Comparative Genome Analysis Reveals Cyanidiococcus gen. nov., A New Extremophilic Red Algal Genus Sister to Cyanidioschyzon (Cyanidioschyzonaceae,Rhodophyta)

The taxonomic placement of strains belonging to the extremophilic red alga Galdieria maxima has been controversial due to the inconsistent phylogenetic position inferred from molecular phylogenetic analyses. Galdieria maxima nom. inval. was classified in this genus based on morphology and molecular data in the early work, but some subsequent molecular phylogenetic analyses have inferred strains of G. maxima to be closely related to the genus Cyanidioschyzon. To address this controversy, an isolated strain identified as G. maxima using the rbcL gene sequence as the genetic barcode was examined using a comprehensive analysis across morphological, physiological, and genomic traits. Herein are reported the chloroplast-, mitochondrion-, and chromosome-level nuclear genome assemblies. Comparative analysis of orthologous gene clusters and genome arrangements suggested that the genome structure of this strain was more similar to that of the generitype of Cyanidioschyzon, C. merolae than to the generitype of Galdieria, G. sulphuraria. While the ability to uptake various forms of organic carbon for growth is an important physiological trait of Galdieria, this strain was identified as an ecologically obligate photoautotroph (i.e., the inability to utilize the natural concentrations of organic carbons) and lacked various gene models predicted as sugar transporters. Based on the genomic, morphological, and physiological traits, we propose this strain to be a new genus and species, Cyanidiococcus yangmingshanensis. Re-evaluation of the 18S rRNA and rbcL gene sequences of the authentic strain of G. maxima, IPPAS-P507, with those of C. yangmingshanensis suggests that the rbcL sequences of “G. maxima” deposited in GenBank correspond to misidentified isolates.     

The occurrence of ice nucleation activity in pathogenic bacterial species

We tested the nucleation activity (INA) of 122 strains of plant pathogenic bacteria (12 varieties and 15 subspecies) stored in collection of the University of Göttingen (GSPB). The strains are isolates from diverse host plants and different geographic regions. One-hundred and seven isolates belong to the Pseudomonads, nine to the genus Erwinia and six to the Xanthomonas. The INA was analysed by ?3°, ?5°, ?7° and ?9°C. The observed value of INA cells ranged from non-detectable to a maximum concentration of ice nuclei in a range from ?7.85 at ?5°C to ?2.63 at ?3°C in 1.82 × 10³ cfu to 3.3 × 10³ cfu per ml. The data indicated that 71 (58.2%) of the 122 strains had INA cells, and 51 (41.8%) were inactive. The highest amount of strains with INA cells we found in Pseudomonads (69). In comparison only one strain was active at Erwinia and at Xanthomonas, 46 strains were isolated from the genus Phaseolus vulgaris and 6 from the genus Beta vulgaris. The other isolates with ice active cells belonged to the 13 other plant species. The 51 inactive pathovars were isolated from 21 different culture plants. The pathogens under test were isolated in 16 different countries, mainly in Germany and USA.     

Establishment of endolithic populations of extremophilic Cyanidiales (Rhodophyta)

Background  

Cyanidiales are unicellular extremophilic red algae that inhabit acidic and high temperature sites around hot springs and have also adapted to life in endolithic and interlithic habitats. Comparative genomic analysis of Cyanidioschyzon merolae and Galdieria sulphuraria predicts that the latter may be more broadly distributed in extreme environments because its genome contains membrane transporters involved in the uptake of reduced carbon compounds that are absent from C. merolae. Analysis of an endolithic site in the Phlegrean Fields near Naples, Italy is consistent with this prediction showing this population to be comprised solely of the newly described lineage Galdieria -B and C. merolae to be limited to humid habitats. Here, we conducted an environmental PCR survey of another extreme environment in Tuscany, Italy and contrasted Cyanidiales population structure at endolithic and interlithic habitats in Naples and Tuscany.     

