Biomass Support Research Articles

Production of biodiesel fuel from soybean oil catalyzed by fungus whole-cell biocatalysts in ionic liquids

The methanolysis of soybean oil to produce a fatty acid methyl ester (ME, i.e., biodiesel fuel) was catalyzed by lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst in the presence of ionic liquids. We used four types of whole-cell biocatalysts: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL), recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL), Candida antarctica lipase B (r-CALB), and mono- and diacylglycerol lipase from A. oryzae (r-mdlB). w-ROL gave the high yield of fatty acid methyl ester (ME) in ionic liquid [Emim][BF 4] or [Bmim][BF 4] biphasic systems following a 24 h reaction. While lipases are known to be severely deactivated by an excess amount of methanol (e.g. 1.5 Mequiv. of methanol against oil) in a conventional system, methanolysis successfully proceeded even with a methanol/oil ratio of 4 in the ionic liquid biphasic system, where the ionic liquids would work as a reservoir of methanol to suppress the enzyme deactivation. When only w-ROL was used as a biocatalyst for methanolysis, unreacted mono-glyceride remained due to the 1,3-positional specificity of R. oryzae lipase. High ME conversion was attained by the combined use of two types of whole-cell biocatalysts, w-ROL and r-mdlB. In a stability test, the activity of w-ROL was reduced to one-third of its original value after incubation in [Bmim][BF 4] for 72 h. The stability of w-ROL in [Bmim][BF 4] was greatly enhanced by cross-linking the biocatalyst with glutaraldehyde. The present study demonstrated that ionic liquids are promising candidates for use as the second solvent in biodiesel fuel production by whole-cell biocatalysts.

Immobilization of Escherichia coli cells using polyethyleneimine-coated porous support particles for l-aspartic acid production

Escherichia coli cells were efficiently immobilized in reticulated polyvinyl formal resin biomass support particles (BSPs) that had been simply autoclaved with a solution of a cationic polymer such as polyethyleneimine. When the immobilized E. coli cells containing aspartase were used as whole cell biocatalyst for l-aspartic acid production, they showed specific aspartase activity comparable to that of non-immobilized cells.

Groundwater remediation by an in situ biobarrier: A bench scale feasibility test for methyl tert-butyl ether and other gasoline compounds

Most gasoline contains high percentages of methyl tert-butyl ether (MTBE) as an additive. The physico-chemical properties of this substance (high water solubility, low sorption in soil) result in high mobility and dissolved concentrations in soil. In situ permeable biological barriers (biobarriers, BBs) can remediate MTBE polluted groundwater by allowing pure cultures or microbial consortia to degrade MTBE when aerobic conditions are present, either by direct metabolism or cometabolism. Lab-scale batch and column tests were carried out to assess a selected microbial consortium in biodegrading MTBE and other gasoline compounds (benzene B, toluene T, ethylbenzene E, xylenes X) and to measure the parameters affecting the efficacy of a BB treatment of polluted groundwater. During the aerobic phase of the batch tests, the simultaneous biodegradation of MTBE, tert-butyl alcohol (TBA), B, T, E and o-X was observed. The rapid biodegradation of BTEXs resulted in decreased oxygen availability, but MTBE degradation was nevertheless measured in the presence of BTEXs. Stationary concentrations of MTBE and TBA were measured when anoxic conditions occurred in the systems. Values for a first order kinetic removal process were obtained for MTBE (0.031 ± 0.001 d −1), B (0.045 ± 0.002 d −1) and T (0.080 ± 0.004 d −1) in the inoculated column tests. The estimate of the BB design parameters suggested that inoculation could significantly modify (double) the longitudinal dispersivity value of the biomass support medium. No effect was observed in the retardation factors for MTBE, B and T.

Preparation and comparative characterization of immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase for enzymatic biodiesel production

In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn-2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn-2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn-2 to the sn-1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production.

