Isolation, Cellulase Activity Test and Molecular Identification of Selected Cellulolytic Bacteria Indigenous Rice Bran

Rice bran is the waste product of rice milling which is abundant in Indonesia, it can be used as a raw material for the manufacture of bioethanol by fermentation. Before being fermented, rice bran must be hydrolyzed into glucose by biomass degrading. This study was aimed to isolate indigenous cellulolytic bacteria from rice bran as producer of cellulolytic enzymes and resulted in 22 bacterial isolates that demonstrated cellulolytic activity being identified. Among them, BE 8 and BE 14 isolates showed the highest endoglucanase activity at 2.16 and 1.31 U/mL respectively. Identification of the 16S rDNA showed that BE 8 belongs to Bacillus subtilis and BE 14 in Bacillus cereus.

Download Full-text

KINETIKA REAKSI ENZIMATIS EKSTRAK KASAR ENZIM SELULASE BAKTERI SELULOLITIK HASIL ISOLASI DARI BEKATUL

ALCHEMY ◽

10.18860/al.v0i0.2297 ◽

2013 ◽

Cited By ~ 2

Author(s):

Dyah Ayu Saropah ◽

Akyunul Jannah ◽

Anik Maunatin

Keyword(s):

Rice Bran ◽

Enzymatic Reaction ◽

Milling Process ◽

Cellulolytic Enzyme ◽

Cellulose Content ◽

Optimum Conditions ◽

Cellulolytic Bacteria ◽

Cellulase Enzymes ◽

Rice Milling

Bran rice is a by-product of rice into rice milling process, the cellulose content of 40-60%, so the potential as a carbon source for the growth of microorganisms such as bacteria to produce enzymes particularly cellulolytic bacteria. The purpose of the study was to determine the diversity of the characters from the cellulolytic bacterial isolates and optimum conditions enzyme (cellulase enzymes rough) so that they can hydrolyze the cellulose to glucose with either rice bran. The characterization includes the determination of pH, temperature and time of optimum crude extract of bacterial cellulolytic enzyme cellulase, determination of Vmax and Km and molecular mass determination of cellulase.Research methods include making media, regeneration of isolates, bacterial growth curve manufacturing, production of cellulase enzymes from bacterial cellulolytic rough at the optimum conditions, the kinetics of enzymatic reaction: substrate concentration factor of the reaction rate (with variation of the concentration of 0.50%, 0.75%, 1 , 00%, 1.25% and 1.50% (w / v)) followed by calculating the Vmax and Km.The results showed that the enzyme cellulase of cellulolytic bacteria isolated from rice bran result that has optimum conditions at pH 7.5, temperature 50 ° C, 40 min incubation time to produce Vmax 0.0086 units / mL and Km 1.694%.

Download Full-text

Cellulosomes localise on the surface of membrane vesicles from the cellulolytic bacterium Clostridium thermocellum

FEMS Microbiology Letters ◽

10.1093/femsle/fnz145 ◽

2019 ◽

Vol 366 (12) ◽

Cited By ~ 2

Author(s):

Shunsuke Ichikawa ◽

Satoru Ogawa ◽

Ayami Nishida ◽

Yuzuki Kobayashi ◽

Toshihito Kurosawa ◽

...

Keyword(s):

Clostridium Thermocellum ◽

Cell Communication ◽

Membrane Vesicles ◽

Cellulolytic Enzymes ◽

Crystalline Cellulose ◽

Cellulolytic Bacterium ◽

Cellulolytic Activity ◽

Cellulolytic Bacteria ◽

Degradation Activity ◽

Enzyme Complexes

ABSTRACTMembrane vesicles released from bacteria contribute to cell–cell communication by carrying various cargos such as proteins, nucleic acids and signaling molecules. Cellulolytic bacteria have been isolated from many environments, yet the function of membrane vesicles for cellulolytic ability has been rarely described. Here, we show that a Gram-positive cellulolytic bacterium Clostridium thermocellum released membrane vesicles, each approximately 50–300 nm in diameter, into the broth. The observations with immunoelectron microscopy also revealed that cellulosomes, which are carbohydrate-active enzyme complexes that give C. thermocellum high cellulolytic activity, localized on the surface of the membrane vesicles. The membrane vesicles collected by ultracentrifugation maintained the cellulolytic activity. Supplementation with the biosurfactant surfactin or sonication treatment disrupted the membrane vesicles in the exoproteome of C. thermocellum and significantly decreased the degradation activity of the exoproteome for microcrystalline cellulose. However, these did not affect the degradation activity for soluble carboxymethyl cellulose. These results suggest a novel function of membrane vesicles: C. thermocellum releases cellulolytic enzymes on the surface of membrane vesicles to enhance the cellulolytic activity of C. thermocellum for crystalline cellulose.