Molecular variation in Galdieria sulphuraria (Galdieri) Merola and its bearing on taxonomy

Cyanidium caldarium, Cyanidioschyzon merolae and Galdieria sulphuraria are three unicellular algae characteristic, of acid thermal environments. Recently, on the basis of morphological characters, three new species of Galdieria (G. partita, G. daedala, G. maxima ) isolated from acid-thermal springs in Russia have been instituted. A selected region of rbcL and the sequence of the intergenic spacer between the rbcL and rbcS have been amplified and sequenced from different Galdieria species and strains, in order to define molecular relationship among these interesting algae. The obtained cladogram shows that Cyanidium caldarium and Cyanidioschyzon merolae form a sister group which, in turn, is in a sister group relationship with Galdieria. This last genus is divided in two clades, one of which includes G. sulphuraria accessions from Naples (Italy), California, and Yellowstone and the other one includes G. sulphuraria accessions from Java (Indonesia) and from the Russian species. These results support the status of the genus Galdieria and suggest that G. daedala, G. maxima and G. partita are three very similar strains of G. sulphuraria; the rbcL variation within Galdieria accessions has a pattern which is broadly connected to the geographial distribution. The data obtained from the intergenic rbcL-rbcS spacer partly confirm those from the rbcL analysis.     

Physiological and genomic characterization of a new ‘Candidatus Nitrotoga’ isolate

Oxidation of nitrite to nitrate is an important process in the global nitrogen cycle. Recent molecular biology-based studies have revealed that the widespread nitrite-oxidizing bacteria (NOB) belonging to the genus ‘Candidatus Nitrotoga’ may be highly important for the environment. However, the insufficient availability of pure Nitrotoga cultures has limited our understanding of their physiological and genomic characteristics. Here, we isolated the ‘Ca. Nitrotoga’ sp. strain AM1P, from a previously enriched Nitrotoga culture, using an improved isolation strategy. Although ‘Ca. Nitrotoga’ have been recognized as cold-adapted NOB, the strain AM1P had a slightly higher optimum growth temperature at 23°C. Strain AM1P showed a pH optimum of 8.3 and was not inhibited even at high nitrite concentrations (20 mM). We obtained the complete genome of the strain and compared the genome profile to five previously sequenced ‘Ca. Nitrotoga’ strains. Comparative genomics suggested that lactate dehydrogenase may be only encoded in the strain AM1P and closely related genomes. While the growth yield of AM1P did not change, we observed faster growth in the presence of lactate in comparison to purely chemolithoautotrophic growth. The characterization of the new strain AM1P sheds light on the physiological adaptation of this environmentally important, but understudied genus ‘Ca. Nitrotoga’.     

Strain diversity of plant-associated Lactiplantibacillus plantarum

Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) is a lactic acid bacteria species found on plants that is essential for many plant food fermentations. In this study, we investigated the intraspecific phenotypic and genetic diversity of 13 L. plantarum strains isolated from different plant foods, including fermented olives and tomatoes, cactus fruit, teff injera, wheat boza and wheat sourdough starter. We found that strains from the same or similar plant food types frequently exhibited similar carbohydrate metabolism and stress tolerance responses. The isolates from acidic, brine-containing ferments (olives and tomatoes) were more resistant to MRS adjusted to pH 3.5 or containing 4% w/v NaCl, than those recovered from grain fermentations. Strains from fermented olives grew robustly on raffinose as the sole carbon source and were better able to grow in the presence of ethanol (8% v/v or sequential exposure of 8% (v/v) and then 12% (v/v) ethanol) than most isolates from other plant types and the reference strain NCIMB8826R. Cell free culture supernatants from the olive-associated strains were also more effective at inhibiting growth of an olive spoilage strain of Saccharomyces cerevisiae. Multi-locus sequence typing and comparative genomics indicated that isolates from the same source tended to be genetically related. However, despite these similarities, other traits were highly variable between strains from the same plant source, including the capacity for biofilm formation and survival at pH 2 or 50°C. Genomic comparisons were unable to resolve strain differences, with the exception of the most phenotypically impaired and robust isolates, highlighting the importance of utilizing phenotypic studies to investigate differences between strains of L. plantarum. The findings show that L. plantarum is adapted for growth on specific plants or plant food types, but that intraspecific variation may be important for ecological fitness and strain coexistence within individual habitats.     

