Different species belonging to the genus Cellulomonas namely C. biazotea, C. cellusea, C. fimi and C. flavigena, previously isolated in these laboratories , were screened for production of celluloytic enzymes in both solid and aqueous media containing various cellulosic substrates. Six basic parameters were used to evaluated the organisms; growth on solid media ; growth in aqueous culture media to product celluslases; resistance of the cellulases/ xylanases to end-product inhibition; thermal stability of the enzymes; ability of the organisms for enhanced production of cellulases and xylanases with changes in the growth conditions and saccharification efficiency. All strains grew rapidly on defined media containing carboxymethly cellulose â€“Na salt (CMC), esculin, sigmacell 100 or cellobiose in the presence or absence of glucose. The criteria used is based on the secretion of enzymes namely CMCcase, Î²-glucosidase and Avicelase (Sigmacellase) and cellobiase. These studies and liquid culture studies revealed that C. biazotea was the fastest growing and the best producer of theses enzymes. These also showed some resistance to end-product inhibition.
Liquid culture screening for production of cellulases and grew rapidly in liquid media and produced cellulases equivalent to or better than those of other well studied celluloytic microbes (1.86 IU FPase/m1). The use of Î±-cellulose, filter paper, cotton wool or sigma cell 100 resulted in increased yields of Fpase followed by Kallar grass (Leptochloa fusca L. Kunth) rasied from saline lands, while xylanase activities were detected when Kallar grass was used inducer. FPase, CMCase and xylanase accumulated extracellularly while Î²-xylosidase were mainly cell-bound. The greatest enzyme production occurred at 30 oC, pH 7.3 after 3 days of incubation both from realistic cullulosic substrates viz, filter paper Î±-cellulose or sigmacell 100 or lignocellulosic substrates . C biazotes was selected as the best producer of enzymes and was studied in greater detail.
The enzymes titres were characterized for some of the properties related to saccharification. The standard assay temperature pH were 40oC and 7 respectively. The change in assay temperature and pH changed the activities. The enzyme activities from C. biazotea showed the temperature optima between 35-50oC. The enzyme system was stable at 25 oC for 7 days in the presence of growth substrate. The cullulases and xylanases exhibited a broad PH activity profile; with optimal activity for Î²-glucosidase glucosidase and Î²-xylosidase in the range of 3-7 while CMCase, FPase and Xylanases activities showed pH optima of 8-7.5. This could be an important attribute from the practical application stand point as pH control would not be as critical as observed with many other cellulose preparations.
Another attribute of the cellulose/ xylanase complex was its resistance to end-product inhibition e.g maximum deactivation of FPase, CMCase and Î²-glucosidase occurred by 12, 30 and 27 % respectively in the presence of 10 % glucose when exposed for 1 hour. Similar was the effect of cellobiose, xylose or thanol. The enzyme complex was activated by Co. Mn, S, Ca, Mg and Fe and repressed by Hg or Zn salts.
The conditions for enzyme production were optimized. In plated tests 0.25% sigmacell 100 for Avicelase production while aqueous culture studies indicated that 1 % carbohydrate concentration in cellulosic or LC substrates was optimum. Yeast extract could not be replaced by peptones or other growth promoters; pH 7.3 and temperature 30oC were found optimum for production of cellulases and xylanases. Dubos salts were found optimum to provide ionic strength ; other media used by other workers to produce these enzymes were not suitable for enhancing production of cellulases and xylanases. Nitrates were the best nitrogen sources and supported greater cell mass and enzyme production. Inoculum size, between 80 and nitrate addition enhanced production of cellulases and hemicelluases. All enzymes were produced from Kallar grass when glucose, glycerol, galactose, lactose, sucrose, cellobiose or sorbose was also present in the liquid medium.
The maximum activities of FPase, CMCase, Xylanase, Î²-glucosidase and Î²-4.6, 16.1, 0.81, 1.45 IU/mg protein respectively.