Use of mono- and diacylglycerol lipase as immobilized fungal whole cells to convert residual partial glycerides enzymatically into fatty acid methyl esters

The accumulation of partial glycerides such as monoglyceride (MG) and diglyceride (DG) is one of the rate-limiting steps in plant oil methanolysis catalyzed by Rhizopus oryzae producing triacylglycerol lipase. To convert partial glycerides efficiently into their corresponding methyl esters (MEs), we attempted to use a mono- and diacylglycerol lipase (mdlB) derived from Aspergillus oryzae. By considering cost efficiency, R. oryzae and recombinant mdlB-producing A. oryzae were immobilized independently within polyurethane foam biomass support particles and directly utilized as a whole-cell biocatalyst. The mdlB-producing A. oryzae effectively exhibited substrate specificity toward MG and DG and was then used for the methanolysis of intermediate products (approximately 82% ME), which were produced using R. oryzae. In the presence of 5% water, the use of mdlB-producing A. oryzae resulted in less than 0.1% of MG and DG, whereas a considerable amount of triglyceride was present in the final reaction mixture. On the basis of these results, we developed a packed-bed reactor (PBR) system, which consists of the first column with R. oryzae and the second column containing both R. oryzae and mdlB-producing A. oryzae. Ten repeated-batch methanolysis cycles in the PBR maintained a high ME content of over 90% with MG and DG at 0.08–0.69 and 0.22–1.45%, respectively, indicating that the PBR system can be used for long-term repeated-batch methanolysis with partial glycerides at low levels. The proposed method is therefore effective for improving enzymatic biodiesel production.

Whole-cell biocatalysts for biodiesel fuel production

Biodiesel fuel (BDF), which refers to fatty acid alkyl esters, has attracted considerable attention as an environmentally friendly alternative fuel for diesel engines. Alkali catalysis is widely applied for the commercial production of BDF. However, enzymatic transesterification offers considerable advantages, including reducing process operations in biodiesel fuel production and an easy separation of the glycerol byproduct. The high cost of the lipase enzyme is the main obstacle for a commercially feasible enzymatic production of biodiesel fuels. To reduce enzyme associated process costs, the immobilization of fungal mycelium within biomass support particles (BSPs) as well as expression of the lipase enzyme on the surface of yeast cells has been developed to generate whole-cell biocatalysts for industrial applications.

Rhizopus oryzae Whole-Cell-Catalyzed Biodiesel Production from Oleic Acid in tert-Butanol Medium

During the usage of Rhizopus oryzae whole cell instead of immobilized enzyme for biodiesel production, the intracellular lipase was found to have 1,3-positional specificity when used to catalyze methanolysis of triglycerides. Thus, the application of R. oryzae whole cell in biodiesel production from triglycerides is restrained to some extent. However, it might be a promising catalyst for biodiesel production from free fatty acids (FFAs). In this paper, R. oryzae IFO4697 whole cell [immobilized within biomass support particles (BSPs)] catalyzed biodiesel production from oleic acid was studied systematically. It has been demonstrated that, in a tert-butanol system, R. oryzae whole cell exhibited both better methanol endurance and better stability than that in a solvent-free system. Molecular sieves (3 A) were added into the reaction mixture to online remove the produced water, and a much higher biodiesel yield could be achieved (biodiesel yield reached 90% at 48 h).

Can the environmental benefits of biomass support agriculture?—The case of cereals for electricity and bioethanol production in Northern Spain

Recent policy documents, such as the EC Communication on an Energy Policy for Europe (January 2007) make emphasis on the opportunities that energy applications can offer certain agricultural commodities, especially in the framework of a progressive dismantling of the Common Agricultural Policy. This paper analyses whether this can be true for wheat and barley farmers, using the real example of a straw-based power plant in Northern Spain and a theoretical factory for bioethanol production fed with cereal grain. The outcomes of such an exercise, in which their relative environmental benefits vis-à-vis fossil fuel alternatives are worked out with the aid of a simplified life-cycle approach, show that the characteristics of the electricity and biomass markets, the baseline scenario and the fuel prices are crucial for the future of the sector.

Enzymatic production of biodiesel from Jatropha oil: A comparative study of immobilized-whole cell and commercial lipases as a biocatalyst

The large percentage of biodiesel fuel (BDF) cost associated with feedstock oil and enzyme. In order to reduce the cost of BDF production, the lipase producing whole cells of Rhizopus oryzae (ROL) immobilized onto biomass support particles (BSPs) was used for the production of BDF from relatively low cost non-edible oil from the seeds of Jatropha curcas. The activity of ROL was compared with that of commercially available most effective lipase (Novozym 435). Different alcohols as a hydroxyl donor are tested, and methanolysis of Jatropha oil progresses faster than other alcoholysis regardless of lipases used. The maximum methyl esters content in the reaction mixture reaches 80 wt.% after 60 h using ROL, whereas it is 76% after 90 h using Novozym 435. Both the lipases can be used for repeated batches and both lipases exhibit more than 90% of their initial activities after five cycles. Our results suggest that whole-cell ROL immobilized on BSP is a promising biocatalyst for producing BDF from oil.

