Madihah Md. Salleh

A High Molecular-Mass Anoxybacillus sp. SK3-4 Amylopullulanase: Characterization and Its Relationship in Carbohydrate Utilization

An amylopullulanase of the thermophilic Anoxybacillus sp. SK3-4 (ApuASK) was purified to homogeneity and characterized. Though amylopullulanases larger than 200 kDa are rare, the molecular mass of purified ApuASK appears to be approximately 225 kDa, on both SDS-PAGE analyses and native-PAGE analyses. ApuASK was stable between pH 6.0 and pH 8.0 and exhibited optimal activity at pH 7.5. The optimal temperature for ApuASK enzyme activity was 60 °C, and it retained 54% of its total activity for 240 min at 65 °C. ApuASK reacts with pullulan, starch, glycogen, and dextrin, yielding glucose, maltose, and maltotriose. Interestingly, most of the previously described amylopullulanases are unable to produce glucose and maltose from these substrates. Thus, ApuASK is a novel, high molecular-mass amylopullulanase able to produce glucose, maltose, and maltotriose from pullulan and starch. Based on whole genome sequencing data, ApuASK appeared to be the largest protein present in Anoxybacillus sp. SK3-4. The α-amylase catalytic domain present in all of the amylase superfamily members is present in ApuASK, located between the cyclodextrin (CD)-pullulan-degrading N-terminus and the α-amylase catalytic C-terminus (amyC) domains. In addition, the existence of a S-layer homology (SLH) domain indicates that ApuASK might function as a cell-anchoring enzyme and be important for carbohydrate utilization in a streaming hot spring.

Isolation and characterization of pullulan-degrading Anoxybacillus species isolated from Malaysian hot springs

Two thermophilic bacteria (SK3-4 and DT3-1) were isolated from the Sungai Klah (SK) and Dusun Tua (DT) hot springs in Malaysia. The cells from both strains were rod-shaped, stained Gram positive and formed endospores. The optimal growth of both strains was observed at 55°C and pH 7. Strain DT3-1 exhibited a higher tolerance to chloramphenicol (100 μ g ml−1) but showed a lower tolerance to sodium chloride (2%, w/v) compared to strain SK3-4. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains belong to the genus Anoxybacillus. High concentrations of 15:0 iso in the fatty acid profiles support the conclusion that both strains belong to the genus Anoxybacillus and exhibit unique fatty acid compositions and percentages compared to other Anoxybacillus species. The DNA G+C contents were 42.0 mol% and 41.8 mol% for strains SK3-4 and DT3-1, respectively. Strains SK3-4 and DT3-1 were able to degrade pullulan and to produce maltotriose and glucose, respectively, as their main end products. Based on phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequences, and the DNA G+C content, we propose that strains SK3-4 and DT3-1 are new pullulan-degrading Anoxybacillus strains.

Effect of Agitation and Aeration Rates on Chitinase Production Using Trichoderma virens UKM1 in 2-l Stirred Tank Reactor

Shrimps have been a popular raw material for the burgeoning marine and food industry contributing to increasing marine waste. Shrimp waste, which is rich in organic compounds is an abundant source of chitin, a natural polymer of N-acetyl-d-glucosamine (GluNac), a reducing sugar. For this respect, chitinase-producing fungi have been extensively studied as biocontrol agents. Locally isolated Trichoderma virens UKM1 was used in this study. The effect of agitation and aeration rates using colloidal chitin as control substrate in a 2-l stirred tank reactor gave the best agitation and aeration rates at 200xa0rpm and 0.33xa0vvm with 4.1xa0U/l per hour and 5.97xa0U/l per hour of maximum volumetric chitinase activity obtained, respectively. Microscopic observations showed shear sensitivity at higher agitation rate of the above system. The oxygen uptake rate during the highest chitinase productivity obtained using sun-dried ground shrimp waste of 1.74xa0mg of dissolved oxygen per gram of fungal biomass per hour at the kLa of 8.34 per hour.

