Simultaneous production of citric acid and erythritol from crude glycerol by Yarrowia lipolytica Wratislavia K1

This study shows a possible microbial process for utilization of crude glycerol generated by the biodiesel industry for citric acid and erythritol production. Simultaneous production of citric acid and erythritol under nitrogen-limited conditions with glycerol as the carbon source was achieved with an acetate negative mutant of Y. lipolytica Wratislavia K1 in fed-batch cultivations. The effect of the initial glycerol concentration (from 30–180 g dm⁻³) on the citrate and erythritol production was investigated. As a result of the experiments, maximum citric acid production (110 g dm⁻³) and a very high amount of erythritol (81 g dm⁻³) were determined after 168 h of fed-batch cultivation with the initial glycerol concentration of 150 g dm⁻³ and the total glycerol concentration of 250 g dm⁻³. In addition, the citric acid to isocitric acid ratio of the products from this strain was 35.5:1. Presented at the 34th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 21–25 May 2007.     

Expression of Trichoderma reesei exo-cellobiohydrolase I in transgenic tobacco leaves and calli

Expression of Trichoderma reesei exo-cellobiohydrolase I (CBHI) gene in transgenic tobacco was under the control of CaMV 35S promoter. In transgenic leaf tissues, CBHI activity up to 66.1 μmol/h/g total protein was observed. In transgenic calli, the highest CBHI activity was 83.6 μmol h/g total protein. Protein immunoblot analysis confirms the presence of CBHI enzyme in both transgenic calli and leaf tissues. CBHI expression levels accounted for about 0.11% and 0.082% of total protein in transgenic leaf tissues and calli, respectively, Furthermore, expression of CBHI gene did not affect normal growth and development of transgenic plants.     

Adsorption of a Trichoderma reesei endoglucanase and cellobiohydrolase onto bleached Kraft fibres

Cellulases can be used to modify pulp fibres. For the development of biotechnical applications, a better understanding of the adsorption of cellulases onto commercial wood fibres is needed. In this work, the adsorption behaviour of purified CBH I and EG II on bleached Kraft fibres was investigated. Three variables were studied with respect to their effect on adsorption: fibre type (hardwood or softwood), fibre history (never-dried or once-dried), and ionic strength. The results showed that fibre history had the largest influence on the extent of adsorption of each enzyme. The effect of ionic strength was shown to be dependent on the enzyme and fibre type. At high ionic strength, CBH I exhibited a higher affinity for both once-dried and never-dried fibres at low enzyme concentrations; however, salt was shown to decrease the extent of adsorption at higher enzyme dosages. In contrast, salt increased the maximum adsorption of EG II, most notably on the once-dried hardwood fibres. Fibre type was also shown to affect adsorption behaviour. CBH I had a higher affinity for softwood fibres than for hardwood fibres at low enzyme concentrations. The maximum adsorption of EG II onto once-dried softwood fibres increased by 80% compared to the once-dried hardwood fibres. Interestingly, this did not correlate to in creased fibre hydrolysis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Citric acid production from raw glycerol by acetate mutants of Yarrowia lipolytica

Three acetate mutants of the yeast species Yarrowia lipolytica were screened using batch cultivation. The strain Y. lipolytica 1.31 was found to be the most suitable for citric acid production from raw glycerol, a by-product of biodiesel production from rapeseed oil. At the initial concentration of glycerol of 200 g dm⁻³, the citric acid production of 124.5 g dm⁻³, yield of 0.62 g g⁻¹, and productivity of 0.88 g dm⁻³ h⁻¹ were achieved. Presented at the 33rd International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 22–26 May 2006.     

