Studies on Production of L-Lysine by Corynebacterium glutamicum and Its Use in Poultry Feed

Abstract

A local soil isolate of Corynebacten'um glutamicum (MRL strain, GenBank accession nos. HM368259) was studied for biochemical, molecular and L-lysine production parameters. Identification of the bacterium was done on the basis of morphology, microscopic appearance, biochemical tests and two gene sequences (165 rDNA & lysC). Biochemical tests revealed the isolate to be a glutamic acid producing Corynebacterium similar with the reactions reported for C glutamicum except a positive test result for esculine (~-glucosidase production). Comparison of 165 rDNA gene sequences with closely related taxa revealed that the local bacterium was originating from same ancestor belonging to the genus Corynebacten·um. Similar analysis of aspartate kinase producing gene (lysC) sequence showed its close phylogenetic position to C glutamicum ATCC 13032 and C glutamicum type R. BLAST results from NCBI data showed a 96-97% similarity among various glutamic acid producing Corynebacten'um strains and our strain (165 rRNA gene sequence bases). Minimum doubling time of wild type C glutamicum (MRL) was observed at 30 ' C in complete medium CASO and also in fermentation medium CGXII whereas in minimal medium (MM1) it was at 35' C. So, optimum growth temperature for our wild type strain is 30'C. Growth limit of C glutamicum (MRL) at 39'C and 40 'C was tested. C glutamicum (MRL) showed minimal doubling time in CASO at 39'C in shake flask culture followed by C efficiens and C glutamicum ATCC 13032, respectively. At 39' C C glutamicum (MRL) strain behaved more closely to C efficiens rather than C glutamicum ATCC 13032. At 40' C, none of the strains showed satisfactory growth. Potential of C glutamicum (MRL) for glutamic acid and lysine production at 30' , 35 ' and 37' C was tested in fermentation medium (CGXII). Production showed increasing xiii trend up to 36 hours of incubation at 30 0 & 35°C and then declined, but continuous increase was observed at 37°C up to 60 hours of incubation and then became almost static. It was evident from the data that glutamic acid & lysine production and its extracellular excretion were not related to high growth (OD600). Maximum growth (OD600) was noted at 30°C but maximum production of both glutamic acid and lysine were observed at 37°C. Mutants of C glutamicum are the best known for production of L-Iysine. To get a potent L-Iysine producer, mutation was induced in wild type local strain C glutamicum (MRL). A lethality level of 99.9% was achieved after UV exposure for 180 seconds from 15 cm distance. Selection of homoserine auxotrophic mutants was done by growing cells (surviving after UV exposure) in penicillin G enriched broth (MM1). Non auxotrophs grow in MM1 and die under the effect of penicillin, but auxotrophs survive as quillicent cells. After washing, these were poured to grow on minimal agar media (MM1) plates containing 10 mM AEC, 1 mM threonine, 0.01 % L-homoserine. Thus only those auxotrophs which were able to grow in presence of AEC were selected for qualitative and quantitative screening of lysine producing cells. Maximum L-lysine producing mutant (MRL116; 8.2g1L) was selected for shake flask optimization and for production in stirred tank fermentor. Optimized conditions for the best production of L-lysine (12.5g1L) in basal medium were found at pH value of 7.6,30° C temperature and 300 rpm. Laboratory grade glucose, and two industrial by products i.e., sugar cane molasses and com starch hydrolysate were used as carbon source. Maximum lysine production was achieved around 9% available sugars regardless of the source provided. Ammonium sulphate concentration from 2.5 to 4.5% level was tested in all three fermentation media. Optimum level in FM3 medium was 3.5% and for FM1 and FM2 it was around 4%. A reason for a bit lower requirement in FM3 medium is due to fulfillment of the need of amino group form organic sources (amino acids) present in com steep liquor. xiv Present study was further conducted to estimate in-vivo safety and efficacy of locally produced L-Iysine (H2S04/HCI acidified) in crude form (without purification through ion exchange process). L-Iysine enriched supernatant (2Sg L-Iysine /L) and commercial crystalline L-lysine (HCI) was used in poultry feed at different concentrations and growth performance of broiler chicks was compared. Significantly higher (P<O.OOl) weight gain values were observed in all treatment groups as compared to negative control at 21 and 42 days of age. No significant difference in feed: gain values of positive control (B) compared to treatment groups providing same level of L-Iysine through fermentation liquid was indicative of the efficacy of our local product. There was also no significant difference in feed intake and mortality among all experimental groups till 21 days of age. L-Iysine supplementation significantly (P<O.OS) reduced feed intake and feed: gain in all treatment groups as compared to negative control (group A) during 6 weeks trial. Analysis of variance showed no negative impact of treatments on immunity. There was no damage to visceral organs. Our findings showed that local product was safe to use as poultry feed supplement and there was no significant difference in bioavailability and growth response parameters when liquid L-Iysine enriched broth was used in HCI or sulphate forms. Thus it can be concluded that the local L-Iysine enriched fermentation broth can be substituted with imported crystalline L-Iysine. With use of local raw material for production of L-Iysine by local soil bacteria, this is an indigenous solution to cut import bill ofL-lysine.