Biomass Support Particles Research Articles

Enhancing the production and immobilization of cell‐bound lipase from yeast‐like fungus Magnusiomyces capitatusA4C for sustainable biodiesel production in a packed bed reactor

AbstractMagnusiomyces capitatus A4C, a mycelium‐forming and lipase‐producing yeast‐like fungus, was employed in a five‐level factorial design to optimize the collective and interactive influences of carbon, nitrogen and emulsifying sources and their possible effects on cell‐bound lipase (CBL) and cell biomass production. The cell culture of M. capitatus A4C was incubated along with biomass support particles (BSPs) to immobilize the enzyme while anchoring CBL on their surfaces. Among the BSPs tested, CBL immobilized on loofah sponge under optimized conditions showed a substantial hydrolytic activity of 12.7 U mL−1 and a cell‐loading capacity of 0.61 g g−1 of BSPs. Immobilized CBL was applied for biodiesel production via transesterification and esterification. The conversion percentage of triacylglycerides was approximately 100% at 24 h with the addition of water at 1:1 (v/v). The conversion of oleic acid into biodiesel via esterification was 100% at 48 h in the presence of 15% (v/v) isooctane. Further, biodiesel production was scaled up using a packed bed reactor. The batch production of biodiesel in a packed bed reactor through transesterification was 96.2%, with a circulation flow rate of 5.5 mL min−1 for 18 h. On the other hand, oleic acid conversion into biodiesel via esterification was 99.5%, with a circulation flow rate of 5.5 mL min−1 for 24 h. Further investigation revealed that the immobilized biocatalyst exhibited higher stability with esterification (85.3% fatty acid methyl ester) after ten repeated cycles.

Growth kinetic of Rhizopus sp. immobilised on loofah sponge for whole-cell biocatalyst in different cultivation media

Immobilisation of filamentous fungi such as Rhizopus sp. onto biomass support particles (BSP) surface was studied as whole-cell biocatalyst. The function was to aid fermentation process. By using the microbial immobilisation process, the complex procedures of isolation, purification and immobilisation of extracellular enzyme can be avoided. Loofa sponge was selected as the BSP to aid in the immobilisation of the cells. In this study, the growth of immobilised Rhizopus sp. on loofa sponge was compared in four different cultivation media and the attachment of Rhizopus sp. on loofa sponge was investigated using scanning electron microscopy (SEM). The media used in this study were hydrolysed cassava starch, cassava dextrose, potato dextrose, and soy dextrose. The process condition and other parameters which were temperature, pH, inoculum dilution, and weight of loofa sponge, fixed at 30 °C, 7, 20 mL, and 2 g, respectively. The highest and lowest of maximum growth (ymax) of the immobilised cells were determined from potato dextrose and soy powder mixed with dextrose media, respectively at 1.5281 g/g and 1.0370 g/g. Whilst, the highest and lowest observed rate constant (k) were obtained from cassava starch mixed with dextrose and soy powder mixed with dextrose, which respectively at 2.9403 day−1 and −0.8763 day−1. SEM images showed the presence of mycelia attached to the loofah sponge after immobilisation process. In conclusion, Rhizopus sp. has been successfully immobilised on loofah sponge as a whole-cell biocatalyst.

Palm Oil Decanter Cake Wastes as Alternative Nutrient Sources and Biomass Support Particles for Production of Fungal Whole-Cell Lipase and Application as Low-Cost Biocatalyst for Biodiesel Production

This is the first report on the possible use of decanter cake waste (DCW) from palm oil industry as alternative nutrient sources and biomass support particles for whole-cell lipase production under solid-state fermentation (SSF) by newly isolated fungal Aspergillus sp. MS15 and their application as a low-cost and environment-friendly biocatalyst for biodiesel production. The results found that DCW supplemented with 0.1% K2HPO4, 0.05% MgSO4·7H2O, 1% peptone and 2% urea and pH adjusted to 6.0 was optimal for whole-cell lipase production. The optimal moisture content and fermentation temperature was 60% and 37.5 °C, respectively. Environmentally friendly biodiesel production, through either esterification or transesterification using whole-cell lipase immobilized on DCW as a biocatalyst, was optimized. The optimal reaction temperature for both reactions was 37 °C. The whole-cell lipase effectively esterified oleic acid into >95% biodiesel yield through esterification under optimal water activity at 0.71 and an optimal methanol to oleic acid molar ratio of 2:1, and also effectively transesterified palm oil under optimal water activity at 0.81 and an optimal methanol to oil molar ratio of 3:1. The fuel properties of produced biodiesel are close to the international biodiesel standards. These results have shown the circular utilization of palm oil mill waste for the low-cost production of an effective biocatalyst, and may contribute greatly to the sustainability of renewable bioenergy production.