β-Glucosidase production by Trichoderma reesei

The hydrolysis of cellulose to the water-soluble products cellobiose and glucose is achieved via synergistic action of cellulolytic proteins. The three types of enzymes involved in this process are endoglucanases, cellobiohydrolases, and β-glucosidases. One of the best fungal cellulase producers is Trichoderma reesei RUT C30. However, the amount of β-glucosidases secreted by this fungus is insufficient for effective cellulose conversion. We investigated the production of cellulases and β-glucosidases in shake-flask cultures by applying three pH-controlling strategies: (1) the pH of the production medium was adjusted to 5.8 after the addition of seed culture with no additional pH adjustment performed, (2) the pH was adjusted to 6.0 daily, and (3) the pH was maintained at 6.0 by the addition of Tris-maleate buffer to the growth medium. Different carbon sources—Solka Floc 200, glucose, lactose, and sorbitol—were added to standard Mandels nutrients. The lowest β-glucosidase activities were obtained when no pH adjustment was done regardless of the carbon source employed. Somewhat higher levels of β-glucosidase were measured in the culture filtrates when daily pH adjustment was carried out. The effect of buffering the culture medium on β-glucosidase liberation was most prominent when a carbon source inducing the production of other cellulases was applied.     

Electrochemical study of heavy metals and metallothionein in yeast Yarrowia lipolytica

The bioaccumulation of heavy metals (cadmium, nickel, cobalt and zinc) and the effect of these metals on the production of metallothionein and metallothionein-like proteins (MT) in Yarrowia lipolytica was studied by electrochemical methods. The concentrations of heavy metals were determined by differential pulse voltammetry (DPV). A combination of the constant current chronopotentiometric stripping analysis (CPSA) and adsorptive transfer stripping technique (AdTS) was used to determine the content of MT in cells. Both the bioaccumulation of heavy metals and the production of MT in different cell compartments of Y. lipolytica exposed to heavy metals were monitored. The LD(50) of each metal was determined from the number of viable cells in yeast cultures: LD(50)Cd (37.5 microM), LD(50)Ni (570 microM), LD(50)Co (700 microM), and LD(50)Zn (1800 microM). The highest concentrations of heavy metals were found in the cell wall and membrane debris while the lowest concentrations were detected in the cytoplasm. Cadmium and nickel showed the most significant effect on the production of MT. This study provides new insights into the ecophysiology of microorganisms and demonstrates the potential use of these electrochemical methods in biotechnology.     

Polykaryon formation using a swollen conidium of Trichoderma reesei

The cellulolytic fungus, Trichoderma has oval and mononucleate conidia. When these conidia are incubated in a liquid medium, they begin to swell and their shape becomes spherical followed by an increase in inner space. In such swollen conidia, it is possible to produce a larger autopolyploid nucleus using a mitotic arrester compared with the case of the original conidia. In this study, polykaryon formation was attempted using these swollen conidia. Dried mature green conidia of Trichoderma reesei QM6a (IFO 31326) were incubated in Mandel's medium in order to swell. The swollen conidia were treated with a mitotic arrester, colchicine, for autopolyploidization. After autopolyploidization, polykary on formation was carried out using the swollen conidia. After the treatment, multiple smaller nuclei whose diameter was almost the same as that of the original strain were generated from an autopolyploid nucleus in a swollen conidium. A cellulase hyperproducer without decrease in growth rate could be selected using such swollen conidia.     

Active nuclear shuffling system using a swollen conidium of Trichoderma reesei

Cellulase hyperproducers of Trichoderma reesei can be constructed using autopolyploidization and haploidization techniques. To increase the efficiency of this method, the active nuclear shuffling system in a swollen conidium was effective. A dried mature green conidium of a model strain, T. reesei QM6a (IFO 31326), was swollen to make room for a larger autopolyploid nucleus. After colchicine treatment, a larger autopolyploid nucleus was produced in such a swollen conidium. Benomyl treatment of swollen conidia generated multiple smaller nuclei from one larger autopolyploid nucleus. Those smaller nuclei were transported through conidia to mycelia after germination. This system could contribute to increasing the efficiency of genetic shuffling.     

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources—including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucosegrown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO₂ evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO₂ evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO₂ evolution are analyzed and discussed herein.     

