution to this. One of the mechanisms involved in killing of microorganisms by MW is by altering the permeability of the cell membrane. The alteration in the permeability of the membrane of the cells is reflected by cell shape changes observed under electron microscopy or by detecting leakage of intracellular protein or DNA using spectroscopy [Chen et al., 2007]. Due to the paucity of information regarding the exact mechanism involved in ‘nonthermal microwave effects’ (also referred to as athermal effects or MW specific effects), their existence is highly controversial. [Kozempel et al., 2000] attempted at detection of nonthermal effects of MW energy on microbes at low temperature in various test fluids. To separate thermal and nonthermal effects in a system, they developed a continuous experimental microwave process combining rapid energy input to the food system using microwave, with rapid removal of thermal energy utilising an efficient heat exchanger design. A continuous MW treatment (7 kW, 2450 MHz) was given to the test organisms in various test fluids like water, liquid egg, beer, apple juice, and tomato juice. They concluded that MW energy in the absence of other stresses did not kill microorganisms at low temperatures and there was no convincing evidence that MW energy could kill microorganisms without thermal energy. [Dreyfuss and Chipley, 1980] studied the effect of MW at sub-lethal temperature on Staphylococcus aureus and suggested the existence of a phenomenon different from thermal heating resulting in altered activity of various metabolic enzymes. There has been a plenty of reports favoring the existence of non-thermal effects [Dreyfuss and Chipley, 1980; Copty et al., 2006; Carta and Desogus, 2012], but the studies refusing the possibility of athermal effects [Vela and Wu, 1979; Kozempel et al., 2000; Shazman et al. 2007] can also not be neglected.

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Figure 2.1 the orientation of dipoles in absence (a) and in presence (b) of electric field

[Williams , 2009]

2.4 Biological effects of MW radiation

Research is being done worldwide on thermal and athermal effects of MW on different biological systems. Phormidium spp. Kutzing ISC31 (a cyanobacterium) grown in BG-11 medium was treated at a frequency of 2450 MHz using a microwave oven by combining five different intensities (180, 360, 540, 720 and 900 W/cm2) and three pretreatments (10, 20 and 30 s). The content of chlorophyll a decreased with increase in intensity and exposure time. Synthesis of phycobiliproteins, phycocyanin, phycoerythrin, and allophycocyanin increased in all exposures except in 720 and 900 W/cm2 (30 s). Photosynthetic rate compared to nitrogenase activity increased by all microwave exposures except at 180 W (10 s) and 720 W (10 s) as compared to control [Asadi et al., 2011]. Studies on E. coli and S. aureus suggested that physical damage caused by MW led to alterations in membrane permeability and consequently influx of extracellular Ca2+. An increase in cell permeability of upto 89.8% and 19.7% was obtained after MW treatment in S. aureus and E. coli respectively [Chen et al. 2007]. Effect of MW on transformation efficiency was studied by Fregel et al. (2008), where calcium chloride competent cells of E. coli were given MW treatment at 180 W for 1 min, and the transformation efficiency was increased three-fold as compared to the classical method. Otludil et al., (2004) studied the effect of microwave (2450 MHz, 55 W) on the cellular differentiation of Bacillus subtilis YB 886 and its Rec derivatives YB 886 A4. It was found that organism’s growth and amount of DNA decreased after MW exposure by 4% and 27% respectively, whereas the amount of RNA and plasmid was enhanced by 6.5% and 21% respectively. They noted an increase in the amount of specific protein synthesized during DNA damage by SOS repair system, and its binding to din C promoter region, following MW exposure in rec+ bacteria.

2.5 Microwave Mutagenesis

A number of studies reveal that effects of MW can reach up to genetic level and can even result in stable mutation. Genetically stable mutant strains with higher nitrogenase activity and phosphate solubilizing capabilities of Klebsiella pneumoniae RSN19 were obtained by microwave (250W, 36 s) mutagenesis [Li et al., 2011]. Mutagenic potential of MW has also been demonstrated with respect to cellulase production in Trichoderma viride, wherein a compound mutagenesis by MW (700W, 15-195 s) and ultraviolet radiation was employed, and the mutants were found to be stable up to 9 generations [Li et al., 2009]. Lin et al. (2012) studied the effect of MW (400 W for 3 min) on Lactobacillus rhamnosus which induced >50% increase in L-lactic acid production than the parent strain, and the mutant generated was found to be stable up to 9 generations. Such studies together indicate the potential of MW as a substantial tool for strain improvement through mutagenesis. MW till now has not been exploited as widely UV has been as a mutagenic radiation. Identifying the frequencies, power range and exposure duration in MW region, which are most suitable for mutagenesis among microbes will certainly be of interest to fermentation industries.

