Anode biofilm is a crucial component in microbial fuel cells (MFCs) for electrogenesis. Better knowledge about the biofilm development process on electrode surface is believed to improve MFC performance. In this study, double-chamber microbial fuel cell was operated with diluted POME (initial COD = 1,000 mg L(-1)) and polyacrylonitrile carbon felt was used as electrode. The maximum power density, COD removal efficiency and Coulombic efficiency were found as 22 mW m(-2), 70 and 24 %, respectively. FTIR and TGA analysis confirmed the formation of biofilm on the electrode surface during MFC operation. The impact of anode biofilm on anodic polarization resistance was investigated using electrochemical impedance spectroscopy (EIS) and microbial community changes during MFC operation using denaturing gradient gel electrophoresis (DGGE). The EIS-simulated results showed the reduction of charge transfer resistance (R ct) by 16.9 % after 14 days of operation of the cell, which confirms that the development of the microbial biofilm on the anode decreases the R ct and therefore improves power generation. DGGE analysis showed the variation in the biofilm composition during the biofilm growth until it forms an initial stable microbial community, thereafter the change in the diversity would be less. The power density showed was directly dependent on the biofilm development and increased significantly during the initial biofilm development period. Furthermore, DGGE patterns obtained from 7th and 14th day suggest the presence of less diversity and probable functional redundancy within the anodic communities possibly responsible for the stable MFC performance in changing environmental conditions.
This paper demonstrates Pseudomonas cepacia lipase catalyzed hydrolysis of p-nitrophenyl palmitate under irradiation of light with wavelengths of 250-750 nm. The reaction follows Michaelis-Menten Kinetics and the light irradiation increases the overall rate of hydrolysis. Using Lineweaver-Burk plot K M and V max values for the reaction in presence of light are found to be 39.07 and 66.67 mM/min/g, respectively; while for the same reaction under dark condition, the values are 7.08 and 10.21 mM/min/g. The linear form of enzyme dependent rate of reaction confirms that no mass-transfer limitations are present and the reaction is a kinetically controlled enzymatic reaction.
Pleurotus pulmonarius F043, a fungus collected from tropical rain forest, was used to degrade pyrene, a four-rings polycyclic aromatic hydrocarbons (PAHs), in a mineral medium broth. A maximum degradation rate of pyrene (90 %) was occurred at pH 3 and the lowest degradation rate was found in the culture at pH 10 (2 %). More than 90 % pyrene degradation was achieved at pH ranged from 3 to 5, whereas the degradation rate significantly declined when the pH was >5. The degradation of pyrene increased from 2 to 96 % when the temperature rose from 4 to 25 °C. When the temperature was increased to 60 °C resulting the lowest degradation rate into 7 %. Among the agitation rates tested, 120 rpm was the best with 95 % degradation, followed by 100 rpm (90 %). The optimum agitation range for pyrene degradation by P. pulmonarius F043 was 100-120 rpm. Among all the concentrations tested, 0.5 % Tween 80 was the best with 98 % degradation, followed by 1 % Tween 80 (90 %). The optimum concentration of Tween 80 for pyrene degradation by P. pulmonarius F043 was 0.5-1 %. The degradation rate decreased, while the concentration of Tween 80 was increased. The metabolic product was found during degradation process through the identification of gentisic acid by TLC, UV-Spectrophotometer, and GC-MS.
A xylanase gene (xyn2) from Trichoderma reesei ATCC 58350 was previously cloned and expressed in Kluyveromyces lactis GG799. The production of the recombinant xylanase was conducted in a developed medium with an optimised batch and with fed-batches that were processed with glucose. The glucose served as a carbon source for cell growth and as an inducer for xylanase production. In a 1-L batch system, a glucose concentration of 20 g L(-1) and 80 % dissolved oxygen were found to provide the best conditions for the tested ranges. A xylanase activity of 75.53 U mL(-1) was obtained. However, in the batch mode, glucose depletions reduced the synthesis of recombinant xylanase by K. lactis GG799. To maximise the production of xylanase, further optimisation was performed using exponential feeding. We investigated the effects of various nitrogen sources combined with the carbon to nitrogen (C/N) molar ratio on the production of xylanase. Of the various nitrogen sources, yeast extract was found to be the most useful for recombinant xylanase production. The highest xylanase production (110.13 U mL(-1)) was measured at a C/N ratio of 50.08. These conditions led to a 45.8 % increase in xylanase activity compared with the batch cultures. Interestingly, the further addition of 500 g L(-1) glucose led to a 6.2-fold increase (465.07 U mL(-1)) in recombinant xylanase activity. These findings, together with those of the exponential feeding strategy, indicate that the composition of the C/N molar ratio has a substantial impact on recombinant protein production in K. lactis.