Utilizing palm oil mill effluent (POME) for the immobilization of Aspergillus oryzae whole‐cell lipase strains for biodiesel synthesis

AbstractPalm oil mill effluent (POME) is a palm industrial waste by‐product that is readily available and possesses high quantities of the organic compounds that are necessary for microbial growth. This study investigated the potential of immobilized Aspergillus oryzae whole cells expressing Candida antartica lipase B (r‐CALB) when using a POME‐based medium as a low‐cost carbon source within biomass support particles. The dry cell weight, hydrolytic activity, and remaining protein were measured for a variety of POME (0–2% w/v) and carbon source concentrations (0–2% w/v) with an incubation time of 96 h. Using POME in the culture medium as a carbon source instead of glucose doubled the retention of cell weight from 1.16 ± 0.03 to 2.36 ± 0.02 g within the biomass support particles and improved the hydrolytic activity from 1.61 ± 0.02 to 2.23 ± 0.02 U mg−1 in whole‐cell lipase. This immobilized r‐CALB was used as a catalyst to esterify POME into biodiesel. This synthesis produced an ester content of 97.52 ± 0.21% w/w after 96 h. Immobilized r‐CALB was reusable for more than ten batches, and resulted in an ethyl ester content of more than 90% w/w even after 10 cycles. The present study demonstrated that POME could serve as a sustainable carbon source for industrial microbial culture with the additional incentive of being a treatment that is inexpensive and sustainable. © 2021 Society of Chemical Industry and John Wiley & Sons, Ltd

Nutrient (sulphate) removal from wastewater in inverse fluidized bed biofilm reactor

Sulphate is one of the inorganic nutrient pollutants present in wastewater which needs to be treated due to present day global stringent emission norms. Among the several available biological means to treat industrial wastewaters, the use of inverse fluidized bed biofilm reactors (IFBBRs) has gained much attention in recent years over other fluidized bed reactors. In this work, the feasibility of removal of sulphate from synthetic wastewater having initial concentrations of 13.096, 32.74, and 65.48 mg/l was studied using mixed bacterial culture. Spherical polypropylene balls were taken as biomass support particles having density of 920 kg/m3 and diameter of 5.63 mm. Three different bed-volume to reactor-volume (Vb/Vr) ratios i.e., 0.304, 0.380, & 0.445 and three different superficial air velocities i.e., 0.0065, 0.0085, & 0.0106 m/s were considered for experimental runs. The reactor inoculum was prepared from the sludge collected from local steel industry’s wastewater treatment plant. The effects of different input parameters such as Vb/Vr ratio, superficial air velocities, and initial concentration of sulphate in synthetic wastewater on the output sulphate concentration were studied. Temperature of 30–32 °C and pH of 8.3–8.8 were maintained throughout the degradation studies. The reactor was operated in semi-batch recirculation mode with superficial water velocity of 0.0021 m/s. Sulphate concentration was measured by turbidity method using UV spectrophotometer at 420 nm which is as per APHA standards. The maximum removal efficiency of sulphate was found to be 72–75% which was achieved at a Vb/Vr ratio of 0.380 and the superficial gas velocity of 0.0085 m/s observed at a time of 40 h. Higher sulphate reduction was not achieved due to presence of more inorganic salts in the synthetic wastewater. At higher initial sulphate concentration, reactor operational difficulties were observed.

The production of immobilized whole-cell lipase from Aspergillus nomius ST57 and the enhancement of the synthesis of fatty acid methyl esters using a two-step reaction