凡口铅锌矿酸性底泥可培养微生物资源的探索分离

通过改良优化多种传统分离培养基,对凡口铅锌尾矿的酸性底泥可培养微生物资源进行分离,并结合多种分子标记基因对分离资源进行系统发育地位分析。结果表明,凡口铅锌尾底泥中分离获得细菌41株,分属于放线菌门(Actinomycetes)、变形菌门(Proteobacteria)和厚壁菌门(Firmicutes)的11目,18科,18属;真菌8株,分属于2门,5目,5科,5属;藻类1株,属于Galdieria属的红藻。改良ISP 2和改良9号培养基的分离效果较好,分离获得的微生物多样性较为良好。研究结果为矿山酸性废水微生物多样性研究提供参考,为酸性废水的修复及生态治理研究提供物质基础。     

Obligately and facultatively autotrophic,sulfur- and hydrogen-oxidizing thermophilic bacteria isolated from hot composts

A variety of autotrophic, sulfur- and hydrogen-oxidizing thermophilic bacteria were isolated from thermogenic composts at temperatures of 60–80° C. All were penicillin G sensitive, which proves that they belong to the Bacteria domain. The obligately autotrophic, non-spore-forming strains were gram-negative rods growing at 60–80°C, with an optimum at 70–75°C, but only under microaerophilic conditions (5 kPa oxygen). These strains had similar DNA G+C content (34.7–37.6 mol%) and showed a high DNA:DNA homology (70–87%) with Hydrogenobacter strains isolated from geothermal areas. The facultatively autotrophic strains isolated from hot composts were gram-variable rods that formed spherical and terminal endospores, except for one strain. The strains grew at 55–75° C, with an optimum at 65–70° C. These bacteria were able to grow heterotrophically, or autotrophically with hydrogen; however, they oxidized thiosulfate under mixotrophic growth conditions (e.g. pyruvate or hydrogen plus thiosulfate). These strains had similar DNA G+C content (60–64 mol%) to and high DNA:DNA homology (> 75%) with the reference strain of Bacillus schlegelii. This is the first report of thermogenic composts as habitats of thermophilic sulfur- and hydrogen-oxidizing bacteria, which to date have been known only from geothermal manifestations. This contrasts with the generally held belief that thermogenic composts at temperatures above 60° C support only a very low diversity of obligatory heterotrophic thermophiles related to Bacillus stearothermophilus. Received: 20 July 1995 / Accepted: 25 September 1995     

Methanogenium marinum sp. nov., a H2-using methanogen from Skan Bay,Alaska, and kinetics of H2 utilization

A methanogen, strain AK-1, was isolated from permanently cold marine sediments, 38- to 45-cm below the sediment surface at Skan Bay, Alaska. The cells were highly irregular, nonmotile coccoids (diameter, 1 to 1.2 μm), occurring singly. Cells grew by reducing CO₂ with H₂ or formate as electron donor. Growth on formate was much slower than that on H₂. Acetate, methanol, ethanol, 1- or 2-propanol, 1- or 2-butanol and trimethylamine were not catabolized. The cells required acetate, thiamine, riboflavin, a high concentration of vitamin B₁₂, and peptones for growth; yeast extract stimulated growth but was not required. The cells grew fastest at 25 °C (range 5 °C to 25 °C), at a pH of 6.0 – 6.6 (growth range, pH 5.5 – 7.5), and at a salinity of 0.25 – 1.25 M Na⁺. Cells of this and other H₂-using methanogens from saline environments metabolized H₂ to a very low threshold pressure (less than 1 Pa) that was dependent on the methane partial pressure. We propose that the threshold pressure may be limited by the energetics of catabolism. The sequence of the 16S rDNA gene of strain AK-1 was most similar (98%) to the sequences of Methanogenium cariaci JR-1 and Methanogenium frigidum Ace-2. DNA–DNA hybridization between strain AK-1 and these two strains showed only 34.9% similarity to strain JR-1 and 56.5% similarity to strain Ace-2. These analyses indicated strain AK-1 should be classified as a new species within the genus Methanogenium. Phenotypic differences between strain AK-1 and these strains (including growth temperature, salinity range, pH range, and nutrient requirements) support this. Therefore, a new species, Methanogenium marinum, is proposed with strain AK-1 as type strain. This revised version was published online in August 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">Microbulbifer arenaceous</Emphasis> sp. nov., a New Endolithic Bacterium Isolated from the Inside of Red Sandstone