Polyporus sp. S133 decolorized the Amaranth in 72 h (30 mg L(-1)) under static and shaking conditions. Liquid medium containing glucose has shown the highest decolorization of Amaranth by Polyporus sp. S133. When the effect of increasing inoculum concentration on decolorization of Amaranth was studied, maximum decolorization was observed with 15 % inoculum concentration. Significant increase in the enzyme production of laccase (102.2 U L(-1)) was observed over the period of Amaranth decolorization compared to lignin peroxidase and manganese peroxidase. Germination rate of Sorghum vulgare and Triticum aestivum was less with Amaranth treatment as compared to metabolites obtained after its decolorization. Based on the metabolites detected by GC-MS, it was proposed that Amaranth was bio-transformed into two intermediates, 1-hydroxy-2-naphthoic acid and 1,4-naphthaquinone. Overall findings suggested the ability of Polyporus sp. S133 for the decolorization of azo dye and ensured the ecofriendly degradation of Amaranth.
The effects of structure and concentration of surfactants on the biodegradation of fluoranthene, a three rings polycyclic aromatic hydrocarbon in the aqueous phase, as well as their effects on the biodegradation and enzyme activity were investigated. The toxicity ranking of studied surfactants is: non-ionic Tween 80 4,500 mg/L) was showed by Tween 80 (10 mg/L) culture, manifesting that the non-ionic surfactant present in the culture were beneficial to the fungal growth. Laccase showed the highest enzymes activity in all surfactants culture. Non-ionic Tween 80 showed a significant result for laccase activity (1,902 U/L) in the Armillaria sp. F022 culture. The increased enzymes cumulative activity may stem directly from the rising fluoranthene biodegradability as addition of appropriate surfactants. The biotransformation of fluoranthene was greatly improved by Tween 80, and totally fluoranthene degradation was obtained as Tween 80 was 10 mg/L. Two fluoranthene metabolites were isolated from the culture medium and analyzed by a thin layer chromatography, UV visible spectrometer and gas chromatography-mass spectrometry (GC-MS). The oxidation of fluoranthene is initiated by oxygenation at the C-2,3 positions resulting 9-fluorenone. At the end of experiment, one metabolite was detected in the culture extract and identified as phthalic acid. Evidently, Armillaria sp. F022 seems efficient, high effective and deserves further application on the enhanced bioremediation technologies for the treatment of fluoranthene-contaminated soil.
Microbial strain optimization focuses on improving technological properties of the strain of microorganisms. However, the complexities of the metabolic networks, which lead to data ambiguity, often cause genetic modification on the desirable phenotypes difficult to predict. Furthermore, vast number of reactions in cellular metabolism lead to the combinatorial problem in obtaining optimal gene deletion strategy. Consequently, the computation time increases exponentially with the increase in the size of the problem. Hence, we propose an extension of a hybrid of Bees Algorithm and Flux Balance Analysis (BAFBA) by integrating OptKnock into BAFBA to validate the result. This paper presents a number of computational experiments to test on the performance and capability of BAFBA. Escherichia coli, Bacillus subtilis and Clostridium thermocellum are the model organisms in this paper. Also included is the identification of potential reactions to improve the production of succinic acid, lactic acid and ethanol, plus the discussion on the changes in the flux distribution of the predicted mutants. BAFBA shows potential in suggesting the non-intuitive gene knockout strategies and a low variability among the several runs. The results show that BAFBA is suitable, reliable and applicable in predicting optimal gene knockout strategy.
Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L(-1 )days(-1), or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L(-1).
