The project undertaken aimed at the exploitation of locally isolated bacteria for the production of amino acids. For this purpose, six strains of amino acid producing-bacteria were isolated from local soils and water samples by employing nutrient agar, Leifson's medium (18), MacConkey's bi1esalt-lactose-peptone liquid and agar media (20), and EMB agar. The methods employed in the screening and isolation procedures were microscopic examination of unstained and stained slide preparations, plating methods, presumptive coliform test and Eijkman test.
Cultural morphological and physiological studies showed that the strains isolated were Escherichia coli, Aerobacter aerogenes, Klebsiella czaenae, Pseudomonas fluorescens, Bacillus megaterium and Bacillus subtilis, representing families Enterobacteriaceae, Pseudomonadaceae and Bacillaceae respectively.
Cultural characteristics were studied by observing size, shape, margin, elevation, consistancy, odour, optical features and pigmentation of colonies on agar plates (nutrient agar, MacConkey agar, EMB agar, Endo agar and ammonium salt glucose agar), as well as growth patterns on agar slants, gelatin stabs, potatoes and nutrient broth. Streak, pour and spread plate techniques (17,24) were used in the preparation or agar plates.
Cultural characteristic studies revealed that E.coli, A. aerogenes and K. ozaenae were lactose fermenters and produced pink Colonies of different shades, having different consistancies, on MacConkey agar. The colonies of A.aerogenes resembled those of E.coli in every respect except for the slightly mucoid character exhibited by the former. K.ozaenae, though a lactose fermenter, produced much bigger and highly mucoid colonies which coalesced and became golden-brown with age. The colonies of P. fluorescens resembled those of E.coli and A.aerogenes in shape, smoothness, edge and elevation, and produced good growth on MacConkey agar. However the organisms were found to be non-Iactose-fermenters and chromogenetic, and made the dark brown colour of MacConkey agar dirty due to the excretion of greenish colour into the medium:. B.subtilis and B.megaterium failed to grow on MacConkey agar even after prolonged incubation, under optimum growth conditions. Thus MacConkey agar was found to be an excellent differential as well as selective medium which enabled not only the separation of Gram-negative fromGram-positive bacilli, but also helped to distinguish lactose fermenting Gram-negative bacilli from non-lactose fermenters.
Morphological characteristi~s were studied by means of Gram reaction (25,312), acid fastness (Ziel-Neelson's method), motility test (26,27) and spore test (heat resistant method) with modifications where required.
Gram reaction revealed that E.coli, A.aerogenes, K.ozaenae and P.fluorescens were Gram-negative and species of Bacillus were Gram-positive. The cells of E. coli, A.aerogenes and p.fluorescens were seen to be short rods of 0.5 by 1.5 microns, 0.8 by 1.9 microns and 0.28 by 1 microns respectively. The cells of K.ozaenae were found to been capsulated plump rods of 0.9 by 2.8 microns. All these Gram-negative bacilli were found to be non-spore formers and arranged mostly singly and rarely in pairs'. The cells of the species of Bacillus, appeared to be straight rectangular rods of 1.5 to 2 by 5.5 to 6 microns, occurring singly and also in pairs, and arranged end to end in chains'. They were found to be sporeformers - the spores being oval in-shape and central in position and nearly of the same cross diameter as the bacilli. Microscopic examination was done under oil immersion at a magnification of 2000 times by means of bright field microscopy. Test for acid fastness showed that the bacteria tested were all non-acid fast. The motility test revealed that E.coli and P. fluorescens were actively motile, A.aerognes slightly motile and K.ozaenae and species of Bacillus non-motile. It was seen from the spore test that only the species of Bacillus bore spores and the rest were non-spore formers.
Physiological studies performed included Catalase test (29,30), Oxidase test (32,33,34,35), Oxidation fermentation test (O.F.test) (37), Indole production test (40,41,42), Methyl red test (44), Voges Proskaur test (45,47,306,310), Citrate utilization test (48), Haemolysin production test (50,51), Oxygen relation (54,161), Hydrogen-sulphide production test (55,56), Urease activity test (57,58), temperature relation (159,160), pH for growth (170,171, 172,173), Nitrate reduction test (304), Gluconate test (307), Carbohydrate break-doM1 test (309,311).