Download Full-text

Ultrastructural Localization of Cellulase in Diplodia Maydis and in D. Maydis-Infected Com Stalk Tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079711 ◽

1977 ◽

Vol 35 ◽

pp. 454-455

Author(s):

Judith A. Murphy ◽

Mary R. Thompson ◽

A.J. Pappelis

Keyword(s):

Culture Medium ◽

Cupric Oxide ◽

Cellulase Activity ◽

Growth Period ◽

Ultrastructural Localization ◽

Ultrastructural Level ◽

Low Ph ◽

Cellulolytic Enzymes ◽

Cellulolytic Activity ◽

X Ray

BeMiller et.al.(l) found that D. maydis did not have the solubilizing enzyme C1. They reported that D- maydis exhibited cellulolytic activity constitutively, and hypothesized that the cellulolytic enzymes were attached to fungal hyphal surfaces because they found cellulase released to the culture medium only after the growth period, when available cellulose had been used up.The purpose of this study was to determine the location of cellulolytic enzymes (EC 3.2.1.4; beta-1,4-glucan glucanohydrolase) in D. maydis and D. maydis-infected corn tissue at the ultrastructural level.Cellulase activity produces glucose as an end product which will reduce cupric oxide and can be visualized with an EM because it is electron dense and the Cu component can be verified with x-ray analysis(Figs.l,2). After thorough washing, samples fixed in aldehydes are incubated in a substrate mixture at a low pH. The enzyme is activated and reducing sugar is released. The sample is then reacted with Benedict's solution at a high temperature, allowing CuO crystals to be deposited at the site of reaction.

Download Full-text

Location of cellulolytic enzyme activity in the marine fungus Trichocladium achrasporum

Canadian Journal of Microbiology ◽

10.1139/m85-028 ◽

1985 ◽

Vol 31 (2) ◽

pp. 145-148 ◽

Cited By ~ 8

Author(s):

Malcolm J. MacDonald ◽

Donna L. Hartley ◽

Marilyn K. Speedie

Keyword(s):

Cellulase Activity ◽

Marine Fungus ◽

Particulate Fraction ◽

Cellulolytic Enzymes ◽

Crystalline Cellulose ◽

Cellulolytic Enzyme ◽

Active Growth ◽

Endoglucanase Activity ◽

Produce Cell ◽

Soluble Cell

Cellulase activity in Trichocladium achrasporum was demonstrated by its ability to produce cell-associated and extracellular cellulolytic enzymes when grown on a crystalline cellulose substrate. In addition, azure dye was solubilized from dyed crystalline cellulose, appearing in the growth medium during the phase of cell lysis. Exoglucanase activity was highest in the culture filtrate, with slight activity in the cell fractions, while endoglucanase was associated only with the mycelium. No desorbable exoglucanase nor endoglucanase activity could be released by sonication of residual cellulose particles removed from actively growing cultures. β-Glucosidase activity was located only in the cell-associated fractions during active growth. All enzymes had optimal activity at 50 °C; in the particulate fraction β-glucosidase exhibited a second optimum at 30 °C. In the filtrate, soluble intracellular and particulate fractions optimal exoglucanase activity occurred at pH 6.4, 7.0, and 5.8, respectively. Endoglucanase activity was optimal at pH 5.8 in the soluble cell fraction, and at pH 5.4 in the particulate fraction.

Download Full-text

BIODIVERSITY AND ENZYME ACTIVITY OF INDIGENOUS CELLULOLYTIC AND AMYLOLYTIC BACTERIAS IN DECAYED MANGROVE STEM WASTE PRODUCT AT WAAI SEASHORE, AMBON ISLAND

KnE Life Sciences ◽

10.18502/kls.v2i1.188 ◽

2015 ◽

Vol 2 (1) ◽

pp. 433

Author(s):

Utami Sri Hastuti ◽

Kristin Sangur ◽

Henny Nurul Khasanah

Keyword(s):