The production of immobilized whole-cell lipase from Aspergillus nomius ST57 was optimized by cultivation in 50mL of growth medium with the addition of 100, 0.5×0.5×0.5cm3 pieces of biomass support particles at 150rpm, 30°C for 84h. The hydrolytic activity of the immobilized whole-cell lipase was 30.77% higher than the activity obtained from free biomass (based on 1mg dry cell weight). Response surface methodology with a central composite design, which consisted of three parameters: the amount of the immobilized whole-cell lipase, the reaction temperature and the water content, was applied to optimize the synthesis of fatty acid methyl esters (FAME) from palm oil in a solvent-free system by the transesterification reaction using the immobilized whole-cell lipase from A. nomius ST57 as a biocatalyst. The highest percentage of FAME (78.23%) was obtained from a reaction mixture containing 1g palm oil, 1:4 molar ratio of oil to methanol, 23% (w/w of palm oil) of 50mM sodium acetate buffer pH 5 and 16 pieces of immobilized whole-cell lipase at 40°C and 1200rpm for 4h. The water-free transesterified palm oil mixture was then esterified using the immobilized whole-cell lipase. The highest percentage of FAME (94.77%) was obtained at 1g transesterified palm oil mixture, 16 pieces of immobilized whole-cell lipase and 0.06g of methanol for 2h. The immobilized whole-cell lipase retained 90.95% transesterification activity and 100% esterification activity after use for 10 batches.

Biodiesel synthesis from microalgae using immobilized Aspergillus niger whole cell lipase biocatalyst

Whole cell lipase catalysis and microalgal feedstocks make overall biodiesel synthesis greener and sustainable. In this study, a novel approach of whole cell lipase-catalyzed conversion of Scenedesmus obliquus lipids was investigated for biodiesel synthesis. Microalgal biodiesel was characterized for its fuel properties. Optimization of process parameters for immobilized Aspergillus niger whole cell lipase-catalyzed biodiesel synthesis was carried out. Highest biodiesel conversion of 53.76% was achieved from S. obliquus lipids at 35 °C, methanol to oil ratio of 5:1 and 2.5% water content based on oil weight with 6 BSPs (Biomass support particles). Step-wise methanol addition was applied to account for methanol tolerance, which improved biodiesel conversion upto 80.97% and gave 90.82 ± 1.43% yield. Immobilized A. niger lipase can be used for 2 batches without significant loss in conversion efficiency. Most of the fuel properties of biodiesel met the specifications set by international standards.

Two-step biocatalytic process using lipase and whole cell catalysts for biodiesel production from unrefined jatropha oil.

To avoid lipase deactivation by methanol in the enzymatic transesterification process, a two-step biocatalytic process for biodiesel production from unrefined jatropha oil was developed. Unrefined jatropha oil was first hydrolyzed to free fatty acids (FFAs) by the commercial enzyme Candida rugosa lipase. The maximum yield achieved of FFAs 90.3% at 40 °C, water/oil ratio 0.75:1 (v/v), lipase content 2% (w/w) after 8 h reaction. After hydrolysis, the FFAs were separated and converted to biodiesel by using Rhizopus oryzae IFO4697 cells immobilized within biomass support particles as a whole-cell biocatalyst. Molecular sieves (3 Å) were added to the esterification reaction mixture to remove the byproduct water. The maximum fatty acid methyl ester yield reached 88.6% at 35 °C, molar ratio of methanol to FFAs 1.2:1, molecular sieves (3 Å) content 60% (w/w) after 42 h. In addition, both C. rugosa lipase and R. oryzae whole cell catalyst in the process showed excellent reusability, retaining 89 and 79% yields, respectively, even after six batches of reactions. This novel process, combining the advantages of enzyme and whole cell catalysts, saved the consumption of commercial enzyme and avoid enzyme deactivation by methanol.

Biodiesel Production from Waste Cooking Oils by Using Immobilized Microorganisms as Whole Cell Catalysts

The main hurdle to the commercialization of lipase-catalyzed production of biodiesel is the cost of enzyme and feedstock oil. In order to reduce the cost of biodiesel production, the lipase-producing whole cells ofAspergillus nigerand immobilized onto biomass support particles (BSPs) were used for the production of biodiesel from waste cooking oil. This article studies this technological process, focusing on optimization of several process parameters, including the water content, catalyst loading and molar ratio of methanol to waste cooking oil. The results indicate that the water content of 20%(based on oil weight), BSPs-immobilized cell catalysts of 6% and methanol/oil molar ratio of 4:1 are the optimum conditions for biodiesel production from waste cooking oil. Under the optimum conditions, the maximum methyl ester (ME) content in the reaction mixture reaches 84.7 wt.% after 72 h. In addition, the whole-cell biocatalysts showed excellent reusability, retaining 73% productivity after 6 batches. Our results suggest that whole-cell A. niger immobilized on BSP is a promising biocatalyst for biodiesel production from waste cooking oil.