Bioemulsifier production in batch culture using glucose as carbon source by Candida lipolytica

The yeast Candida lipolytica IA 1055 produced an inducible extracellular emulsification activity while utilizing glucose at different concentrations as carbon source during batch fermentation at 27°C. In all glucose concentrations studied, maximum production of emulsification activity was detected in the stationary phase of growth, after pH reached minimal values. The bioemulsifier isolated was a complex biopolymer constituting proteins, carbohydrates, and lipids. The results obtained in this work show that the biosynthesis of a bioemulsifier is not simply a prerequisite for the degradation of extracellular hydrocarbon.     

Comparison of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Lipase Immobilization Yield of Entrapment,Adsorption, and Covalent Bond Techniques

The purpose of this study was to immobilize lipase from Yarrowia lipolytica using three methods including inclusion, adsorption, and covalent bond to study enzyme leaching, storage, and catalytic properties. Sodium alginate and chitosan were the polymers selected to immobilize lipase by inclusion. The beads of each polymer were dried by freeze drying and fluidization. The results show that chitosan was more adapted to the inclusion of lipase. Even though freeze dried, bead activity was low compared to that of fluidized beads. The freeze-drying process seems to produce suitable beads for storage at 4 and 20 degrees C. The immobilization by adsorption was carried out on both celite and silica gel. Maximum immobilization yield of 76% was obtained with celite followed by 43% in silica gel. The enzyme adsorbed on the two supports exhibited greater stability at a certain temperature (50 degrees C) and in no polar solvents (Isooctane, n-heptane, and n-hexane). In addition, the lipase immobilized by covalent bond retained residual activity equitable to 70%. It was demonstrated that the enzyme immobilized by covalent bond showed greater activity (80%) after 5 months of storage.     

Comparison of different strains of the yeastYarrowia lipolytica for citric acid production from glucose hydrol

Four commercial strains and two mutants of the yeast species Yarrowia lipolytica were screened using batch fermentation. Strain Y. lipolytica A-101-1.14 (induced with UV irradiation) was found to be the most suitable for citric acid production from glucose hydrol (39.9% glucose and 2.1% other sugars), a byproduct of glucose production from potato starch. The specific rate of total citric and isocitric acid production was 0.138 g/g.h, the yield on consumed glucose 0.93 g/g, and the productivity achieved was as high as 1.25 g/L.h. All of the tested yeast strains were able to utilize only the glucose from the glucose hydrol medium. Thus, some residual higher oligosaccharides remained in the process effluent.     

Effect of acetic acid and furfural on cellulase production of Trichoderma reesei RUT C30

Because of the high temperature applied in the steam pretreatment of lignocellulosic materials, different types of inhibiting degradation products of saccharides and lignin, such as acetic acid and furfural, are formed. The main objective of the present study was to examine the effect of acetic acid and furfural on the cellulase production of a filamentous fungus Trichoderma reesei RUT C30, which is known to be one of the best cellulase-producing strains. Mandels’s mineral medium, supplemented with steam-pretreated willow as the carbon source at a concentration corresponding to 10 g/L of carbohydrate, was used. Four different concentration levels of acetic acid (0–3.0 g/L) and furfural (0–1.2 g/L) were applied alone as well as in certain combinations. Two enzyme activities, cellulase and β-glucosidase, were measured. The highest cellulase activity obtained after a 7-d incubation was 1.55 FPU/mL with 1.0 g/L of acetic acid and 0.8 g/L of furfural added to the medium. This was 17% higher than that obtained without acetic acid and furfural. Furthermore, the results showed that acetic acid alone did not influence the cellulase activity even at the highest concentration. However, β-glucosidase activity was increased with increasing acetic acid concentration. Furfural proved to be an inhibiting agent causing a significant decrease in both cellulase and β-glucosidase production.     

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.     

A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A) and endoglucanase I (cel7B) of Trichoderma reesei

It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.     