2.6 MW and cell phones

Within last decade, the world population has adopted the use of cell phones in a horrendous way. Increasing use of radiofrequency devices has become a trend as well as a need in a large section of society. Cellular phones transmit radiofrequency waves of very low intensity and there has been a lot of discussion on possible adverse effects of these radiations on health. Effect of electromagnetic radiation from communication towers on human and other forms of biodiversity has been a matter of concern. Panagopoulos and Margaritis (2002) exposed fruit flies to cell phone radiation (Global System for Mobile communication-GSM-900 MHz) at very low levels (3-7 mW/cm2) for 6 min/day before hatching. The exposed groups in their adult life showed a loss in reproductive activity varying between 15-60%. Frey (1962) reported that exposure of heads of volunteers to low level of radar from a radar horn resulted in headaches, and that low level of radar can interact with nerve tissue to cause dysaesthesiae. Hocking and Westerman (2001) reported a neurological abnormality in a patient after accidental exposure of left side of the face to cellular phone radiation, which led to long-lasting loss of tactile sensitivity of the facial skin, unilateral left blurred vision, and pupil constriction. The worker took virtually 6 months to recover.

2.7 Effect of MW on growth and enzyme activity

MW effects on growth and other cellular activities are being studied for atleast more than 50 years. Growth rate of yeast Saccharomycetes cerevisiae either increased upto 15% or decreased up to 29%, when exposed to MW radiation having narrow frequency range of 41.8-42.0 GHz [Grundler et al., 1977]. When S. aureus culture was exposed in a controlled temperature experiment to microwave radiation (24 GHz) for 10, 20, 30, and 40 s, the activity of various enzymes like malate dehydrogenase, cytoplasmic adenosine triphosphatase, glucose-6-phosphate dehydrogenase, and cytochrome oxidase increased in microwave treated cells than microwave non treated cells; whereas membrane adenosine triphosphate, alkaline phosphatase and lactate dehydrogenase activity remained unaffected [Dreyfuss and Chipley, 1980]. The effect of MW radiation on 94 strains of Enterobacteriaceae was studied and it was found that microwave irradiation increased the enzyme activity of bacteria in suspension [Spencer et al., 1985]. Low power MW treatment (2450 MHz; 90 W; 2 min exposure) on Aeromonas hydrophila decreased its total protease activity by 33%. Urease activity and aflatoxin production in S. aureus and Aspergillus parasiticus respectively was completely inhibited by MW exposure [Dholiya et al. 2012]. Low power (100 W, 60 s) microwave radiation reduced not only the cell number but also the acid resistance and verocytotoxin productivity in enterohemorrhagic E. coli [Tsuji and Yokoigawa, 2011]. Komarova et al. (2008) investigated influence of MW on soil bacteria. They observed both the suppression and the stimulation of the growth for different bacterial species under the impact of microwaves. Spore suspensions responded to microwave radiation upon a shorter time of exposure than suspensions of vegetative bacterial cells. The influence of microwave radiation on the biomass accumulation and the intensity of other physiological processes in streptomycetes species led to changes in the number and activity of these microorganisms in the soil microbial complex.

2.8 Lactic acid

Lactic acid is one of the useful compounds utilized in food, pharmaceutical and chemical industries. Stereoselective two isomers exist for having a chiral carbon. It can be polymerized to biodegradable plastics, i.e. poly lactic acid, which has great potential for replacing petrochemical plastics (Datta et al., 1995).

Applications

The demand on production of Lactic acid is increasing almost daily. Some of the most important uses of Lactic acid are listed below [Narayanan et. al, 2004;Naveena et. al 2004].

Food industries:

FDA has classified lactic acid as GRAS for use in food industries as general-purpose food additive in US. L (+) isomer, one of the stereoisomers of Lactic acid is preferred in food and dairy industries due to the presence of L (+) Lactate dehydrogenase in human being.

1) Lactic acid is added to margarine, butter, yogurts etc. for its pleasant taste (taste enhancer).
2) Lactic acid is used as pickling agent for olives and pickled vegetables.
3) It is also used as jelling agent for jams and jellies.
4) Ca-lactate is added to milk and other sports drink as mineral supplement
5) Lactic acid and its salt can increase the shelf life of food products like sausages, hams, poultry, fish, etc. by 30 to 50 %.
6) A large mass fraction of (>50%) fermentation grade Lactic acid isused to produce emulsifying agents such as sodium and calcium stearoyl lactate in bakery goods.
7) Calcium salt of this acid is a good dough conditioner and the sodium salt is both conditioner and emulsifier for Yeast leavened bakery products.

Pharmaceutical Industries

Poly lactic acid polymers are biocompatible, biodegradable and restorable materials used in medical application as sutures, orthopaedic implants, controlled drug release etc. Polymers of Lactic acid, after adjusting the composition and the molecular wt., can control the degradation of biodegradable transparent thermoplastics. Other applications in this industry are formulation of ointments, lotions, anti-acne solutions and dialysis applications. Ca-lactate can be used for calcium deficiency therapy and as anti carries agents.

Chemical Industries

Lactic acid is used as acidulant in Leather tanning industries and in small scale operations like pH adjustments, hardening baths for cellophanes used in food packaging, terminating agent for phenol formaldehyde resins, alkyl resin modifiers, solder flux, lithographic and textile printing developers, adhesive formulations, in electroplating and electro polishing baths, detergent builders etc. Lactic acid esters like ethyl/butyl lactates can be used as green solvents. They are high boiling, non-toxic and degradable components.