Development of Clay Foam Ceramic as a Support for Fungi Immobilization for Biodiesel Production

Biodiesel is an attractive alternative fuel because of its nontoxicity and biodegradability. Biodiesel is produced through transesterification of vegetable oils' triglyceride. It is obtained from vegetable oils or fats either by chemical or enzyme-cataly zed transesterification with methanol or ethanol. Use of whole-cell biocatalyst immobilized within biomass support particles (BSPs) can overcome the obstacle of high cost of enzymatic catalyst. The objective of this research is to produce clay foam ceramic as BSP by replica method from raw materials such as clay, sodium silicate and sodium tripolyphosphate. To prepare the whole cell biocatalyst Rhizopus oryzae fungi (PTCC 5174) was immobilized on the ceramic foam with bulk density and porosity of 0.3 g/cm 3 and 88.8%, respectively. The deposited biomass on clay foam particles can be observed through scanning electronic microscopy (SEM). A packed-bed reactor (PBR) system using whole-cell biocatalyst was developed for biodiesel production by pretreated used cooking oil (UCO). Clay foam ceramic seems to be more suitable compared to polyurethane foam for supporting fungi immobilization because it shows high mechanical strength and reduces damaged microorganisms.

Nutrient removal and microbial communities' development in a young unplanted constructed wetland using Bauxsol™ pellets to treat wastewater

Municipal wastewater was treated over a six month period in an unplanted constructed wetland with a lower soil layer and an upper Bauxsol™ pellet layer. The interactions between Bauxsol™ pellets, soil, effluent and microbial communities demonstrated a positive influence on contaminant removal. Bauxsol™ treated effluent showed >95% phosphate removal and ~26% nitrogen removal during the trial. Substantial quantities of nitrate, trace-metals and Colwell P were bound to the pellets, whereas only ammonium was bound to the soil. The structure of microbial communities analysed by denaturing gradient gel electrophoresis (DGGE) showed distinct bacterial communities attached to Bauxsol™ pellets and soil owing to differences in geochemistry and micro-environmental conditions. Polymerase chain reaction (PCR) amplification of specific marker genes (i.e. bacterial and archaeal amoA genes, nosZ gene, and hzo gene) was used to evaluate the presence of microbial communities associated with nitrogen transformation. Data revealed the co-existence of aerobic ammonia-oxidising bacteria, anaerobic ammonia-oxidising bacteria (anammox) and denitrifiers attached to Bauxsol™ pellets and ammonia-oxidising bacteria and archaea attached to soil. This study successfully demonstrates that Bauxsol™ pellets are a suited alternative media for constructed wetland to treat wastewater effectively removing phosphate and serving as biomass support particles for bacterial communities associated with nitrogen-cycling.

Diversity of microbial communities in an attached-growth system using Bauxsol™ pellets for wastewater treatment

Columns of Bauxsol™ pellets were used in a field experiment as biomass support particle for wastewater microbial communities. The attached microbial community structure was analysed using denaturing gradient gel electrophoresis (DGGE), targeting the 16S rDNA gene's V3 region. DGGE profiles showed that the type and composition of support particles used (i.e. Bauxsol™ pellets or gravel) had a significant impact on the attached bacterial communities (64% dissimilarity). In addition, ecological indices revealed a more heterogeneous bacterial community structure on the Bauxsol™ pellets. TOC/TN ratios post-experiment (6.5–9.3) suggested a good level of biological activity (i.e. active biofilm) in the Bauxsol™ columns. Moreover, Bauxsol™ pellets were mostly made of inorganic carbon, suggesting insoluble carbonate biomineralisation. Polymerase chain reaction (PCR) amplification of specific marker genes (i.e. bacterial and archaeal amoA genes, nosZ gene, and hzo gene) were used to identify the presence of attached bacterial communities associated with nitrogen transformation. The results along with geochemical data (i.e. up to 50% nitrogen removal) revealed co-existence of ammonia-oxidising bacteria, denitrifiers, and anammox organisms. This study conclusively demonstrates that microbial communities are well-adapted to Bauxsol™ pellets and bacterial communities involved in the nitrogen cycle are present.

