Bambang Prasetya

From mannan to bioethanol: cell surface co-display of β-mannanase and β-mannosidase on yeast Saccharomyces cerevisiae.

BackgroundMannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. However, the utilization of mannans as sustainable resources has been less advanced in sustainable biofuel development. Based on a yeast cell surface-display technology that enables the immobilization of multiple enzymes on the yeast cell walls, we constructed a recombinant Saccharomyces cerevisiae strain that co-displays β-mannanase and β-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source.ResultsParental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered β-mannanase and β-mannosidase; co-display of the two enzymes on the cell surface was confirmed by immunofluorescence staining and enzyme activity assays. The constructed yeast cells successfully hydrolyzed 1,4-β-d-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-β-d-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate.ConclusionsWe successfully displayed β-mannanase and β-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying β-mannanase and β-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering β-mannanase and β-mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials containing mannan components.

Potential utilization of cassava pulp for ethanol production in Indonesia

Cassava is one of the major crops produced in Indonesia. Cassava grows in all provinces in Indonesia. In the last decade, even though cassava plantation area is decreased, cassava production and its productivity in Indonesia have been on the increase steadily. The tendency of using cassava for ethanol production would affect supply of cassava for food. Cassava pulp, a by- product of tapioca industry is one of the potential biomass that can be used for ethanol production because it contains starch and fiber in significant amounts which could be further converted to ethanol. A large scale of tapioca plant having production capacity of 20 ton tapioca flour per day has a potency to produce 8.7 kL of ethanol per day. Conversion of cassava pulp to ethanol can be accomplished through different kinds of processes such as physical, chemical, biological process or combinations of those processes. The utilization of cassava pulp for ethanol production would be beneficial since the material is abundantly and continuously available in many big tapioca industries and could help in solving the problem of waste disposal of tapioca industry. However, comprehensive studies are still needed for establishment of bioethanol industry from cassava pulp. Key words: Cassava, cassava pulp, utilization, starch, fiber, ethanol. INTRODUCTION Cassava ( Manihot esculenta ) is one of the important crops in the world. Global production of cassava reached 228.14 million tons in 2007 (Wuttiwai, 2009). Nigeria and Brazil were the two most leading cassava producers. Cassava is also one of the major crops in Southeast Asia, especially in Thailand and in Indonesia. According to Wuttiwai (2009), Thailand was so far the third largest cassava producers with a total production of 26 million tons. On the other hand, Indonesia produced around 20 million tons of cassava per year with total area of plantation around 1.2 million ha (Ministry of Agriculture of Republic of Indonesia, 2009). Recently, there is a

Fermentation Characteristics and Microbial Diversity of Tropical Grass-legumes Silages

Calliandra calothyrsus preserved in silage is an alternative method for improving the crude protein content of feeds for sustainable ruminant production. The aim of this research was to evaluate the quality of silage which contained different levels of C. calothyrsus by examining the fermentation characteristics and microbial diversity. Silage was made in a completely randomized design consisting of five treatments with three replications i.e.: R0, Pennisetum purpureum 100%; R1, P. purpureum 75%+C. calothyrsus 25%;, R2, P. purpureum 50%+C. calothyrsus 50%; R3, P. purpureum 25%+C. calothyrsus 75%; and R4, C. calothyrsus 100%. All silages were prepared using plastic jar silos (600 g) and incubated at room temperature for 30 days. Silages were analyzed for fermentation characteristics and microbial diversity. Increased levels of C. calothyrsus in silage had a significant effect (p<0.01) on the fermentation characteristics. The microbial diversity index decreased and activity was inhibited with increasing levels of C. calothyrsus. The microbial community indicated that there was a population of Lactobacillus plantarum, L. casei, L. brevis, Lactococcus lactis, Chryseobacterium sp., and uncultured bacteria. The result confirmed that silage with a combination of grass and C. calothyrsus had good fermentation characteristics and microbial communities were dominated by L. plantarum.