Isolation, Screening, and Identification of Potential Cellulolytic and Xylanolytic Producers for Biodegradation of Untreated Oil Palm Trunk and Its Application in Saccharification of Lemongrass Leaves

This study presents the isolation and screening of fungi with excellent ability to degrade untreated oil palm trunk (OPT) in a solid-state fermentation system (SSF). Qualitative assay of cellulases and xylanase indicates notable secretion of both enzymes by 12 fungal strains from a laboratory collection and 5 strains isolated from a contaminated wooden board. High production of these enzymes was subsequently quantified in OPT in SSF. Aspergillus fumigates SK1 isolated from cow dung gives the highest xylanolytic activity (648.448 U g−1), generally high cellulolytic activities (CMCase: 48.006, FPase: 6.860, beta-glucosidase: 16.328 U g−1) and moderate lignin peroxidase activity (4.820 U/g), and highest xylanolytic activity. The xylanase encoding gene of Aspergillus fumigates SK1 was screened using polymerase chain reaction by a pair of degenerate primers. Through multiple alignment of the SK1 strains xylanase nucleotide sequences with other published xylanases, it was confirmed that the gene belonged to the xylanase glycoside hydrolase family 11 (GH11) with a protein size of 24.49 kD. Saccharification of lemongrass leaves using crude cellulases and xylanase gives the maximum reducing sugars production of 6.84 g/L with glucose as the major end product and traces of phenylpropanic compounds (vanillic acid, p-coumaric acid, and ferulic acid).

Screening factors influencing the production of astaxanthin from freshwater and marine microalgae

Astaxanthin, a carotenoid pigment found in several aquatic organisms, is responsible for the red colour of salmon, trout and crustaceans. In this study, astaxanthin production from freshwater microalga Chlorella sorokiniana and marine microalga Tetraselmis sp. was investigated. Cell growth and astaxanthin production were determined spectrophotometrically at 620 and 480xa0nm, respectively. Astaxanthin was extracted using acetone and measured subsequent to biomass removal. Aerated conditions favoured astaxanthin production in C. sorokiniana, whereas Tetraselmis sp. was best cultured under unaerated conditions. C. sorokiniana produced more astaxanthin with the highest yield reached at 7.83xa0mg/l in 6.0xa0mM in nitrate containing medium compared to Tetraselmis sp. which recorded the highest yield of only 1.96xa0mg/l in 1.5xa0mM nitrate containing medium. Production in C. sorokiniana started at the early exponential phase, indicating that astaxanthin may be a growth-associated product in this microalga. Further optimization of astaxanthin production was performed using C. sorokiniana through a 23 full factorial experimental design, and a yield of 8.39xa0mg/l was achieved. Overall, the study has shown that both microalgae are capable of producing astaxanthin. Additionally, this research has highlighted C. sorokiniana as a potential astaxanthin producer that could serve as a natural astaxanthin source in the current market.

16S rRNA metagenomic analysis of the symbiotic community structures of bacteria in foregut, midgut, and hindgut of the wood-feeding termite Bulbitermes sp.

The termite gut is a highly structured microhabitat with physicochemically distinct regions. It is generally separated into the foregut, midgut and hindgut. The distribution of gut microbiota is greatly influenced by varying physicochemical conditions within the gut. Thus, each gut compartment has a unique microbial population structure. In this study, the bacterial communities of foregut, midgut and hindgut of wood-feeding higher termite, Bulbitermes sp. were analyzed in detail via metagenomic sequencing of the 16S rRNA V3-V4 region. While the microbiomes of the foregut and midgut shared a similar taxonomic pattern, the hindgut possessed more diverse bacterial phylotypes. The communities in the foregut and midgut were dominated by members of the group Bacilli and Clostridia (Firmicutes) as well as taxon Actinomycetales (Actinobacteria). The main bacterial lineage found in hindgut was Spirochaetaceae (Spirochaetes). The significant difference among the three guts was the relative abundance of the potential lignin-degrading bacteria, Actinomycetales, in both the foregut and midgut. This suggests that lignin modification was probably held in the anterior part of termite gut. Predictive functional profiles of the metagenomes using 16S rRNA marker gene showed that cell motility, energy metabolism and metabolism of cofactors and vitamins were found predominantly in hindgut microbiota, whereas xenobiotics degradation and metabolism mostly occurred in the foregut segment. This was compatible with our 16S rRNA metagenomic results showing that the lignocellulose degradation process was initiated by lignin disruption, increasing the accessibility of celluloses and hemicelluloses.