Effects of ammonia fiber explosion treatment on activity of endoglucanase from Acidothermus cellulolyticus in transgenic plant

A critical parameter affecting the economic feasibility of lignocellulosic bioconversion is the production of inexpensive and highly active cellulase enzymes in bulk quantity. A promising approach to reduce enzyme costs is to genetically transform plants with the genes of these enzymes, thereby producing the desired cellulases in the plants themselves. Extraction and recovery of active proteins or release of active cellulase from the plants during bioconversion could have a significant positive impact on overall lignocellulose conversion economics. The effects of ammonia fiber explosion (AFEX) pretreatment variables (treatment temperature, moisture content, and ammonia loading) on the activity of plant-produced heterologous cellulase enzyme were individually investigated via heat treatmett or ammonia treatment. Finally, we studied the effects of all these variables in concert through the AFEX process. The plant materials included transgenic tobacco plants expressing E1 (endoglucanase from Acidothermus cellulolyticus). The E1 activity was measured in untreated and AFEX-treated tobacco leaves to investigate the effects of the treatment on the activity of this enzyme. The maximum observed activity retention in AFEX-treated transgenic tobacco samples compared with untreated samples was approx 35% (at 60°C, 0.5∶1 ammonia loading, and 40% moisture). Based on these findings, it is our opinion that AFEX pretreatment is not a suitable option for releasing cellulase enzyme from transgenic plants.     

The effect of additional autopolyploidization in a slow growing cellulase hyperproducer of Trichoderma

M14-2 is a cellulase hyperproducer derived from Trichderma recesei QM 6a, but with a growth rate lower than that of the original strain. When M14-2 was autopolyploidized followed by haploidization and selection, the strain with both a higher cellulase productivity per mycelia and a higher growth rate could be obtained as M14-2B. This strain seemed to be constructed using gene sources amplified by additional autopolyploidization.     

Utilization of methyloleate in production of microbial lipase

In this article, we report the development and optimization of an industrial culture medium for the production of extracellular lipase in the yeast Yarrowia lipolytica. Until now olive oil in combination with glucose was used as the carbon source and inducer for the production of lipase. Our results demonstrate that methyloleate, a cheap hydrophobic compound, could efficiently substitute olive oil as the inducer and carbon source for lipase production. A new process of lipase production was developed yielding a twofold increase in the level of production compared with the levels in previous reports.     

Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems

Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa. The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up.     

Influence of biomass and gold salt concentration on nanoparticle synthesis by the tropical marine yeast Yarrowia lipolytica NCIM 3589

Cell-associated gold nanoparticles and nanoplates were produced when varying number of Yarrowia lipolytica cells were incubated with different concentrations of chloroauric acid (HAuCl₄) at pH 4.5. With 10⁹ cells ml⁻¹ and 0.5 or 1.0 mM of the gold salt, the reaction mixtures developed a purple or golden red colour, respectively, and gold nanoparticles were synthesized. Nanoparticles of varying sizes were produced when 10¹⁰ cells ml⁻¹ were incubated with 0.5, 1.0 or 2.0 mM chloroauric acid salt. With 3.0, 4.0 or 5.0 mM HAuCl₄, nanoplates were also observed. With 10¹¹ cells ml⁻¹ nanoparticles were synthesized with almost all the gold salt concentrations. The cell-associated particles were released outside when nanoparticle-loaded cells were incubated at low temperature (20 °C) for 48 h. With increasing salt concentrations and a fixed number of cells, the size of the nanoparticles progressively increased. On the other hand, with increasing cell numbers and a constant gold salt concentration, the size of nanoparticles decreased. These results indicate that by varying the number of cells and the gold salt concentration, a variety of nanoparticles and nanoplates can be synthesized. Fourier transform infrared (FTIR) spectroscopy revealed the possible involvement of carboxyl, hydroxyl and amide groups on the cell surfaces in nanoparticle synthesis.