Glutathione production from mannan-based bioresource by mannanase/mannosidase expressing Saccharomyces cerevisiae

This work aims to produce glutathione directly from mannan-based bioresources using engineered Saccharomyces cerevisiae. Mannan proved to be a valuable carbon source for glutathione production by this organism. Mannan-hydrolyzing S. cerevisiae was developed by heterologous expression of mannanase/mannosidase on its cell surface. This strain efficiently produced glutathione from mannose polysaccharide, β-1,4-mannan. Furthermore, it produced glutathione from locust bean gum (LBG), a highly dense and inexpensive mannan-based bioresource, as sole carbon source. Glutathione productivity from LBG was enhanced by engineering the glutathione metabolism of mannan-hydrolyzing S. cerevisiae. Expression of extracellular mannanase/mannosidase protein combined with intracellular metabolic engineering is potentially applicable to the efficient, environmentally friendly bioproduction of targeted products from mannan-based bioresources.

GH-10 and GH-11 Endo-1,4-β-xylanase enzymes from Kitasatospora sp. produce xylose and xylooligosaccharides from sugarcane bagasse with no xylose inhibition

A novel strategy for the low-cost, high-yield co-production of xylose and xylooligosaccharides together with no xylose inhibition was developed using a novel heterologous expression of XYN10Ks_480 endo-1,4-β-xylanase with a ricin-type β-trefoil type of domain and XYN11Ks_480 endo-1,4-β-xylanase with a CBM 2 superfamily from the Kitasatospora sp in an actinomycetes expression system. Xylose is the main building block for hemicellulose xylan. Our findings demonstrated high levels of expression and catalytic activity for XYN10Ks_480 during hydrolysis of the extracted xylan of bagasse, and three types of xylan-based substrates were used to produce xylose and xylooligosaccharides. However, hydrolysis by XYN11Ks_480 produced xylooligosaccharides without xylose formation. This study demonstrated how integrating sodium hypochlorite-extracted xylan and enzymatic hydrolysis could provide an alternative strategy for the generation of XOS from lignocellulosic material.

Optimization of simultaneous saccharification and fermentation in bioethanol production from sugarcane bagasse hydrolyse by Saccharomyces cerevisiae BTCC 3 using response surface methodology

The response surface method (RSM) was used to investigate the effects of bioethanol fermentation parameters, including sugarcane bagasse concentrations (0-5 % (w/v), enzyme types (cellulase and hemicellulase), enzyme concentrations (0-1 FPU/g biomass), medium pH, and incubation period. Bioethanol production was conducted by simultaneous saccharification and fermentation (SSF) of sugarcane bagasse hydrolyse using Saccharomyces cerevisiae BTCC 3. The experiment results showed that the maximum bioethanol production by using the SSF method was the same as RSM prediction. The optimum conditions for bioethanol production were 5% (w/v) of sugarcane bagasse, a mixture of cellulase and hemicellulase at 1 FPU/g of sugarcane bagasse for each enzyme, pH medium 6.0, and fermentation period 72 hours. The bioethanol yield of 2.43 g/L was obtained under these conditions.

Xylanase and feruloyl esterase from actinomycetes cultures could enhance sugarcane bagasse hydrolysis in the production of fermentable sugars

Abstract The addition of enzymes that are capable of degrading hemicellulose has a potential to reduce the need for commercial enzymes during biomass hydrolysis in the production of fermentable sugars. In this study, a high xylanase producing actinomycete strain (Kitasatospora sp. ID06-480) and the first ethyl ferulate producing actinomycete strain (Nonomuraea sp. ID06-094) were selected from 797 rare actinomycetes, respectively, which were isolated in Indonesia. The addition (30%, v/v) of a crude enzyme supernatant from the selected strains in sugarcane bagasse hydrolysis with low-level loading (1 FPU/g-biomass) of Cellic® CTec2 enhanced both the released amount of glucose and reducing sugars. When the reaction with Ctec2 was combined with crude enzymes containing either xylanase or feruloyl esterase, high conversion yield of glucose from cellulose at 60.5% could be achieved after 72 h-saccharification. Xylanase and feruloyl esterase produced by actinomycetes that cotanined in culture supernatants could enhance sugarcane bagasse hydrolysis with low loading commercial CTec2 enzyme.