The use of biomaterials or microorganisms in PAHs degradation had presented an eye-catching performance. Pleurotus eryngii is a white rot fungus, which is easily isolated from the decayed woods in the tropical rain forest, used to determine the capability to utilize naphthalene, a two-ring polycyclic aromatic hydrocarbon as source of carbon and energy. In the meantime, biotransformation of naphthalene to intermediates and other by-products during degradation was investigated in this study. Pleurotus eryngii had been incubated in liquid medium formulated with naphthalene for 14 days. The presence of metabolites of naphthalene suggests that Pleurotus eryngii begin the ring cleavage by dioxygenation on C1 and C4 position to give 1,4-naphthaquinone. 1,4-Naphthaquinone was further degraded to benzoic acid, where the proposed terepthalic acid is absent in the cultured extract. Further degradation of benzoic acid by Pleurotus eryngii shows the existence of catechol as a result of the combination of decarboxylation and hydroxylation process. Unfortunately, phthalic acid was not detected in this study. Several enzymes, including manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase and 2,3-dioxygenase are enzymes responsible for naphthalene degradation. Reduction of naphthalene and the presence of metabolites in liquid medium showed the ability of Pleurotus eryngii to utilize naphthalene as carbon source instead of a limited glucose amount.
This review is focused on the production of microbial lipases by high cell density fermentation. Lipases are among the most widely used of the enzyme catalysts. Although lipases are produced by animals and plants, industrial lipases are sourced almost exclusively from microorganisms. Many of the commercial lipases are produced using recombinant species. Microbial lipases are mostly produced by batch and fed-batch fermentation. Lipases are generally secreted by the cell into the extracellular environment. Thus, a crude preparation of lipases can be obtained by removing the microbial cells from the fermentation broth. This crude cell-free broth may be further concentrated and used as is, or lipases may be purified from it to various levels. For many large volume applications, lipases must be produced at extremely low cost. High cell density fermentation is a promising method for low-cost production: it allows a high concentration of the biomass and the enzyme to be attained rapidly and this eases the downstream recovery of the enzyme. High density fermentation enhances enzyme productivity compared with the traditional submerged culture batch fermentation. In production of enzymes, a high cell density is generally achieved through fed-batch operation, not through perfusion culture which is cumbersome. The feeding strategies used in fed-batch fermentations for producing lipases and the implications of these strategies are discussed. Most lipase-producing microbial fermentations require oxygen. Oxygen transfer in such fermentations is discussed.
Armillaria sp. F022 is a white-rot fungus isolated from a tropical rain forest in Indonesia that is capable of utilizing pyrene as a source of carbon and energy. Enzymes production during the degradation process by Armillaria sp. F022 was certainly related to the increase in biomass. In the first week after incubation, the growth rate rapidly increased, but enzyme production decreased. After 7 days of incubation, rapid growth was observed, whereas, the enzymes were produced only after a good amount of biomass was generated. About 63 % of pyrene underwent biodegradation when incubated with this fungus in a liquid medium on a rotary shaker (120 rpm, 25 °C) for 30 days; during this period, pyrene was transformed to five stable metabolic products. These metabolites were extracted in ethyl acetate, isolated by column chromatography, and then identified using thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). 1-Hydroxypyrene was directly identified by GC-MS, while 4-phenanthroic acid, 1-hydroxy-2-naphthoic acid, phthalic acid, and protocatechuic acid were identified to be present in their derivatized forms (methylated forms and silylated forms). Protocatechuic acid was the end product of pyrene degradation by Armillaria sp. F022. Dynamic profiles of two key enzymes, namely laccase and 1,2-dioxygenase, were revealed during the degradation process, and the results indicated the presence of a complicated mechanism in the regulation of pyrene-degrading enzymes. In conclusion, Armillaria sp. F022 is a white-rot fungus with potential for application in the degradation of polycyclic aromatic hydrocarbons such as pyrene in the environment.
Armillaria sp. F022, a white-rot fungus isolated from decayed wood in tropical rain forest was used to biodegrade anthracene in cultured medium. The percentage of anthracene removal by Armillaria sp. F022 reached 13 % after 7 days and at the end of the experiment, anthracene removal level was at 87 %. The anthracene removal through sorption and transformation was investigated. 69 % of eliminated anthracene was transformed by Armillaria sp. F022 to form other organic structure, while only 18 % was absorbed in the mycelia. In the kinetic experiment, anthracene dissipation will not stop even though the biomass had stopped growing. Anthracene removal by Armillaria sp. F022 was correlated with protein concentration (whole biomass) in the culture. The production of enzyme was affected by biomass production. Anthracene was transformed to two stable metabolic products. The metabolites were extracted in ethyl-acetate, isolated by column chromatography, and then identified using gas chromatography-mass spectrometry (GC-MS).