An endolithic bacterium, strain RSBr-1, was isolated from the inside of a piece of red sandstone from coastal areas of Scotland. RSBr-1 was Gram negative, oxidase and catalase positive, and cells were non-motile rods. Sodium was required for growth. The optimum sodium chloride concentration and pH for growth were 4% and pH 8.0, respectively. Eumelanin was produced in marine broth and in BY medium. RSBr-1 hydrolyzes chitin, esculin, gelatin, and starch, but not agar. Nitrate reduction is positive. Taxonomic characterization of this strain indicated that it belongs to the genus Microbulbifer. The difference between the aligned 16S rDNA sequences of RSBr-1 and the closest relative, M. elongata, is greater than the difference between the 16S rDNA sequences of M. hydrolyticus and M. elongata. On the basis of the phenotypic and genotypic comparison of this isolate with the other strains, RSBr-1 is proposed as a new species, Microbulbifer arenaceous, with type strain RSBr-1.     

<Emphasis Type="Italic">Rhizobium sphaerophysae</Emphasis> sp. nov., a novel species isolated from root nodules of <Emphasis Type="Italic">Sphaerophysa salsula</Emphasis> in China

Four gram-negative, aerobic, motile, non-spore, forming rods with a wide pH and temperature range for growth (pH 7.0–11.0, optimum pH 8.0; 20–45°C, optimum 28°C) strains were isolated from root nodules of Sphaerophysa salsula and characterized by means of a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the four strains formed a new lineage related to the genus Rhizobium and the sequence similarities between the isolate and the most related type strain Rhizobium giardinii was 96.5%. These strains also formed a distinctive group from the reference strains for defined Rhizobium species based on housekeeping gene sequences (atpD and recA), BOX-PCR fingerprinting, phenotypic features and symbiotic properties. The representative strain CCNWGS0238^T has DNA-DNA relatedness of less than 33.4% with the most closely related species R. giardinii. It is therefore proposed as a new species, Rhizobium sphaerophysae sp. nov., with isolate CCNWGS0238^T (=ACCC17498^T = HAMBI3074^T) as the type strain.     

Psychrotrophic amylolytic bacteria from deep sea sediment of Prydz Bay, Antarctic: diversity and characterization of amylases

Seventeen psychrotrophic bacteria with cold-adaptive amylolytic, lipolytic or proteolytic activity were isolated from deep sea sediment of Prydz Bay, Antarctic. They were affiliated with γ-Proteobacteria (12 strains) and gram-positive bacteria (5 strains) as determined by 16S rDNA sequencing. The amylase-producing strains belonged to genus Pseudomonas, Rhodococcus, and Nocardiopsis. Two Pseudomonas strains, 7193 and 7197, which showed highest amylolytic activity were chosen for further study. The optimal temperatures for their growth and amylase-producing were between 15 and 20°C. Both of the purified amylases showed highest activity at 40°C and pH 9.0, and retained 50% activity at 5°C. The SDS-PAGE and zymogram activity staining showed that the molecular mass of strain 7193 and 7197 amylases were about 60 and 50 kDa respectively. The Pseudomonas sp. 7193 amylase hydrolyzed soluble starch into glucose, maltose, maltotriose, and maltotetraose, indicating that it had both activities of α-amylase and glucoamylase. The product hydrolyzed by Pseudomonas sp. 7197 amylase was meltotetraose.     

Iron Demand by Thermophilic and Mesophilic Bacteria Isolated from an Antarctic Geothermal Soil

The thermophilic bacterial strain MP4 assigned to a new species, likely of the genus Alicyclobacillus, was isolated from geothermal soils on the NW slope of Mount Melbourne, Antarctica. These soils have high iron concentrations and the strain MP4 requires iron additions for growth. Four mesophilic bacterial strains Paenibacillus validus MP5, MP8, and MP10, and P. apiarius MP7, isolated from the same site, need iron supply for growth depending on the medium. Growth temperature of thermophilic strain ranges from 42 to 70 °C, and that one of mesophiles from 25 to 44 °C. Thermophilic and mesophilic strains shared microenvironments with temperature of 42–44 °C and showed optima of pH values ranging from 5.5 to 6.0. The thermophilic strain MP4 reached values of 10⁶ CFU ml⁻¹ in aqueous soil extract from the NW slope of Mt. Melbourne, and 10⁵ CFU ml⁻¹ in water extracts from other geothermal Antarctic areas (Mt. Rittmann and Cryptogam Ridge). Growth of thermophilic bacteria in aqueous extracts of the NW slope of Mount Melbourne soils caused a reduction of 50% of soluble iron content, which was recovered in bacterial biomass. These results suggest a possible involvement of the thermophilic strain MP4 in iron bioavailability in these geothermal soils.     