Rigid polyurethane foams obtained from tall oil and filled with natural fibers: Application as a support for immobilization of lignin-degrading microorganisms

The forest biomass represents an abundant, renewable, non-food competition, and low-cost resource that can play an alternative role to petro-resources. The first topic of the research activity is focused on the use of wood and a pulp mill by-product—tall oil —as raw materials for the production of rigid polyurethane foams. The maximum content of the renewable resource in ready foams is 26%. By using biopolymers as a matrix, a natural way is to reinforce them with natural fibers. Further advantages are significant weight and cost savings and, at the same time, replacement of petrochemical raw materials. Three different natural fibers (cellulose, wood, and modified cellulose) were tested as a filler of foams. Rigid polyurethane foams was used as biomass support particles for immobilization of microorganisms. Suspension cultures of the organism with biomass support particles can promote the adhesion of cells to the porous matrix surface, and subsequently the cells become immobilized during the cultivation. This method for cell immobilization has a great potential for enhancing the production of proteins or chemicals in culture supernatants. The presence of natural fibers in the matrix promotes the enzyme production because the material not only functions as an attachment place but also supplies some nutrients to the microorganism and induces the production of ligninolytic enzymes. This paper discusses the studies into the use of tall oil as a renewable source in rigid polyurethane foam production

Screening, immobilization and utilization of whole cell biocatalysts to mediate the ethanolysis of babassu oil

Abstract The screening, biomass growth of lipase-producing fungus isolated from different sources and available at URM (University Recife Mycologia), as well as, the immobilization and utilization of the whole cells for the transesterification of babassu oil were investigated. Rhizopus oryzae (URM 3231, 4692), Mucor circinelloides (URM 4140, 4182) and Penicillium citrinum URM 4216 were considered to be good intracellular lipase producers whereas those from Mucor hiemalis URM 4144 and Mucor piriformis URM 4145 were weaker. Fungi biomass containing high lipase activities was immobilized on different biomass support particles (BSPs) and with the exception of Penicillium citrinum URM 4216 all the other fungi strains exhibited high lipase activity (20–50 U g −1 ) when immobilized in situ using polyurethane foam particles. Transesterification activities of the immobilized whole cells were evaluated in the ethanolysis reaction with babassu oil and the highest performance was attained by M. circinelloides URM 4182 giving 83.22 ± 3.68% ester yield in less than 96 h reaction. The biocatalyst operational stability was also assessed and an inactivation profile was found to follow the Arrhenius model, revealing values of 26 days and 2.6 × 10 −2 day −1 , for half-life and a deactivation coefficient, respectively. The purified product (biodiesel) exhibited viscosity (6.63 cSt) close to the value to attend specifications by the ASTM D6751 to be used as biofuel. Results are favorable compared with data already reported in the literature and demonstrated that M. circinelloides URM 4182 whole cells is a cheaper biocatalyst that can be used in the biodiesel synthesis.

Improved performance of a packed-bed reactor for biodiesel production through whole-cell biocatalysis employing a high-lipase-expression system

To improve enzymatic biodiesel production, we developed a packed-bed reactor (PBR) system using recombinant cells, into which a strong enolase promoter and 5′ untranslated region were introduced. Aspergillus oryzae expressing Fusarium heterosporum lipase was immobilized within biomass support particles (BSPs) during cultivation and used directly as a whole-cell biocatalyst. BSP-immobilized A. oryzae carrying three copies of the expression cassette showed a higher performance than previously developed cell systems, resulting in 87.5% conversion after 10 passes in PBR. The performance was also affected by operational factors including residence time and methanol feeding. After optimization, the PBR system attained a final methyl ester content of 96.1% with a residence time of 140min per pass and stepwise addition of 4.25molar equivalents of methanol to oil for 6 passes. Moreover, lipase activity was maintained for 5 batch cycles. Therefore, the developed PBR employing a high-lipase-expression system is considered useful for improving enzymatic biodiesel production.

Transesterification of phosphatidylcholine in sn-1 position through direct use of lipase-producing Rhizopus oryzae cells as whole-cell biocatalyst

The enzymatic process presents an advantage of producing specified phospholipids that rarely exist in nature. In this study, we investigated the regiospecific modification of phosphatidylcholine (PC) in the sn-1 position using immobilized Rhizopus oryzae. In a reaction mixture containing egg yolk PC and exogenous lauric acid (LA) in n-hexane, lipase-producing R. oryzae cells immobilized within biomass support particles (BSPs) showed a much higher transesterification activity than lipase powders. To improve the product yield, several parameters including substrate ratio and reaction time were investigated, resulting in the incorporation of 44.2% LA into the product PC after a 48-h reaction. The analysis of the molecular structure showed that a large proportion of exogenous LA (>90%) was incorporated in the sn-1 position of the enzymatically modified PC. Moreover, the BSP-immobilized R. oryzae maintained its activity for more than 12 batch cycles. The presented results, therefore, suggest the applicability of BSP-immobilized R. oryzae as a whole-cell biocatalyst for the regiospecific modification of phospholipids.