Bacillus Cereus ◽

Amylase Activity ◽

Micrococcus Luteus ◽

Waste Product ◽

Cellulase Activity ◽

Chemistry Laboratory ◽

State University ◽

Microbiology Laboratory ◽

Cellulolytic Bacteria ◽

Nutrient Agar Medium

Mangrove (Sonneratia spp.) could be found at Waai seashore, Ambon Island. The remainder of the mangrove stem will be decayed and become the waste product. Some indigenous bacteria species that live in the decayed mangrove stem waste product have cellulolytic and amylolytic characters. The objectives of this research were to: 1) identify and determine the cellulolytic bacteria species; 2) identify and determine the amylolytic bacteria spesies; 3) determine the cellulolytic bacteria species that have the highest cellulase activity; 4) determine the amylolytic bacteria species that have the highest amylase activity. This research was conduct at the Microbiology Laboratory-Biology Departement-State University of Malang, Microbiology Laboratory-Faculty of Medicine-Brawijaya University, Chemistry Laboratory-Muhammadiyah Malang University. Twenty five grams sample were ground and diluted in 225 ml nutrient broth to get 10-1 suspension. Then the suspension were diluted gradually until 10-10. The suspension were inoculated on nutrient agar medium 0.1 ml each, and incubated in 370C during 1x24 hours. Each bacteria colonies were isolated and determine to know which one were the cellulolytic and the amylolytic bacteria. The bacteria isolates were identify. Afterward the cellulase activity as well as the amylase activity were analyzed. The research results shows that: 1) there are 4 cellulolytic bacteria spesies, i.e: Micrococcus luteus, Bacillus pumilus, Planococcus citreus, and Bacillus cereus; 2) there are 4 amylolytic bacteria species, i.e: Bacillus firmus, Nitrobacter sp., Bacillus mycoides, and Pseudomonas aeruginosa; 3) Bacillus cereus has the highest cellulase activity; 4) Nitrobacter sp. has the highest amylase activity. Keywords: biodiversity, cellulolytic bacteria, amylolytic bacteria, decayed mangrove stem waste product.

Download Full-text

Produksi Crude Selulase dari Limbah Kayu Mahoni Menggunakan Phanerochaete chrysosporium

Jurnal Teknik Kimia dan Lingkungan ◽

10.33795/jtkl.v3i1.86 ◽

2019 ◽

Vol 3 (1) ◽

pp. 39 ◽

Cited By ~ 1

Author(s):

Sri Rulianah ◽

Christyfani Sindhuwati ◽

Prayitno Prayitno

Keyword(s):

Phanerochaete Chrysosporium ◽

Incubation Time ◽

Economic Value ◽

Cellulase Activity ◽

Cellulase Production ◽

Operating Conditions ◽

Size Reduction ◽

Raw Material ◽

Fermentation Time ◽

Activity Test

Limbah kayu mahoni dapat dikategorikan sebagai limbah lignoselulosa yang dapat dimanfaatkan sebagai bahan baku pembuatan biofuel, seperti bioetanol. Selulosa pada limbah kayu mahoni dapat dimanfaatkan sebagai bahan baku produksi crude selulase dengan bantuan kapang Phanerochaete chrysosporium. Crude selulase yang dihasilkan memiliki nilai ekonomis yang tinggi dan dapat dimanfaatkan dalam berbagai bidang. Tujuan dari penelitian ini adalah untuk mengetahui pengaruh waktu fermentasi dan penambahan konsentrasi serbuk kayu mahoni terhadap aktivitas crude selulase, dan untuk mengetahui kondisi operasi terbaik sehingga diperoleh crude selulase dengan aktivitas yang tertinggi. Produksi crude selulase dari limbah kayu mahoni melalui beberapa tahapan yaitu, size reduction, peremajaan dan pembuatan inokulum kapang Phanerocheate chrysosporium, produksi crude selulase dan uji aktivitas selulase dengan metode DNS. Variabel berubah pada penelitian ini adalah % penambahan serbuk kayu mahoni pada media pembuatan crude selulase yaitu 5%, 6% dan 7%, dan waktu inkubasi pembuatan crude selulase yaitu 9, 11, 13, 15 dan 17 hari. Hasil penelitian menunjukkan, semakin lama waktu fermentasi, dan semakin tinggi jumlah penambahan serbuk kayu mahoni, maka aktivitas selulase yang dihasilkan semakin tinggi. Kondisi operasi terbaik diperoleh pada waktu inkubasi selama 17 hari, dan jumlah penambahan serbuk kayu mahoni 7% , diperoleh aktivitas crude selulase sebesar 39,034 U/ml.Mahogany waste can be categorized as lignocelluloses waste which can be used as raw material of biofuel such as bioethanol. Cellulose in mahogany can also be utilized as crude cellulose raw material with the help of Phanerochaete chrysosporium. Crude selulase produced has high economic value and can be utilized in many sectors. This research is aim to determine the effect of fermentation time and the addition of mahogany concentration on crude cellulase activity, and to determine the best operating conditions. Crude cellulase production from waste of mahogany through several steps, those are size reduction, rejuvenation and inoculum production of Phanerocheate chrysosporium, crude cellulase production and activity test with DNS method. The variable in this experiment was the precentage of mahogany powder added on crude cellulase production media which was 5%, 6% and 7%, and incubation time of crude cellulase production which were 9, 11, 13, 15 and 17 days. The experiment shows that the highest cellulase activity was at concentration of mahogany powder of 7% with incubation time of 17days as 39,034 U/ml.