Enzymatic biodiesel fuel production using whole-cell biocatalysts immobilized within biomass support particles

Enzymatic biodiesel fuel production using whole-cell biocatalysts immobilized within biomass support particles

Immobilization of Escherichia coli cells using porous support particles coated with cationic polymers

The technique of cell immobilization using porous biomass support particles (BSPs), which is attractive from the point of view of simplicity and convenience, relies on the inherent ability of adhesive cells, as a consequence of their growth, to form films around the support material or the ability of flocculent cells to create flocs within the porous structure. In the present study, the immobilization of Escherichia coli cells using BSPs was investigated in shake-flask culture. The density of the cells immobilized within the BSPs was evaluated by measuring their intracellular lactate dehydrogenase (LDH) activity. Since E. coli K12 cells were not successfully retained within reticulated polyvinyl formal (PVF) resin BSPs with matrices of relatively small pores (pore diameter 60 microm), coating the surface of the BSPs with various polymers was examined as a way of promoting cell attachment. When positively charged polyamino acids such as poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly-L-ornithine were adsorbed onto the particle surface, they were found to increase the immobilized cell density, while neutral and negatively charged polyamino acids including poly-L-asparagine and poly-L-glutamic acid were not effective. These results indicate that E. coli cells can be efficiently immobilized in PVF resin BSPs by electrostatic interaction between the negatively charged ions of the cell surface and the positively charged polymers adsorbed onto the BSP surface. A significantly high immobilized cell density was also achieved by coating the surface of the BSPs with the synthetic polymeric amine polyethyleneimine.

Immobilized recombinant Aspergillus oryzae expressing heterologous lipase: An efficient whole-cell biocatalyst for enantioselective transesterification in non-aqueous medium

Organic esters are used in various industries such as perfumery, flavour and pharmaceutical intermediates. The use of biocatalysts for esterification and transesterification reaction under ambient reaction conditions gives better products for use in flavour and fragrance industries. In the current study, enantioselective transesterification reaction was developed by using recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica. The recombinant fungal cells were immobilized on Biomass Support Particles (BSPs) to facilitate the reusability of whole-cell biocatalyst. The immobilized CALB expressing whole-cell biocatalyst was used for the optical resolution of ( RS)-1-phenylethanol by enantioselective transesterification with vinyl acetate as acyl donor. The activity of the whole-cell biocatalyst was optimized and compared with the other whole-biocatalysts E. coli displaying CALB, S. cerevisiae displaying ROL and A. oryzae whole-cell biocatalyst expressing tglA lipase. The initial activity of immobilized CALB expressing A. oryzae was at least 15-folds higher than that of A. oryzae expressing tglA lipase. The maximum yield of ( R)-1-phenylethyl acetate reached 88.1% with an enantiomeric excess (ee) of >99% after 3.5 h reaction, while tglA lipase expressing A. oryzae showed 90% yield and 95% ee after 48 h. The recombinant A. oryzae retained its activity in hexane, heptane, toluene, cyclohexane and octane. Moreover, whole-cell biocatalyst maintained its activity for more than 15 batch reaction cycles. Current study demonstrated the applicability of recombinant whole-cell biocatalyst to bioconversion process in non-aqueous medium.

Production of bionanocapsules in immobilized insect cell culture using porous biomass support particles

L particles, composed of the L protein of the hepatitis B virus surface antigen, are candidates for a specific gene and drug delivery system. We previously constructed stably transfected insect cells for L particle production. In this study, the cells were successfully immobilized within porous biomass support particles (BSPs) in shake-flask culture. The immobilized cells showed a high specific productivity, comparable to the maximum productivities in static and shake-flask cultures of nonimmobilized cells.

