By Stephen Adeniyi 1319897 June, 2015 Isolation and Characterization of Cellulose Degrading Bacteria (CDB) from Lake and River By Stephen Adeniyi 1319897 June, 2015

Introduction Bacteria Degrade cellulosic substances for biofuel production. High synergy between cellulases - effective cellulose hydrolysis (Bhat and Bhat, 1997). endoglucanase homo-polysaccharide comprised of glucose units, linked by β-(1→4) glycosidic bonds that accounts for about 50% dry weight of biomass(Haruta et al., 2002). Hundreds of bacteria species responsible have been discovered (Elberson et al., 2000). Exoglucanases active on cellulose crystalline regions Endoglucanases, whose activity can be assayed using soluble cellulose substrates i.e., the carboxymethylcellulase assay (CMCase), are typically active on the more soluble cellulose amorphous region (Maki et al., 2009). Hence, high degree of interaction occurs between endoglucanases and exoglucanases, and such synergy is necessary for effective hydrolysis of cellulose (Bhat and Bhat, 1997). exoglucanase Bio-ethanol (Biofuels)

Objective of study To isolate novel Cellulose Degrading Bacteria (CDB) from Maiden Erlegh Lake and River Thames To characterize by identification of 16S rDNA sequence Investigate if CDB species is similar and/or different. Studies have isolated and characterized wastewaters (Tai et al., 2004), compost systems (Lu et al., 2005), soils (Wirth and Ulrich, 2002), hot-springs (Xue et al., 2001), plant decay from forestry or agricultural waste and in faeces of ruminants (Doi, 2008). Effective screening approaches done in previous and current studies, usually require detection of bacterial cellulase activity among isolated microbes, performed by Congo red flooding typically on the soluble form of cellulose i.e. carboxymethylcellulose (CMC) containing media (Teather and Wood, 1982). Easily recognized zone of hydrolysis are made visible, for extracellular secreting cellulases in bacteria. Aim Screen and isolate novel cellulose-degrading bacteria (CDB) from Maiden Erlegh Lake and River Thames. Identify by 16S rDNA sequencing and phylogenetic analysis if there were similar and/or different species of CDB isolated.

Sampling site of Maiden Erlegh Lake site and River Thames Isolation of CDB from Maiden Erlegh Lake and River Thames

Isolation of CDB Maiden Erlegh Lake (ME) River Thames (TR) Aliquots Aliquots Carboxymethylcellulose (CMC) media 72 h incubation at 15 oC, 25 oC and 37 oC Colony forming units (CFU) per site Replica plating on fresh CMC (Lederberg and Lederberg,1952) 24 – 48 h incubation Cellulase detection assay - Congo red flooding (Teather and Wood, 1982) Cellulase detection assay (Teather and Wood, 1982) Maiden Erlegh Lake (ME)- 2 of 80 CFU at 15 oC, 11 of 189 CFU at 25 oC and 10 of 37 at 37 oC. River Thames (TR)- 11 of 108 at15 oC, 15 of 211 at 25 oC and 15 of 64 at 37 oC Zone of clearing 15 – 30 mins Isolate and purify CDB Characterize Colony morphology Gram stain 16S rDNA PCR, Sequencing and Bioinformatics 15 – 30 min Zone of clearing = cellulose hydrolysis Confirmation Isolate and purify CDB Characterize CDB 16S rDNA PCR Sequencing Bioinformatics Colony morphology Gram stain

CDB isolates 27 colonies purified Preliminary investigations Confirmation 10 CDB isolates selected further 4 of 9 pure CDB colonies isolated at different temperature from ME were selected and detected for celluase activity (Figure 2). The four isolates selected, were two from the enriched cultures isolated at 25 °C and 37 °C only, from Maiden Erlegh Lake (ME). Thus, selection was based on the zone of clearing by cellulase assay and physical characteristics from preliminary observations (Table 2). 6 of 18 pure CDB colonies isolated at different temperature from TR were selected and detected for celluase activity (Figure 3). The six isolates selected, were two from each of the enriched cultures isolated at 15 °C, 25 °C and 37 °C respectively from River Thames (TR). Thus, selection was based on the zone of clearing by cellulase assay and physical characteristics from preliminary observations (Table 3 and Table 4).

CDB isolated from Maiden Erlegh Lake 08ME25 06ME25 17ME37 15ME37

CDB isolated from River Thames 03TR15 06TR15 09TR25 10TR25 18TR37 22TR37

Gram stain of CDB isolates Maiden Erlegh Lake River Thames 06TR15 03TR15 08ME25 09TR25 10TR25 15ME37 17ME37 18TR37 22TR37

Confirmation of CDB growth on Tryptone soya agar (TSA) and cellulase acitivity at different temperature Maiden Erlegh Lake River Thames Isolates Temperature oC Growth on TSA Cellulase Assay 06ME25 15 ++ + 25 37 -- 50 08ME25 +++ 15ME37 17ME37 Isolates Temperature oC Growth on TSA Cellulase Assay 03TR15 15 ++ 25 37 + 50 -- 06TR15 09TR25 +++ 10TR25 18TR37 22TR37 06ME25 Grew Cellulase activity at 15 °C & 25 °C 08ME25 Cellulase activity at 15 °C, 25 °C, 37 °C & 50 °C Both 15ME37 and 17ME37 Cellulase activity at 25 °C, 37 °C and 50 °C

16S rDNA PCR products of isolates Universal 27F and 519R primers used - 27 CDB isolates produced 16S rDNA PCR amplified products.

Identification of CDB species Maiden Erlegh Lake River Thames 18TR37 96 % identity to B. licheniformis Endoglucanase, β-glucosidase, xylanase 08ME25 99 % identity to Bacillus licheniformis Endoglucanase, β-glucosidase, xylanase Synergy can occur for potential biofuels (Veith et al., 2004). 22TR37 99 % identity to B. amyloliquefaciens subsp. Plantarum 17ME37 99 % identity to B. stratosphericus Exoglucanase, β-glucosidase, endoglucanase, and xylanase (Odisi et al., 2012) 03TR15 97 % identity to Flavobacterium aquidurense 09TR25 95 % identity to F. aquidurense 15ME37 99 % identity to B. siamensis 06TR15 97 % identity to F. saccharophilum 06ME25 99 % identity to Pseudomonas punonensis 10TR25 98 % identity to F. pectinovorum

Conclusion 10 isolates selected further - confirmed cellulose degraders NO novel species identified Further isolation and characterisation of CDB from Maiden Erlegh Lake and River Thames Therefore, Classification of isolates/species - application of 16S–23S rDNA, Quantitative activity assays and, Synergy of bacterial cellulases for potential biofuels. This study identified that there were CDB related to Bacillus species among the microbial communities from both aquatic source of ME and TR, Pseudomonas species from ME and Flavobacterium species from TR. However, there was no novel species identified based on comparison between their 16S rDNA nucleotide sequences of the selected CDB isolates with 16S ribosomal DNA sequences from bacteria GenBank database. Hence, further isolation and characterisation of CDB from both sources could detect similar and/or different species identified in this study. Generally, additional classification of isolates by application of their 16S–23S rDNA intergenic spacer could perhaps confirm their identity to known species, combined with quantitative activity assays and mechanisms involved for synergic relationship of their bacterial cellulases to confirm its function if novel for potential biofuels.

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