Oil palm fronds are the most abundant lignocellulosic biomass in Malaysia. In this study, fronds were tested as the potential renewable biomass for ethanol production. The soaking in aqueous ammonia pretreatment was applied, and the fermentability of pretreated fronds was evaluated using simultaneous saccharification and fermentation. The optimal pretreatment conditions were 7 % (w/w) ammonia, 80 °C, 20 h of pretreatment, and 1:12 S/L ratio, where the enzymatic digestibility was 41.4 % with cellulase of 60 FPU/g-glucan. When increasing the cellulase loading in the hydrolysis of pretreated fronds, the enzymatic digestibility increased until the enzyme loading reached 60 FPU/g-glucan. With 3 % glucan loading in the SSF of pretreated fronds, the ethanol concentration and yield based on the theoretical maximum after 12 and 48 h of the SSF were 7.5 and 9.7 g/L and 43.8 and 56.8 %, respectively. The ethanol productivities found at 12 and 24 h from pretreated fronds were 0.62 and 0.36 g/L/h, respectively.
Anaerobic treatment processes to remove organic matter from palm oil mill effluent (POME) have been used widely in Malaysia. Still the amounts of total organic and total mineral released from POME that may cause degradation of the receiving environment need to be verified. This paper proposes the use of the hydrodynamic equations to estimate performance of the cascaded anaerobic ponds (CAP) and to calculate amounts of total organic matter and total mineral released from POME. The CAP efficiencies to remove biochemical oxygen demands, chemical oxygen demands, total solids and volatile solids (VS) as high as 94.5, 93.6, 96.3 and 98.2 %, respectively, are estimated. The amounts of total organic matter and total mineral as high as 538 kg VS/day and 895 kg FS/day, respectively, released from POME to the receiving water are calculated. The implication of the proposed hydrodynamic equations contributes to more versatile environmental assessment techniques, sometimes replacing laboratory analysis.
Rice bran (RB) and de-oiled rice bran (DRB) have been treated and used as the carbon source in acetone-butanol-ethanol (ABE) production using Clostridium saccharoperbutylacetonicum N1-4. The results showed that pretreated DRB produced more ABE than pretreated RB. Dilute sulfuric acid was the most suitable treatment method among the various pretreatment methods that were applied. The highest ABE obtained was 12.13 g/L, including 7.72 g/L of biobutanol, from sulfuric acid. The enzymatic hydrolysate of DRB (ESADRB), when treated with XAD-4 resin, resulted in an ABE productivity and yield of 0.1 g/L h and 0.44 g/g, respectively. The results also showed that the choice of pretreatment method for RB and DRB is an important factor in butanol production.
A functionalized polystyrene nanofiber (PSNF) immobilized β-galactosidase assembly (PSNF-Gal) was synthesized as a nanobiocatalyst aiming to enhance the biocatalyst stability and functional ability. The PSNF fabricated by electrospinning was functionalized through a chemical oxidation method for enzyme binding. The bioengineering performance of the enzyme carriers was further evaluated for bioconversion of lactose to galacto-oligosaccharides (GOS). The modified PSNF-Gal demonstrated distinguished performances to preserve the same activity as the free β-galactosidase at the optimum pH of 7.0, and to enhance the enzyme stability of PSNF-Gal in an alkaline condition up to pH 10. The PSNF assembly demonstrated improved thermal stability from 37 to 60 °C. The nanobiocatalyst was able to retain 30 % of its initial activity after ninth operation cycles comparing to four cycles with the unmodified counterpart. In contrast with free β-galactosidase, the modified PSNF-Gal enhanced the GOS yield from 14 to 28 %. These findings show the chemically modified PSNF-based nanobiocatalyst may be pertinent for various enzyme-catalysed bioprocessing applications.