Rhizopus oryzae IFO 4697 whole cell catalyzed methanolysis of crude and acidified rapeseed oils for biodiesel production in tert-butanol system

Whole cell Rhizopus oryzae ( R. oryzae) IFO4697 immobilized within biomass support particles (BSPs) was used as catalyst for biodiesel production in tert-butanol, in which the stability of the catalyst could be enhanced significantly. Different feedstocks (refined, crude and acidified rapeseed oils) were adopted further for biodiesel production in tert-butanol system and it was found that when acidified rapeseed oil was used as feedstocks, the reaction rate and final methyl ester (ME) yield were significantly higher than that of refined and crude rapeseed oil. Major differences among the aforementioned oils were found to be the contents of free fatty acid (FFA), water and phospholipids, which showed varied influences on whole cell mediated methanolysis for biodiesel production. The reaction rate increased with the increase of free fatty acid content in oils; water content had varied influence on reaction rate and biodiesel yield; using adsorbent to remove excessive water could increase biodiesel yield significantly (from 73 to 84%); it was also found interestingly that phospholipids contained in oils could increase the reaction rate to a certain extent.

Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica

To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A. oryzae niaD300, which was derived from the wild type strain RIB40, was used as the host strain. The CALB gene was isolated from C. antarctica CBS6678 and expression plasmids were constructed with and without secretion signal peptide. The lipase gene was expressed under the control of improved glaA and pNo-8142 promoters of plasmids pNGA142 and pNAN8142, respectively. The Southern blot analysis demonstrated the successful integration of the CALB gene in the genome of A. oryzae. To determine the role of signal peptide, the expression plasmids were constructed with homologous and heterologous secretion signal sequences of triacylglycerol lipase gene (tglA) from A. oryzae and lipase B (CALB) from C. antarctica, respectively. The C-terminal FLAG tag does not alter the catalytic properties of the lipase enzyme and Western blotting analysis using anti-FLAG antibodies demonstrated the presence of cell wall and membrane bound lipase responsible for the biocatalytic activity of the whole-cell biocatalyst. The resultant recombinant A. oryzae was immobilized within biomass support particles (BSPs) made of polyurethane foam (PUF) and the BSPs were successfully used for the hydrolysis of para-nitrophenol butyrate (p-NPB) and for the optical resolution of (RS)-1-phenyl ethanol by enantioselective transesterification with vinyl acetate as acyl donor.

Optimization of whole cell-catalyzed methanolysis of soybean oil for biodiesel production using response surface methodology

Utilizing whole cell biocatalyst instead of free or immobilized enzyme is a potential way to reduce the cost of catalyst in lipase-catalyzed biodiesel production. Rhizopus oryzae ( R. oryzae) IFO4697 whole cell immobilized within biomass support particles (BSPs) was used for the methanolysis of soybean oil for biodiesel production in this paper. tert-Butanol was demonstrated to be an ideal reaction medium, in which the negative effects caused by substrate methanol could be eliminated effectively. A central composite design was adopted to study the effect of tert-butanol quantity, methanol quantity, water content and dry biomass of the immobilized cell on biodiesel (methyl ester) yield. Each factor was studied in five levels. Using response surface methodology, a quadratic polynomial equation was obtained for methyl ester yield by multiple regression analysis. Biodiesel yield of 72% could be obtained under the optimal conditions and further verification experiments confirmed the validity of the predicted model.

Performance Of Aerobic Bioreactors During Treatment Of Tunning Industry Wastewater

The performance of aerobic bioreactors loaded with novel biomass support (the KMTi?½ support particles) during the treatment of tanning industry wastewater is presented in this paper. The wastewater samples were collected from TANWAT Company in Njombe, which extracts tannins from barks of the wattle tree. The wastewater generated from TANWAT is very acidic with high loading of organic compounds, which are very complex and difficult to biodegrade using conventional methods. The paper introduces a new technology for treatment of heavily loaded industrial wastewater, like tannins, that is the Three-Phase Fluidized Bed Bioreactor (TPFBB) which utilizes the KMTi?½ support particles. Bench scale bioreactors (BSBs) were used to establish the suitable inoculum for use in the TPFBB. To establish the effect of the KMTi?½ support particles, the BSBs were operated with and without the biomass support particles (BSPs). The BSBs were also inoculated with different types of inoculum (waste stabilization ponds (WSP) and rumen fluid (RF) inoculums) to establish the suitable source of microorganisms. Control samples were also implemented accordingly. The results show that the WSP was a suitable inoculum for which high chemical oxygen demand (COD) removal efficiency and tannins concentration reduction efficiency (TCRE) were observed. Tests conducted in the TPFBB with initial COD of about 13,200 ppm shows that the reactor is capable of biodegrading the tannins (at 85% efficiency) after aeration times of up to 7 days in recirculation mode. The final COD values of 1,000 ppm were reached in most cases. For final discharge, however, such a final COD necessitates the use of sub-surface flow constructed wetland (SSFCW) coupled to the TPFBB for final smoothing of the effluent. The TPFBB is recommended for treatment of tannin wastewater to minimize chemical pollution in the receiving water bodies.