The results of these tests revealed that indole was produced only by E.coli whereas acetylmethyl carbinol production was done by A. aerogenes. K.ozaenae and B.subtilies A positive methyl red test was shown by E.coli & K.Ozaenae but citrate was utilized by all organisms but E.coli gattalse production was observed in case or all bacteria but E.coli, and a positive oxidase test was given by K.ozaenae and P.fluorescens only. Hydrogen sulphide was produced by B.subtilis & P.fluorescens but nitrate was reduced by all the bacteria but B. megaterium. Ammonia production was shown by K.ozaenae, P. fluorescens and species of Bacillus. From the results or of test, it was evident that p. fluorescens was oxidative, B.subtilis, was oxidative as well as fermentative, whereas E. coli A.aerogenes and K.ozaenae were fermentative. A wholly positive gluconate test was obtained only in case of A. aerogenes. Acid and gas production varied in carbohydrate-break-down-test. For example acid and gas were produced by E.Coli, A. aerogenes and K. Ozaenae in glucose, sucrose and lactose, whereas acid without gas was produced by B.subtilis, B. megaterium and P. fluorescens in glucose. Acid was produced by all the organisms in mannitol. A partial haemolysis of blood was brought about only by species of Bacillus. Optimum growth temperature in case of all gram-negative bacilli was found to be 37o C but in case of B. subtillis, and B. Megaterium, it ranged between 25o to 35 oC. All organisms under study were found to be aerobic but species of Bacillus were also facultatively anaerobic. pH for growth ranged from 6. 5 to 7.5. Identification of bacteria was achieved by comparing the results obtained, with those recorded in the diagnostic tables given by Cowan and steel (21) as well as with the information given in Bargey's manual of Determinative Bacteriology (60).
An endeavour was also made to derive biochemical mutants by employing physical and chemical methods of mutation. Mutations were induced physically by exposing 10 ml of cell suspension in 0.85% saline water, containing 108 bacterial cells/ml, to ultra violet radiation for 30 minutes in uncovered periodishes of 11. 5 cm dia, at a distance of 40 cm from the source (UV lamp, 220V, 60CC George W. Gates & Co, N. Y) Mutation was completed in five stages, increasing the time of exposure by 10 minutes each time, and. keeping the distance constant from the source, so that the survivals obtained were between 10 to 12 percent. Washing and harvesting of the cells was done by using International centrifuge (size 2, Model V. No. 42386. H) at 15000 rpm for 20-30 minutes.
Mutations were also induced chemically by preparing a bacterial cell suspension, containing 108 cells per ml, in tris-maleate buffer which contained 1. 75 mg/ml of NTG. The cells were allowed to remain suspended for 20 minutes, before they were harvested and washed with 0.85% saline. The washed cells were suspended in tris-maleate buffer without NTG for half an hour before these were inoculated into com plete agar. The treated cells in both the cases were washed and plated on complete agar, and incubated at 37o C for 72 hours and were designated as EM5, AM5, KM5, PM5 and BM5 . Cell concentration was determined by using Brown's opacity tube method (315, 316, 317).
By further mutational treatment auxotrophic mutants viz. BM5_ -Homoserine (a homoserine auxotroph from BM5) E MS- Histidine ( a histidine +. auxotroph of EM5),BM5 - DAP - Met - Ile (an isolucine revertant from a triple auxotroph of EM5), were derived from. BM5 & EM5, which were obtained mean of UV irradiation; and analog resistants viz. BM5 â€“ R -Arghx-RDA (a mutant of B. subtitles resistant to argentine hydroxamate and D-arginine), CM5-RTA (a mutant of Corynebacterium glutamicum resistant to 2-thiazole alanine) were derived from BM5 and CM5, obtained by means of NTG treatment. In order to derive auxotrophic mutants BM5-Homoserine EM5 - Histidine, and EM5 - DAP - Met - IIe), the UV irradiated mutants of B. Subtilis and E. Coli (BM5 and EM5) were cultivated for 72 hours at 37oC in minimal medium supplemented with 100 mg/ml of homoserine, 150 ug/ml of histidine 100 ug/ml of DAP, 75 ug/ml of DL methionine and 25 ug/ml of isoleucine respectively. Colonies grown in 2 to 3 days were tested by replica method of Lederberg and Lederberg (71), and those which grew on the minimal medium supplemented with the deficient nutrients were selected as desired auxotrophs. In certain cases, penicillin enrichment method, according to the procedure of Davis (93, 94) was also applied. L. arginine analog resistant mutants ( BM5 -R-Arghx) and L-histidine analog-resistant-mutants ( CM5- RTA) were derived by spreading from c O. 5 ml of cell suspension (prepared from colonies of BM5 and CM5, obtained on complete agar plates), containing 108 cells/ml on minimal agar, incorporated with L-arginine analog Car ginine hydroxamate) at a concentration of 3 mg/ml and L-histidine analog (2 thiazole alanine) at a concentration of 850 ug/ml. Most of the cells were killed and the colonies obtained after 96 hours of incubation, at 37 C, were selected as analog-resistant mutants and designated as BM5- R-Arghx, and CM5-RTA From the monoanalog- resistant mutant of L-argine (B M5- R-Arghx-RDA) by growing BM5-R-A:rghx in minimal agar containing 3 mg/ml of D-arginine for 96 hours at 370 C and isolating the colonies which survived.