Genetic variability and taxonomic revision of the genus Auxenochlorella (Shihira et Krauss) Kalina et Puncocharova (Trebouxiophyceae,Chlorophyta)

The monotypic genus Auxenochlorella with its type species A. protothecoides is so far only known from specific habitats such as the sap of several tree species. Several varieties were described according to physiological performances in culture on different organic substrates. However, two strains designated as Auxenochlorella were isolated from other habitats (an endosymbiont of Hydra viridis and an aquatic strain from an acidic volcano stream). We studied those isolates and compared them with six strains of Auxenochlorella belonging to different varieties. The integrative approach used in this study revealed that all strains showed similar morphology but differed in their SSU and ITS rDNA sequences. The Hydra endosymbiont formed a sister taxon to A. protothecoides, which included the varieties protothecoides, galactophila, and communis. The variety acidicola is not closely related to Auxenochlorella and represented its own lineage within the Trebouxiophyceae. In view of these results, we propose a new species of Auxenochlorella, A. symbiontica, for the Hydra symbiont, and a new genus Pumiliosphaera, with its type species, P. acidophila, for acidophilic strain. These results are supported by several compensatory base changes in the conserved region of ITS‐2 and ITS‐2 DNA barcodes.     

Thermococcus peptonophilus sp. nov., a fast-growing,extremely thermophilic archaebacterium isolated from deep-sea hydrothermal vents

Two extremely thermophilic archaebacteria, strains OG-1 and SM-2, were isolated from newly discovered deep-sea hydrothermal vent areas in the western Pacific ocean. These strains were cocci, obligately anaerobic Archaea about 0.7–2 μm in diameter. Optimum growth conditions for OG-1 and SM-2 were at 85–90°C (range 60–100°C), pH 6 (range pH 4–8), a NaCl concentration of 3% (range 1–5%), and a nutrient concentration (tryptone plus yeast extract) of 0.2% (range 0.005–5%). Elemental sulfur stimulated the growth rate fourfold. Ammonium slightly stimulated growth. Both tryptone and yeast extract allowed growth as sole carbon sources; these isolates were not able to utilize or grow exclusively on sucrose, glucose, maltose, succinate, pyruvate, propionate, acetate, or free amino acids. OG-1 showed the fastest growth rate within the genus Thermococcus. Growth was inhibited by rifampicin. The DNA G+C content was 52 mol%. Sequencing of their 16S rDNA gene fragment indicated that these isolates belonged to the genus Thermococcus. OG-1 and SM-2 were different than the described Thermococcus species. We propose that OG-1 belongs to a new species: Thermococcus peptonophilus. Received: 8 March 1995 / Accepted: 24 May 1995     

Distribution of P1 Type Nuclease in the Genus Penicillium

P₁ type nuclease, which hydrolyzes RNA and heat-denatured DNA completely into 5’-mononucleotides and also shows 3’-nucleotidase activity, was widely distributed among various species belonging to the genus Penicillium such as P. expansum, P. notatum, P. steckii and P. meleagrinum. P₁ type nucleases isolated from these strains were produced in a form of complex with malonogalactan when molds were grown on wheat bran. These enzymes showed similar characters in heat-stability (stable at 60°C), temperature optimum (60 to 70°C for RNA and heat denatured DNA, and 70°C for 3’-AMP) and sensitivity to EDTA. The enzymes from P. steckii and P. expansum were immunologically co-related to nuclease P₁.

In addition, many strains of Penicillium produced base-nonspecific RNases forming 3’-mononucleotides via 2’: 3 ’-cyclic nucleotides. These RNases showed similarity in heat-lability (completely inactivated at 60°C), temperature optimum (45 to 50°C), sensitivity to Zn²⁺ and Cu²⁺, and relative hydrolysis rate toward 2’: 3’-cyclic nucleotides (A?C>U?G).     