Download Full-text

Development and Application of a PCR-Targeted Gene Disruption Method for Studying CelR Function in Thermobifida fusca

Applied and Environmental Microbiology ◽

10.1128/aem.02626-09 ◽

2010 ◽

Vol 76 (7) ◽

pp. 2098-2106 ◽

Cited By ~ 20

Author(s):

Yu Deng ◽

Stephen S. Fong

Keyword(s):

Gene Expression ◽

Genetic Engineering ◽

Cellulase Activity ◽

Deletion Strain ◽

Lag Phase ◽

Thermobifida Fusca ◽

Cellulolytic Activity ◽

Chromosomal Dna ◽

Endoglucanase Activity ◽

Disruption Method

ABSTRACT Thermobifida fusca is a high-G+C-content, thermophilic, Gram-positive soil actinobacterium with high cellulolytic activity. In T. fusca, CelR is thought to act as the primary regulator of cellulase gene expression by binding to a 14-bp inverted repeat [5′-(T)GGGAGCGCTCCC(A)] that is upstream of many known cellulase genes. Previously, the ability to study the roles and regulation of cellulase genes in T. fusca has been limited largely by a lack of established genetic engineering methods for T. fusca. In this study, we developed an efficient procedure for creating precise chromosomal gene disruptions and demonstrated this procedure by generating a celR deletion strain. The celR deletion strain was then characterized using measurements for growth behavior, cellulase activity, and gene expression. The celR deletion strain of T. fusca exhibited a severely crippled growth phenotype with a prolonged lag phase and decreased cell yields for growth on both glucose and cellobiose. While the maximum endoglucanase activity and cellulase activity were not significantly changed, the endoglucanase activity and cellulase activity per cell were highly elevated. Measurements of mRNA transcript levels in both the celR deletion strain and the wild-type strain indicated that the CelR protein potentially acts as a repressor for some genes and as an activator for other genes. Overall, we established and demonstrated a method for manipulating chromosomal DNA in T. fusca that can be used to study the cellulolytic capabilities of this organism. Components of this method may be useful in developing genetic engineering methods for other currently intractable organisms.

Download Full-text

Deactivation of Red Mud by Primary Aluminum Production Wastes

Materials Science Forum ◽

10.4028/www.scientific.net/msf.1040.109 ◽

2021 ◽

Vol 1040 ◽

pp. 109-116

Author(s):

V.Yu. Piirainen ◽

A.A. Barinkova ◽

V.N. Starovoytov ◽

V.M. Barinkov

Keyword(s):

Carbon Dioxide ◽

Red Mud ◽

Negative Impact ◽

Waste Product ◽

Primary Aluminum ◽

Raw Material ◽

Aluminum Production ◽

Environmentally Safe ◽

Primary Aluminum Production ◽

Knowledge Intensive

Current global environmental challenges and, above all, global warming associated with a change in the carbon balance in the atmosphere has led to the need for urgent and rapid search for ways to reduce greenhouse gas emissions into the atmosphere, which primarily include carbon dioxide as a by-product of human activity and technological progress. One of these ways is the creation of industries with a complete cycle of turnover of carbon dioxide. Aluminum is the most sought-after nonferrous metal in the world, but its production is not environmentally safe, so it constantly requires the development of knowledge-intensive technologies to improve the technological process of cleaning and disposal of production waste, primarily harmful emissions into the atmosphere. Another environmental problem related to aluminum production is the formation and accumulation in mud lagoon of huge amounts of so-called highly alkaline "red mud," which is a waste product of natural bauxite raw material processing into alumina - the feedstock for aluminum production. Commonly known resources and technological methods of neutralizing red mud and working with it as ore materials for further extraction of useful components are still not used because of their low productivity and cost-effectiveness. This article describes the negative impact of waste in the form of "red" mud and carbon dioxide of primary aluminum production on the environment. The results showed that thanks to carbonization of red mud using carbon dioxide, it is possible to achieve rapid curing and its compact formation for safer transportation and storage until further use. Strength tests of concrete samples filled with deactivated red mud were also carried out, which showed the prospects of using concrete with magnesia binder.

