Exploring the flexibility of cellulase cocktail obtained from mutant UV-8 of Talaromyces verruculosus IIPC 324 in depolymerising multiple agro-industrial lignocellulosic feedstocks.

Effective management and the valorization of agro-industrial lignocellulosic feedstocks can only be realized if a versatile cellulase cocktail is developed that can release glucose at affordable cost irrespective of biomass type. In the present study the flexibility of using cellulase cocktail obtained from mutant UV-8 of Talaromyces verruculosus IIPC 324 in depolymerizing multiple agro-industrial lignocellulosic feedstocks was explored. Five different dilute acid pretreated biomasses were evaluated and cellulase loading was done at 25 mg protein/g cellulose content. After 72 h of hydrolysis at 55 °C and pH 4.5, corn cob and rice straw emerged as the easiest and toughest substrates with saccharification yield of 83.9 ± 1.17 and 35.5 ± 1.16% respectively from their cellulose fraction. Addition of PEG 6000 could retain >65% of all mono-component enzymes present in cellulase cocktail. Structural elucidation of biomasses gave an insight about key features responsible for variable recalcitrance in the different agro-industrial feedstock. Cellulose hydrolysis showed a significant negative correlation in the order of Cr I > S/G ratio > ash content. The chemical composition of lignin had a major impact on enzyme-lignin interactions. Higher H lignin content and lower S/G ratio promoted enzyme desorption, thereby increasing the likelihood of their recycling and reuse.

Augmented hydrolysis of acid pretreated sugarcane bagasse by PEG 6000 addition: a case study of Cellic CTec2 with recycling and reuse.

In an integrated lignocellulosic biorefinery, the cost associated with the "cellulases" and "longer duration of cellulose hydrolysis" represents the two most important bottlenecks. Thus, to overcome these barriers, the present study aimed towards augmented hydrolysis of acid pretreated sugarcane bagasse within a short span of 16 h using Cellic CTec2 by addition of PEG 6000. Addition of this surfactant not only enhanced glucose release by twofold within stipulated time, but aided in recovery of Cellic CTec2 which was further recycled and reused for second round of saccharification. During first round of hydrolysis, when Cellic CTec2 was loaded at 25 mg protein/g cellulose content, it resulted in 76.24 ± 2.18% saccharification with a protein recovery of 58.4 ± 1.09%. Filtration through 50KDa PES membrane retained ~ 89% protein in 4.5-fold concentrated form and leads to simultaneous fractionation of ~ 70% glucose in the permeate. Later, the saccharification potential of recycled Cellic CTec2 was assessed for the second round of saccharification using two different approaches. Unfortified enzyme effectively hydrolysed 67% cellulose, whereas 72% glucose release was observed with Cellic CTec2 fortified with 25% fresh protein top-up. Incorporating the use of the recycled enzyme in two-stage hydrolysis could effectively reduce the Cellic CTec2 loading from 25 to 16.8 mg protein/g cellulose. Furthermore, 80% ethanol conversion efficiencies were achieved when glucose-rich permeate obtained after the first and second rounds of saccharification were evaluated using Saccharomyces cerevisiae MTCC 180.

Benchmarking hydrolytic potential of cellulase cocktail obtained from mutant strain of Talaromyces verruculosus IIPC 324 with commercial biofuel enzymes.

In the present study, an attempt was made to benchmark the hydrolytic potential of cellulase cocktail obtained from stable mutant UV-8 of Talaromyces verruculosus IIPC 324 (NFCCI 4117) with three commercially available cellulases. With two experimental approaches, acid-pretreated sugarcane bagasse was subjected to hydrolysis for 72 h, where all the enzymes were dosed on the basis of common protein or common cellulase activity /g cellulose content. Concentrated fungal enzyme (CFE) of mutant UV-8 resulted in ~ 59% and 55% saccharification of acid-pretreated sugarcane bagasse after 72 h at 55 °C and pH 4.5 with respect to reducing sugar release, when dosed at 25 mg protein/g and 500 IU CMC'ase/g cellulose, respectively. On the other hand, at similar dosages, the performance of Cellic CTec2 was best resulting in 77% and 66% saccharification, respectively. When enzyme desorption studies were undertaken by carrying out cellulase activities in saccharified broth after 72 h CFE of UV-8 emerged as the best cellulase cocktail. A minimum of 90% endoglucanase and 60% cellobiohydrolase I was successfully desorbed from residual biomass, thereby increasing the probability of enzyme recycle and reuse for next round of hydrolysis.

Feasibility studies with lignin blocking additives in enhancing saccharification and cellulase recovery: Mutant UV-8 of T. verruculosus IIPC 324 a case study.

The process economics of fermentable sugar production is dependent on the performance of cellulase cocktail on realistic lignocellulosic biomass and their capability to be recovered and recycled. Feasibility studies were conducted to enhance the digestibility of acid pretreated sugarcane bagasse using novel cellulase cocktail obtained from stable mutant UV-8 of Talaromyces verruculosus IIPC 324 in presence of lignin blocking additives. PEG 6000 was shortlisted as the best additive as it could simultaneously enhance saccharification and overall cellulase recoveries namely cellobiohydrolase, endoglucanase and cellobiase. Addition of 0.3 g PEG 6000/g acid-insoluble lignin content, resulted in 55% and 49.2% saccharification yields in terms of reducing sugars and glucose respectively using this cellulase cocktail (25 mg protein/g cellulose content) after 72 h from acid pretreated sugarcane bagasse loaded at 7.5%. The study also suggested that the endoglucanase of this mutant was unique with high desorption capability as 85% activity was observed in the saccharified broth devoid of any lignin blocking additive. At its optimum concentration, PEG 6000 was able to retain 94 ± 0.79% cellobiohydrolase I and 97.97 ± 1.16% cellobiase enzyme in the saccharified broth which were otherwise lost in residual biomass by ∼80%, in the absence of this polymeric additive. These results suggest that PEG 6000 was the most promising facilitator for recycling of cellulases obtained from mutant UV-8 of Talaromyces verruculosus IIPC 324 in particular. It paved a way towards the production of cheaper fermentable sugars which serve as a starting raw material for the production of green chemicals and fuels.