Synthesis of lactic acid

Two possibilities for the synthesis of Lactic acid are chemical synthesis and fermentation of solution that contains carbohydrate. The problem with chemically synthesized acids is their racemic properties. Fermented acids can produce desired isomers like L (+) and D (-) Lactic acids (Jin et. al 2003), which are more important in modern applications for the acids uses such as biodegradable plastics. The properties that are derived from the different forms of the isomer are very different. For instance higher optical purity of the L (+) Lactate polymer leads to higher melting point and crystallinity.

Chemical synthesis:

The commercial process of chemical synthesis is based on lactonitrile (Narayanan et. al 2004). Hydrogen cyanide is added to acetaldehyde in the presence of a base to produce lactonitrile. This process occurs in liquid phase at high pressures. The crude lactonitrile is recovered and purified by distillation. It is then hydrolyzed to lactic acid, either by conc. HCl or by H2SO4 to produce corresponding ammonium salt and lactic acid. Lactic acid is then esterified with methanol to produce methyl lactate, which is removed and purified by distillation and hydrolyzed by water under acid catalyst to produce lactic acid and methanol, which is recycled.

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Ammonium sulphate

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Lactic acid Methanol Methyl lactate

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Microbial production via Fermentation:

Stereo specific lactic acid can be made by carbohydrate fermentation

depending on the microbial strain being used. It can be described by the

reactions (Narayanan 2004):

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Carbohydrate Calcium hydroxide Calcium lactate

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Calcium sulphate

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Methyl lactate

CH3CHOHCOOCH3 + H2O → CH3CHOHCOOH + CH3OH

The broth containing calcium lactate is filtered to remove cells, carbon treated, evaporated and acidified with sulphuric acid to get lactic acid and calcium sulphate. The insoluble calcium sulphate is removed by filtration; lactic acid is obtained by hydrolysis, esterification, distillation and hydrolysis. The stereospecificity of the lactate dehydrogenase and the presence of a Lactate racemase determines whether D (-) /L (+)/DL mixture would be produced. There are two specific routes for fermentation depending upon the microorganisms (Shuler, 2003).

Fermentation Vs Chemical synthesis:

Chemical synthesis suffers from some drawbacks, which are:

1) Chemical synthesis is less efficient in terms of % conversion when compared to fermentation.
2) Chemical synthesis always produces a racemic (a DL mixture) mixture instead of a particular stereoisomer.
3) It employs harsh conditions such as high pressure, which is often unattainable at lab scale.

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Fig :- 3 Overview of the two methods of lactic acid (a) Chemical synthesis and (b) Microbial fermentation [Wee et al,2006].

2.9 Microbial pigment production

Pigments producing microorganisms and microalgae are quite common in Nature. Among the molecules produced are carotenoids, melanins, flavins, quinones and more specifically monascins, violacein, phycocyanin or indigo. However, there is a long way from the Petri dish to the market place as only five productions are operated on an industrial scale. The oldest one is red koji or angkak, fermented rice from the fungus Monascus, used in Asia for centuries as food colorant for red rice wine, red soybean cheese, meat and fish products. A long-established food ingredient for Asian consumers, this pigment is still forbidden in Europe and the USA as a mycotoxin may occur in some batches [Duffose et al,2006].

2.9.1. Pigment produced by Microorganisms

Some of the most important natural pigments are cartenoids, flavonoids, tetrapirroles and some xantophylls as astaxanthin. Micro-organisms which have the ability to produce pigments in high yields include species of Monascus,Paecilomyces, Serratia, chromobacterium,staphylococcus,Cordyceps, Streptomyces and yellow-red and blue compounds produced by Penicillium herquei and Penicillium atrovenetum, Rhodotorula, Sarcina, Cryptococcus, Monascus purpureus, Phaffia rhodozyma, Bacillus sp., Achromobacter, Yarrowia and Phaffia also produce a large number of pigments (Table 1).

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Table 1. List of pigment producing bacteria [Malik et al,2012]

Violacein

Violacein is a violet pigment isolated from Chromobacterium violaceum, a gram-negative betaproteobacterium found in the Amazon River in Brazil. Structure of violacein is shown in fig 2.3. It has been reported to kill bacteria It has antibacterial and antioxidant activity [Duran and Menck, 2001] and induces apoptosis in various types of cancer cells (Carvalho et al, 2006;Duran et al,2007). Due to the widespread presence of drug resistance in the malaria parasite, resulting in dramatically decreased efficacy of available antimalarial drugs [Talisuna et al, 2007].

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Fig. 2.3 Chemical structure of violacein

[...]

• College: Nirma University (Institute of Science)
• Course: M.Sc.
• Grade: A
• Authors: Assistant Professor Vijay Kothari (Author), Haren Gosai (Author), Shreya Raval (Author), Vimla Chaudhary (Author)
• Publication Year: 2014
• Pages: 75
• Catalog Number: V286832
• ISBN (eBook): 9783656871187
• ISBN (Book): 9783656871194
• Language: English
• Tags: altered, microwave, mutagenesis