Chapter 16 – Direct Cellulase Gene Amplification From Hot Spring Using the Guidance of 16S rRNA Amplicon Metagenomics

Abstract Thermostable cellulase plays an important role in the process of converting lignocellulosic waste to biofuel. A culture-dependent approach may not effectively discover new thermostable cellulase, hemicellulase, or lignin-acting enzymes, since many thermophiles are not culturable in general laboratory setups. Due to this limitation, culture-independent techniques—for instance, the metagenomic approach—may be a good technique for finding novel genes. This current chapter demonstrates a method to amplify complete genes from a hot spring complex metagenome without the need to isolate bacterium or perform a clonal library. To achieve this, one needs to perform sampling (water, sediment, or biofilm from hot spring), extract the environmental DNA material, and perform 16S rRNA amplicon sequencing using a next-generation sequencer. From the microbial population data, we can then identify the genera of interest and their cellulase genes, followed by the designing of specific or degenerate primers, and finally amplification of the genes of interest using conventional PCR. This methodology will likely be an economic and powerful approach to discover novel genes that are present in complex environments. This chapter also describes the challenges of using such an approach. Using this method, an interesting heat-tolerant enzyme for biomass degradation was cloned, expressed, and purified. The enzyme exhibited optimum activity at a pH of 5.5 and at 90°C, a much higher temperature than other known enzyme counterparts. This enzyme is highly active on complex polysaccharide carboxymethyl cellulose. This technique will be useful for any gene amplification from hot springs using the guidance of 16S rRNA amplicon metagenomics.

The Effect of Partial Replacement of Dietary Fishmeal with Fermented Prawn Waste Liquor on Juvenile Sea Bass Growth

A feeding trial was conducted for 49 days to evaluate the effect of partially substituting fishmeal with fermented prawn waste liquor (FPWL) in juvenile sea bass diets at 10%, 20%, and 30% of the total diet. Growth performance of sea bass from 16 g up to 40 g fed with FPWL-supplemented diet was not significantly different from the all fishmeal control diet. The most cost-effective diet included FPWL at 30%, with weight gain, feed conversion ratio, and protein efficiency ratio of 180%, 1.78%, and 1.2% respectively.

Solid-state fermentation of oil palm frond petiole for lignin peroxidase and xylanase-rich cocktail production

In current practice, oil palm frond leaflets and stems are re-used for soil nutrient recycling, while the petioles are typically burned. Frond petioles have high commercialization value, attributed to high lignocellulose fiber content and abundant of juice containing free reducing sugars. Pressed petiole fiber is the subject of interest in this study for the production of lignocellulolytic enzyme. The initial characterization showed the combination of 0.125xa0mm frond particle size and 60% moisture content provided a surface area of 42.3xa0m2/g, porosity of 12.8%, and density of 1.2xa0g/cm3, which facilitated fungal solid-state fermentation. Among the several species of Aspergillus and Trichoderma tested, Aspergillus awamori MMS4 yielded the highest xylanase (109xa0IU/g) and cellulase (12xa0IU/g), while Trichoderma virens UKM1 yielded the highest lignin peroxidase (222xa0IU/g). Crude enzyme cocktail also contained various sugar residues, mainly glucose and xylose (0.1–0.4xa0g/L), from the hydrolysis of cellulose and hemicellulose. FT-IR analysis of the fermented petioles observed reduction in cellulose crystallinity (I900/1098), cellulose–lignin (I900/1511), and lignin–hemicellulose (I1511/1738) linkages. The study demonstrated successful bioconversion of chemically untreated frond petioles into lignin peroxidase and xylanase-rich enzyme cocktail under SSF condition.

HC-0C-07: Isolation and Characterisation of Crude Oil Degrading Microorganisms from Petrochemical Wastewater

Bacteria with the abilities to degrade crude oil were isolated from soil, activated sludge and biological treatment lagoon of the local petrochemical industries. For the biodegradation process, n-alkanes, of varying carbon chain length, C16–C38, were used. Out of the 12 cultures of bacteria isolated, 3 of the best oil degraders were partially identified via biochemical tests; 2 of which were Acinetobactor spp while another one belonged to Proteus sp. Degradation of the n-alkanes in crude oil was monitored under agitated and non-agitated condition using gas chromatography technique. Generally, non-agitated cultures showed higher degradation rates. One of the Acinetobacter sp. showed the highest degradation rate, in which 80–100 % of the alkanes (C16–C38) in crude oil was degraded without any addition of organic nitrogen and phosphorus. It is of interest to highlight another of the Acinetobacter sp. which showed the ability to degrade longer chain alkanes more rapidly than shorter ones; C36 and C38 were fully degraded in 2 days. Only one bacterium, Proteus sp showed increased rates of degradation under agitated condition.