Continuous production of phospholipase D using immobilized recombinant Streptomyces lividans

Using biomass support particles (BSPs) as a cell immobilized matrix, immobilized recombinant Streptomyces lividans continuously produced phospholipase D (PLD) in a yield of about 1.5 × 10 4 U/L in each of eight batches. In contrast to the original strain Streptoverticillium cinnamoneum, this heterologous expression system with an immobilization method is capable of producing secretory PLD with an 8-fold greater efficiency. The presence of both glucose and tryptone in the initial culture medium also promoted secretory production, and PLD activity around 3.0 × 10 4 U/L were achieved. In addition, the promoter region of PLD ORF was deduced, and three types of plasmid having different lengths of promoter sequence were constructed. The deduced sequence had same effect on either of PLD production or mycelium immobilization, and the transformants harboring each of three plasmids showed the similar cultivation profiles (3.0 × 10 4 U/L). A combination of the immobilization method with BSPs and S. lividans transformant harboring the deduced plasmid has the potential for producing secretory PLD in the culture supernatant continuously.

Biodiesel-fuel production in a packed-bed reactor using lipase-producing Rhizopus oryzae cells immobilized within biomass support particles

A packed-bed reactor (PBR) system using fungus whole-cell biocatalyst was developed for biodiesel fuel production by plant oil methanolysis. Lipase-producing Rhizopus oryzae cells were immobilized within 6 mm × 6 mm × 3 mm cuboidal polyurethane foam biomass support particles (BSPs) during batch cultivation in a 20-l air-lift bioreactor. Emulsification of the reaction mixture containing soybean oils and water improved the methanolysis reaction rate. Using a high flow rate for the reaction mixture in the PBR caused exfoliation of the immobilized cells from the BSPs, while the inefficient mixing of the reaction mixture at low flow rates allowed the BSPs to be covered with a hydrophilic layer of high methanol concentration, leading to a significant decrease in lipase activity. A high methyl ester content of over 90% was achieved at a flow rate of 25 l/h in the first cycle of repeated batch methanolysis and a high value of around 80% was maintained even after the tenth cycle. Comparison with methanolysis reaction in a shaken bottle suggested that the PBR enhances repeated batch methanolysis by protecting immobilized cells from physical damage and excess amounts of methanol. The process presented here is therefore considered to be promising for industrial biodiesel-fuel production.

Anaerobic biodegradation of chlorinated aliphatic compounds using packed bed reactors

The feasibility of anaerobic packed bed reactors with tezontle (volcanic stone widely available in Mexico) and mineral granular activated carbon (GAC) was explored for the degradation of the main chlorinated aliphatic compounds present in the effluent from chemical industry for ethylene and polyvinyl chloride production. The biofilm on the support materials was developed using as inoculums anaerobic sludge. The biodegradation of the halogenated aliphatic compounds (1,2-dichloroethane; 1, 1, 1 -trichloroethane; trichloroethylene and carbon tetrachloride) was possible without addition of external electron donors. High COD removal was obtained in the reactors with both materials. The use of GAC as a biomass support allows a complete biodegradation of the chlorinated aliphatic compounds at organic loads up to 1.24 kgCOD.m(-3).d(-1), with 94% of COD removal and with a biodegradation rate of 2.062 gCOD.kgGAC(-1).d(-1). The reactor with tezontle also had high biodegradation capacity, but the biodegradation of the 1, 1 -dichloroethene, which appeared as intermediate product, was not reached at the required level in the studied range of organic loads.