Download Full-text

An Efficient and Improved Methodology for the Screening of Industrially Valuable Xylano-Pectino-Cellulolytic Microbes

Enzyme Research ◽

10.1155/2015/725281 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 7

Author(s):

Avtar Singh ◽

Amanjot Kaur ◽

Anita Dua ◽

Ritu Mahajan

Keyword(s):

Low Cost ◽

Cost Effective ◽

Industrial Sector ◽

Agricultural Residues ◽

High Yield ◽

Cellulolytic Enzymes ◽

Cellulolytic Activity ◽

Nutrient Media ◽

First Report ◽

Primary Screening

Xylano-pectino-cellulolytic enzymes are valuable enzymes of the industrial sector. In our earlier study, we have reported a novel and cost effective methodology for the qualitative screening of cellulase-free xylano-pectinolytic microorganisms by replacing the commercial, highly expensive substrates with agricultural residues, but the microorganisms with xylanolytic, pectinolytic, cellulolytic, xylano-pectinolytic, xylano-cellulolytic, pectino-cellulolytic, and xylano-pectino-cellulolytic potential were obtained. The probability of getting the desired combination was low, so efforts were made to further improve this cost effective methodology for obtaining the high yield of the microbes capable of producing desired combination of enzymes. By inclusion of multiple enrichment steps in sequence, using only practically low cost substrates and without any nutrient media till primary screening stage, this improved novel protocol for screening gave only the desired microorganisms with xylano-pectino-cellulolytic activity. Using this rapid, efficient, cost effective, and improved methodology, microbes with required combination of enzymes can be obtained and the probability of getting the desired microorganisms is cent percent. This is the first report presenting the methodology for the isolation of xylano-pectino-cellulolytic positive microorganisms at low cost and consuming less time.

Download Full-text

Ethanol production using vegetable peels medium and the effective role of cellulolytic bacterial (Bacillus subtilis) pre-treatment

F1000Research ◽

10.12688/f1000research.13952.1 ◽

2018 ◽

Vol 7 ◽

pp. 271

Author(s):

Salman Khan Promon ◽

Wasif Kamal ◽

Shafkat Shamim Rahman ◽

M. Mahboob Hossain ◽

Naiyyum Choudhury

Keyword(s):

Bacillus Subtilis ◽

Ethanol Production ◽

Energy Demand ◽

Biochemical Characterization ◽

Raw Materials ◽

Maximum Yield ◽

Clean Energy ◽

Raw Material ◽

Cellulolytic Bacteria ◽

Alcohol Production

Background: The requirement of an alternative clean energy source is increasing with the elevating energy demand of modern age. Bioethanol is considered as an excellent candidate to satiate this demand.Methods:Yeast isolates were used for the production of bioethanol using cellulosic vegetable wastes as substrate. Efficient bioconversion of lignocellulosic biomass into ethanol was achieved by the action of cellulolytic bacteria (Bacillus subtilis). After proper isolation, identification and characterization of stress tolerances (thermo-, ethanol-, pH-, osmo- & sugar tolerance), optimization of physiochemical parameters for ethanol production by the yeast isolates was assessed. Very inexpensive and easily available raw materials (vegetable peels) were used as fermentation media. Fermentation was optimized with respect to temperature, reducing sugar concentration and pH.Results:It was observed that temperatures of 30°C and pH 6.0 were optimum for fermentation with a maximum yield of ethanol. The results indicated an overall increase in yields upon the pretreatment ofBacillus subtilis; maximum ethanol percentages for isolate SC1 obtained after 48-hour incubation under pretreated substrate was 14.17% in contrast to untreated media which yielded 6.21% after the same period. Isolate with the highest ethanol production capability was identified as members of the ethanol-producingSaccharomycesspecies after stress tolerance studies and biochemical characterization using Analytical Profile Index (API) ® 20C AUX and nitrate broth test. Introduction ofBacillus subtilisincreased the alcohol production rate from the fermentation of cellulosic materials.Conclusions:The study suggested that the kitchen waste can serve as an excellent raw material in ethanol fermentation.

Download Full-text