The enzyme system of cellulose complex was produced when C. biazotea was grown on Kallar grass straw in the absence or presence of Î± cellulose. Saccharification efficiency was evaluated using Î±-cellulose, Kallar grass, wheat straw, bagasse, and CMC-Na salts as substrates. At low substrate concentration(1, 2.5, 5% w/v), near theoretical yields of reducing sugars were obtained. At higher substrate concentration, the saccharification efficiency even with concentrated enzyme filtrates declined. The highest saccharifications were obtained from CMC, wheat straw or bagasse e.g, maximum saccharification from 10% Î±-cellusose, Kallar grass, wheat straw, bagasse and CMC was 30, 39, 71 69 and 65% respectively and from 12.5%, this was 28, 39, 59, 57 and 51. Apparently cellulase titres from C. biozotes better saccharified wheat straw or bagasse and liberated sugars were fermented to ethanol with Saccharomyces carlsbergensis. Concentrated enzyme filtrates gave a maximum of 85% theoretical yield of ethanol compared to 96 % obtained from glucose. Whole culture filtrates also saccharified 5% wheat straw or bagasse suspensions and the hydrolyaates were fermentable to yield alcohol with a maximum theoretical yield of 65 % .
Trichoderma spp namely T. reesei and T. harzianum were tested for their abilities to produce cellulases from Kallar grass straw, P. maximum, wheat straw or bagasses in the presence or absence of Î±- cellulose, Î±- cellulose induced cellulases in. greater quantity followed by Kallar grass plus Î±- cellulose The enzyme filtrates produced from the latter treatment were used for saccharification and fermentation studies to produce ethanol. The cellulase titres both from T. harzianum and T. reesei saccharified 5 % (alkali-treated ) Kallar grass and P. maximum Saccharified carried out at 50oC gave 85-96 % theoretical yield of sugar calculated on the basis of total carbohydrates present in the substrates whereas saccharfication at 40 oC released 77-91% sugars, P maxium produced more glucose compared against that from Kallar grass. Sugars from Kallar grasshdrolysate, produced with both the enzyme preparations. 10 % substrate suspension was saccharified with concentrated cellulase titres obtained in solid state fermentation and liberated sugars were fermented to ethanol with yeast with appreciable theoretical yield 987-98).
Micro-organisms have the ability to grow on inexpensive cellulosic agricultural residue/ wastes to produce protein of high biological value for feeding livestock and ruminants. Thus to establish optimum condition of pH, ionic concentration of growth medium, nitrogen source, temperature, substrate, substrate concentration, substrate treatment and carbon and nitrogen ratio for maximum production of mycelial cell protein and mycelial biomass form Kallar grass (found to be a strong induce of cell mass biosynthesis among 14 different carbon sources) through fermentation with C. biazotea (found to be high cell mass produce among four different strains of Cellulomonas) has been attempted.
The optimum conditions determined as above were applied to ferment Kallar grass in fed-batch culture studies to produce SCP in a fermentor to have it in large quantity. The biomass obtained was then analysed for proximate composition to find the profile of essential amino acids in pure culture of Cellulomonas or Cellulomonas S. uvarum. The chemical analysis indicted that all essential amino acids were present at desired levels .
The growth studies with Methanosarchina mazei on emthanol and acetate indicated that the organism can rapidly utilize acetate and methanol for growth. The organism showed a high tolerance to acetate; quantitative yields of methane were obtained from upto 2 % acetate. The cellulase titres produced by C. biazotea from Kallar grass saccharificatied Kallar grass to some extent. The hydrolytsates were converted to methane precursors with Klebsiella sp for subsequent conversion of methane by M. mazei. The gass mixture had 60 % methane and 35% Co2. NaOH treatment gave increased cellulose degradation with concomitant increase in methane production. Maximum concentration of Kallar grass for higher methane production was established alongwith the concentration of exogenous supplements. These studies indicated that upto 10 % carbohydrates of Kallar grass remain unconverted in the digestor. The celluloytic complex including Î²-glucosidease of such organisms needs improvement in order to more effectively gair monomeric sugars.