Biodiesel production using Aspergillus niger as a whole-cell biocatalyst in a packed-bed reactor

Enzymatic lipase transesterification of palm oil to biodiesel in a packed-bed reactor (PBR) using a novel strain of the fungus Aspergillus niger, immobilized within polyurethane biomass support particles (BSPs), was investigated. A three-step addition of methanol was used to reduce lipase inhibition by immiscible methanol. The influence of water content and PBR flow rate was investigated. FAME yield was enhanced with an increase of PBR flow rate in the range of 0.15–30 L h−1, where inefficient mixing of the reaction mixture at lower flow rates resulted in low conversion rates i.e. 69% after 72-h reaction. Adding the third mole equivalent of methanol resulted in lipase inhibition due to methanol migration into the accumulated glycerol layer. Glutaraldehyde (GA) solution (0.5 vol.%) was used to stabilize lipase activity, which led to a high FAME yield (>90%) in the PBR after 72-h of reaction time at a flow rate of 15 L h−1, and a water content of 15%. Moreover, a high conversion rate (>85%) was maintained after four palm oil batch conversion cycles in the PBR. In contrast, lipase activity of non-GA-treated cells decreased with each PBR batch cycle, where only 70% FAME was produced after the forth PBR cycle. Transesterification of palm oil in a PBR system using BSPs-immobilized A. niger as a whole-cell biocatalyst is a viable process for enzymatic biodiesel production.

Ethanolysis of rapeseed oil to produce biodiesel fuel catalyzed by Fusarium heterosporum lipase-expressing fungus immobilized whole-cell biocatalysts

We demonstrated ethanolysis of rapeseed oil to produce biodiesel fuel using lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst. We prepared two types of whole-cell biocatalyst: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL) and recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL). Both w-ROL and r-FHL successfully catalyzed the ethanolysis of rapeseed oil, and the fatty acid ethyl ester yield was as high as 79% (w-ROL) or 94% (r-FHL). In the case of r-FHL, the residual monoglycerides (MGs) and diglycerides (DGs) were no more than 0.73 and 0.18%, respectively. In addition, r-FHL could be recycled for the ethanolysis of rapeseed oil, retaining over 85% fatty acid ethyl ester yield by the fifth cycle. r-FHL was revealed to be a promising catalyst for biodiesel production using rapeseed oil and ethanol.

A newly isolated fungal strain used as whole‐cell biocatalyst for biodiesel production from palm oil

AbstractA strain of Aspergillus niger isolated from atmospherically exposed bread and Jatropha curcas seed was utilized as a whole‐cell biocatalyst for palm oil methanolysis to produce fatty acid methyl esters (FAME), or biodiesel. The A. niger strain had a lipase activity of 212.58 mU mL−1 after 144 h incubation at 25 °C with an initial pH value of 6.5, using 7% polypeptone (w/w on basal medium) as the nitrogen source and 3% olive oil (w/w on basal medium) as a carbon source. The A. niger cells spontaneously immobilized within polyurethane biomass support particles (BSPs) during submerged fermentation. Thereafter, the methanolysis of palm oil was achieved via a three‐step addition of methanol in the presence of BSPs‐immobilized with A. niger cells. The influence of water content, reaction temperature and enzyme concentration on reaction rate was investigated. An 8% water content and a temperature of 40 °C in the presence of 30 immobilized BSPs, resulted in an 87% FAME yield after 72 h.

Immobilization of 293 cells using porous support particles for adenovirus vector production

Adenovirus vector production by anchorage-independent 293 cells immobilized using porous biomass support particles (BSPs) was investigated in static and shake-flask cultures for efficient large-scale production of adenovirus vectors for gene therapy applications. The density of cells immobilized within BSPs was evaluated by measuring their WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) reduction activity. In shake-flask culture, 293-F cells, which were adapted to serum-free suspension culture, were not successfully retained within reticulated polyvinyl formal (PVF) resin BSPs (2×2×2mm cubes) with matrices of relatively small pores (pore diameter 60μm). When the BSPs were coated with a cationic polymer polyethyleneimine, a high cell density of more than 10(7) cells cm(-3)-BSP was achieved in both static and shake-flask cultures with regular replacement of the culture medium. After infection with an adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP), the specific Ad EGFP productivity of the immobilized cells was comparable to the maximal productivity of non-immobilized 293-F cells by maintaining favorable conditions in the culture environment.