When the U V irradiated mutants were cultivated, a delay in their appearance was observed which depended on the type of mutant as well as the kind of medium employed. For example the clones of EM5 and A M5 appeared within 24 hours of incubation at 37Â°C on agar plates, but the appearance of the clones of PM5, KM5 and BM5 was delayed by 48 to 72 hours. Further it was seen that the mutant clones of large size appeared within 16 - 18 hours of incubation and the smaller sized clones usually took 3 to 7 days for their appearance. This delay in appearance was observed to be more pronounced on MacConkey agar.
A comparison of the cultural characteristics of the irradiated population with those of the corresponding wild strains, revealed that BM5 not only produced much smaller colonies on blood agar BM5 produced only 5 â€“ 15 clones on MacConkey agar. These clones showed absolutely no tendency to spread or coalesce, proving thereby that KM5 slime. Moreover KM5, AM5 and EM5 failed to ferment lactose partially or wholly when grown on MacConkey agar and the colonies produced appeared golden yellow instead of the usual pink colour. Mutant strains exhibited quite a number of other variations in cultural characteristics which were observed. (See table12L in the thesis).
Morphological studies of UV irradiated mutants showed that M5 had not only changed into Gram-negative but also the rectangular shape of the bacilli had changed into oval at the ends.
When the results of the biochemical tests, given by the UV irradiated mutants, were compared with those of the wild strains, it was found that KM5 and AM5 failed to utilize citrate and thus were unable to grow in Koser's citrate medium. EM5 and AM5 Appeared to have gained the biochemical efficiency to produce NH3 and hydrolyse Christensen's urea slope, whereas KM5, PM5 and BM5 appeared to have lost their ability to produce NH3. A negative Voges-Proskaur test was given by B. SM5 and a negative methyl red test by EM5 and KM5, AM5 was seen to have gained the biochemical efficiency to utilize citrate, whereas AM5 appeared to have lost its ability to utilize citrate and survive in Koser Citrate, when incubated for 4 days at 37oC. Mutants also exhibited considerable variation in the production of acid and gas in carbohydrate-breakdown test, the most important of which was the change undergone by AM5 and KM5 from lactose to lactose. was studied. For this purpose five batches each consisting of five 30 ml cotton wool plugged flasks, each containing 10 ml of fermentation medium, inoculated with 0.5 ml of 24 hour-old-seed culture of each bacterial strain were set up, and incubated at 300C for 120 hours on a shaker at 150 -200 rpm. (Gallen-hamp shaker bath, Oscillation/min Made in England). After every 24 hours, the amino acid contents of the culture broths were determined by the reduced ninhydrin method of Moore and stein (305) with some modifications.
The cell debris of each bacterial strain, collected by centrifugation for 20 minutes at 15000 rpm, from each set of experiments, was dried in a hot air oven, heating to constant weight, at 100o C â€“ 105o C and hydrolysates were prepared by dissolving 0.1 gram of dried cells from each bacterial strain in 5 ml of 6 N Hcl, contained in ampules which were sealed before the hydrolysis was carried out in Vacuo for 24 hours. Alkaline hydrolysis, using 5 N. NaoH, was carried out for tryptophan assay. Paper chromatographic estimation of the amino acid contents of the hydrolysates was also done according to the reduced nin-hydrin method. The absorbancy was read in PYE, UNICAM, SP 600 Series 2 SPECTROPHOTOMETER.