A recently reported stable and efficient EBPR system at high temperatures around 30 °C has led to characterization of kinetic and stoichiometric parameters of the Activated Sludge Model no. 2d (ASM2d). Firstly, suitable model parameters were selected by identifiability analysis. Next, the model was calibrated and validated. ASM2d was found to represent the processes well at 28 and 32 °C except in polyhyroxyalkanoate (PHA) accumulation of the latter. The values of the kinetic parameters for PHA storage (q PHA), polyphosphate storage (q PP) and growth (μ PAO) of polyphosphate-accumulating organisms (PAOs) at 28 and 32 °C were found to be much higher than those reported by previous studies. Besides, the value of the stoichiometric parameter for the requirement of polyphosphate for PHA storage (Y PO4) was found to decrease as temperature rose from 28 to 32 °C. Values of two other stoichiometric parameters, i.e. the growth yield of heterotrophic organisms (Y H) and PAOs (Y PAO), were high at both temperatures. These calibrated parameters imply that the extremely active PAOs of the study were able to store PHA, store polyphosphate and even utilize PHA for cell growth. Besides, the parameters do not follow the Arrhenius correlation due to the previously reported unique microbial clade at 28 and 32 °C, which actively performs EBPR at high temperatures.
The main aim of this study is to investigate the performance of organic oxidation and denitrification of the system under long-term operation. The MFC reactor was operated in continuous mode for 180 days. Nitrate was successfully demonstrated as terminal electron acceptor, where nitrate was reduced at the cathode using electron provided by acetate oxidation at the anode. The removal efficiencies of chemical oxygen demand (COD) and nitrate were higher in the closed circuit system than in open circuit system. Both COD and nitrate reduction improved with the increase of organic loading and subsequently contributed to higher power output. The maximum nitrate removal efficiency was 88 ± 4 % (influent of 141 ± 14 mg/L). The internal resistant was 50 Ω, which was found to be low for a double chambered MFC. The maximum power density was 669 mW/m(3) with current density of 3487 mA/m(3).
Organic solid waste composting is a complex process that involves many coupled physical, chemical and biological mechanisms. To understand this complexity and to ease in planning, design and management of the composting plant, mathematical model for simulation is usually applied. The aim of this paper is to develop a mathematical model of organic substrate degradation and its performance evaluation in solid waste windrow composting system. The present model is a biomass-dependent model, considering biological growth processes under the limitation of moisture, oxygen and substrate contents, and temperature. The main output of this model is substrate content which was divided into two categories: slowly and rapidly degradable substrates. To validate the model, it was applied to a laboratory scale windrow composting of a mixture of wood chips and dog food. The wastes were filled into a cylindrical reactor of 6 cm diameter and 1 m height. The simulation program was run for 3 weeks with 1 s stepwise. The simulated results were in reasonably good agreement with the experimental results. The MC and temperature of model simulation were found to be matched with those of experiment, but limited for rapidly degradable substrates. Under anaerobic zone, the degradation of rapidly degradable substrate needs to be incorporated into the model to achieve full simulation of a long period static pile composting. This model is a useful tool to estimate the changes of substrate content during composting period, and acts as a basic model for further development of a sophisticated model.
Cresol Red belongs to the triphenylmethane (TPM) class of dyes which are potentially carcinogenic or mutagenic. However, very few studies on biodegradation of Cresol Red were investigated as compared to other type dyes such as azo and anthraquinone dye. The aim of this work is to evaluate triphenylmethane dye Cresol Red degradation by fungal strain isolated from the decayed wood in Johor Bahru, Malaysia. Detailed taxonomic studies identified the organisms as Trichoderma species and designated as strain Trichoderma harzianum M06. In this study, Cresol Red was decolorized up to 88% within 30 days under agitation condition by Trichoderma harzianum M06. Data analysis revealed that a pH value of 3 yielded a highest degradation rate among pH concentrations (73%), salinity concentrations of 100 g/L (73%), and a volume of 0.1 mL of Tween 80 (79%). Induction in the enzyme activities of manganese peroxidase, lignin peroxidase, laccase, 1,2- and 2,3-dioxygenase indicates their involvement in Cresol Red removal. Various analytical studies such as Thin-Layer Chromatography (TLC), UV-Vis spectrophotometer, and Gas chromatography mass spectrometry (GC-MS) confirmed the biotransformation of Cresol Red by the fungus. Two metabolites were identified in the treated medium: 2,4-dihydroxybenzoic acid (t R 7.3 min and m/z 355) and 2-hydroxybenzoic acid (t R 8.6 min and m/z 267). Based on these products, a probable pathway has been proposed for the degradation of Cresol Red by Trichoderma harzianum M06.