Repeated ethanol fermentation from membrane-concentrated sweet sorghum juice using the flocculating yeast Saccharomyces cerevisiae F118 strain

The aim of this study was to construct a cost-effective method for repeated bioethanol production using membrane (ultrafiltration permeation and nanofiltration concentration)-concentrated sweet sorghum juice by using flocculent Saccharomyces cerevisiae F118 strain. With low initial dry cell concentrations at around 0.28-0.35 g L-1, the S. cerevisiae F118 strain provided an ethanol titer of 86.19 ± 1.15 g L-1 (theoretical ethanol yield of 70.77%), which was higher than the non-flocculent S. cerevisiae BY4741 strain at 33.92 ± 0.99 g L-1 after 24 h fermentation. This result was correlated with higher gene expressions of the sucrose-hydrolysing enzyme invertase, sugar phosphorylation, and pyruvate-to-ethanol pathways in the F118 strain compared with the BY4741 strain. Sequential fed-batch fermentation was conducted, and the F118 strain was easily separated from the fermentation broth via the formation of flocs and sediment. After the 5th cycle of fermentation with the F118 strain, the ethanol concentration reached 100.37 g L-1.

Enzymatic hydrolysis of lignocellulosic biomass by Kitasatospora sp. to produce xylo-oligosaccharides (XOS)

The optimizations of enzymatic hydrolysis to produce of xylo-oligosaccharides (XOs) from three different lignocellulosic biomasses were investigated. Sugarcane bagasse, oil palm empty fruit bunch, and rice straw contain rich hemicelluloses especially hetero-xylan which can be hydrolyzes by endo-xylanase enzyme. Enzymatic hydrolysis of sugarcane bagasse by endo-xylanase from Kitasatospora sp. was optimum at temperature hydrolysis 30 °C using 16 U of enzyme concentrations and 4 % substrate concentrations, while oil palm empty fruit bunchwas optimum at temperature hydrolysis 30 °C using 16 U of enzyme concentrations and 5 % substrate concentrations, and rice straw was optimum at 40 °C temperature hydrolysis using 16 U of enzyme concentrations and 4 % substrate concentrations. The hydrolysis products were analyzed by TLC and HPLC. The main product hydrolysis for sugarcane bagasse, oil palm empty fruit bunch and rice straw are xylobiose.The optimizations of enzymatic hydrolysis to produce of xylo-oligosaccharides (XOs) from three different lignocellulosic biomasses were investigated. Sugarcane bagasse, oil palm empty fruit bunch, and rice straw contain rich hemicelluloses especially hetero-xylan which can be hydrolyzes by endo-xylanase enzyme. Enzymatic hydrolysis of sugarcane bagasse by endo-xylanase from Kitasatospora sp. was optimum at temperature hydrolysis 30 °C using 16 U of enzyme concentrations and 4 % substrate concentrations, while oil palm empty fruit bunchwas optimum at temperature hydrolysis 30 °C using 16 U of enzyme concentrations and 5 % substrate concentrations, and rice straw was optimum at 40 °C temperature hydrolysis using 16 U of enzyme concentrations and 4 % substrate concentrations. The hydrolysis products were analyzed by TLC and HPLC. The main product hydrolysis for sugarcane bagasse, oil palm empty fruit bunch and rice straw are xylobiose.

Synthesis and characterization of L-lactide and polylactic acid (PLA) from L-lactic acid for biomedical applications

Lactide is the monomer for the polymer polylactic acid (PLA) from lactic acid through polycondensation and depolymerization process. The properties of PLA strongly depend on the quality of the lactide monomer from which it is synthesized. Optical purity of lactide produced in depolymerization process confirmed to be L-lactide. The highest yield of crude lactide was 38.5% at temperature 210 °C with average molecular weight (Mn) of oligomer was 2389. Ring opening polymerization of lactide using Candida rugosa lipase as biocatalyst to PLLA synthesis has been achieved to generate useful biomedical materials free from heavy metal.