Growth curves of each bacterial strain were constructed from DCW obtained in different media (given in the thesis papers). The amino acids recovered after 120 hours of incubation both wild and mutated, included all the essential amino acids viz. a maximum quantity of 6.12 mg/ml of threonine, 6.3 mg/ml of tryptophan, 4.41 mg/ml of Isoleucine, 3.83 mg/ml of histidine, 1.9 mg/ml of methionine, 5.58 mg/ml of phynylalanine, 3.08 mg/ml of argine, 3.50 mg/ml of lysine. 1.04 mg/ml of valine, 1. 80 mg/ml of leucine and two dispensable amino acids i..e. Serine and Cystine-2.l2 mg/m: and 0.40 mg/ml respectively. It is evident from the scrutiny of the results that a maximum number of eight indispensable amino acids i.e. Threonine, Methionine, Leucine, Isoleucine, Arginine, Lysine, Histidine and Phenylalanine from the broths of wild and mutated strains of Klebsiella and seven i. e. threonine, isoleucine, lysine, arginine, histidine, phenylalanine and tryptophan from the broths of wild and mutated strains of Escherichia have been isolated. It can also been seen that all the bacterial strains in their mutated state, produced comparatively greater quantities of amino acids in all the four media. However the kind of amino acids produced by each strain, varied with the type of media employed. For example M5 produced 6. 12 mg/ml of threonine in N 2 medium as compared to its yield of 4. 51 mg/ml in U-medium, 3.36 mg/ml in F-medium and 2.40 mg/ml in G-mediwn. Similarly 6. 3 mg/ml of typtophan was produced by the same mutant in G2-medium as compared to its yield of only 1. 2 mg/ml in F-medium. Similarly strain AM5 produced 5.58 mg/ml of phenyl-alanine in F medium as compared to its produce of 2. 3 mg/ml in G2 medium. Threonine too was excreted in greater quantity i. e. 4. 80 mg/ml in N2 medium by AM5 as against 2.98 mg/ml in U-medium, 2. 73 mg/ml in F-medium and 2. 50 mg/ml in G-medium. In case of KM5 the maximum quantity of 3.83 mg/ml of histidine was isolated from U2 medium, 2.82 mg/ml of arginine from F medium and 1. 84 mg/ml of Lysine from G-medium as compared to their corresponding yield in other media. PM5 gave a higher yield of 2.42 mg/ml of arginine and 2.86 mg/ml of phenylalanine in N and F media respectively. M5 was no exception to this observation as this strain also produced 3.50 rng/ml of lysine in G-l medium and 3.08 rng/ml of arginine in U 1 medium and 2.56 mg/ml of phenylalanine in F-medium as compared to its yield of 1.66 rng/ml of lysine and 2.40 mg/rnl of arginine in Frmedium and 1.60 mg/ml of phenylalanine in G-medium.
The amino acid producing capacity of the auxotrophic and analog-resistant mutants was found to be rnore pronounced. For example 12.6 mg/ml of lysine in G-l mediurn, 10.5 rng/ml of tryptophan in G-2 medium, 13.2 mg/ml of threon-ine in N2-rnedium, 6.6 mg/ml of arginine in Ul-medium and 5.6 rng/ml of histidine in U2-medium, were recovered from the fermented broths of B M5 Homoserine, EM5-Histidine, EM5-DAP, Met, lle BM5-R-Arghx-RDA and CM5-RTA respectively after 1 20 hours of incubation at 300C. It is pertinent to state here that KM5-RTA was derived and tested for its ability to produce L-histidine but the yield instead of improving decreased (l. 2 mg/ml) and hence CM5RTA was derived and employed in the production of L. histidine. It can thus be concluded from these results that microbial genetics constitutes the rationale for strain improvement programme and it provides an approach for developing other products such as some antibiotics and improvement in the yield.
The effects of various nitrogen and carbon sources and growth factors, on the production of threonine. lysine, tryptophan. arginine and histidine were studied and it was found that the accumulation of a maximum quantity of threonine by wild and mutated strains of E. coli was favoured by an optimal level of 7.5% fructose and 1.4% ammonium sulphate in the presence of 100 g/ml of DAP, 50-75 ug/ml of DL-methionine, 20-25 ug/ml of isoleucine. However in order to obtain maximum quantities of lysine and tryptophan. It was found that the fructose in the medium had to be replaced by 7.5% of glucose. Moreover the inclusion of 150 ug/ml of histidine in the medium for the production of tryptophan, and I00 ug/ml of homoserine, 75 ug/ml of DL-methionine and 20 ug/ml of isolucine for the production of lysine were found to be favourable. The indicated quantities of arginine and histidine could only be recovered from the culture broths when the level of glucose was raised to 10% and 15% and that of ammonium sulphate to 3% and 4% respectively in addition to the inclusion of 0.3% of urea in the fermentation media. Moreover the addition of 20 ug/ml of isoleucine and 30 ug/ml of biotin. And 200 ug/l of biotin and 300 ug/l of thiamin hydro-chloride in the media for the production of arginine and histidine respectively were found to be favourable.
NOTE:- As it had been planned to study the microbial production of amino acids from locally isolated strains (whose amino acid production was low) it had, therefore, become necessary to produce mutants and study their biochemical properties in detail before undertaking investigation on their capability to produce amino acids in enhanced yields.