Evaluating the Potential of Halothiobacillus Bacteria for Sulfur Oxidation and Biomass Production under Saline Soil

Abstract

Salinity negatively affects growth of sulfur-oxidizing bacteria (SOB) and their sulfate production ability, meanwhile decreases the available sulfate for plants in soil. The aim of this study was to isolate and characterize the bacteria of genus Halothiobacillus, as a salt-tolerant SOB, from saline and sulfidic habitats of Iran for the first time and evaluating the effect of salinity on their biomass and sulfate production during the oxidation of different sulfur sources. Isolation process and surveying the morphological, biochemical and 16S rRNA gene analysis resulted into identification of three species (eight strains) of Halothiobacillus genus including H. neapolitanus, H. hydrothermalis and H. halophilus. Salinity (0, 0.5, 1, 2 and 4?M NaCl) had a significant impact (p?≤?0.01) on bacterial biomass and sulfate production during the oxidation of thiosulfate and elemental sulfur. Biomass and sulfate production by strains was accompanied by a decrease in residual content of thiosulfate (RCT) in medium. The amount of produced biomass and sulfate in medium supplemented by thiosulfate was much higher than elemental sulfur. The highest amount of biomass and sulfate was produced by H. neapolitanus strain I19 at 0.5 and 1?M NaCl concentration. The results of this study could be the first step to focus on the application of these bacteria to increase sulfate storage of saline soils and crop production.     

Differential growth of Legionella pneumophila strains within a range of amoebae at various temperatures associated with in-premise plumbing

Aims: The potential effect of in‐premise plumbing temperatures (24, 32, 37 and 41°C) on the growth of five different Legionella pneumophila strains within free‐living amoebae (Acanthamoeba polyphaga, Hartmannella vermiformis and Naegleria fowleri) was examined. Methods and Results: Compared with controls that actively fed on Escherichia coli prey, when Leg. pneumophila was used as prey, strains Lp02 and Bloomington‐2 increased in growth at 30, 32 and 37°C while strains Philadelphia‐1 and Chicago 2 did not grow at any temperature within A. polyphaga. Strains Lp02, Bloomington‐2 and Dallas 1E did not proliferate in the presence of H. vermiformis nor did strain Philadelphia‐1 in the presence of N. fowleri. Yet, strain Bloomington‐2 grew at all temperatures examined within N. fowleri, while strain Lp02 proliferated at all temperatures except 41°C. More intriguing, strain Chicago 2 only grew at 32°C within H. vermiformis and N. fowleri suggesting a limited temperature growth range for this strain. Conclusions: Identifying the presence of pathogenic legionellae may require the use of multiple host amoebae and incubation temperatures. Significance and Impact of the Study: Temperature conditions and species of amoeba host supported in drinking water appear to be important for the selection of human‐pathogenic legionellae and point to future research required to better understand Legionella ecology.     

Cellulase from Bacillus licheniformis and Brucella sp. isolated from mangrove soils of Mahanadi river delta,Odisha, India

In the present study, two cellulose-degrading bacteria (CDB-5 and CDB-12) were isolated from mangrove soils of Mahanadi river delta, based on halo zone formation in Congo red agar medium and evaluation for cellulase production in CMC broth medium. Based on morphological, biochemical and 16S rRNA gene sequencing, the two strains, CDB-5 and CDB-12, were identified as Brucella sp. and Bacillus licheniformis, respectively. The gene bank accession number of the strains CDB-5 and CDB-12 are KR632646 and KR632645, respectively. The strain Brucella sp. and B. licheniformis showed an enzyme activity of 96.37?U/ml and 98.25?U/ml, respectively, after 72?h of incubation period. Enzyme production was optimized under different growth conditions such as pH, temperature, agitation rate, carbon source, sodium chloride (NaCl), and nitrogen sources. Maximum cellulase production by both the strains was obtained in the same parameter condition such as pH (7.0), rpm (150), and NaCl (2%, w/v) which varies for other parameters. The strain, CDB-5, produced maximum cellulase at 35?°C temperature, maltose as a carbon source, and yeast extract as a nitrogen source where as the strain CDB-12 produces maximum cellulase at 45?°C temperature, carboxyl methyl cellulose (CMC) as carbon source and trypton as a nitrogen source. The bacterial crude enzyme was purified by ammonium sulfate precipitation followed by overnight dialysis. SDS-PAGE analysis of the partially purified cellulase enzyme exhibited band sizes of approximately 55 and 72?kDa.