Efficient Transformation of <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> by Electroporation and Conjugation with <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The conjugative self-transmissible plasmid pHT73, harbored in Bacillus thuringiensis var. kurstaki, was demonstrated to be transferred to Cellulomonas flavigena, a cellulolytic bacterium. Both conjugation and transformation procedures yielded resistant colonies; however, chromosomal integration was observed only when bacterial conjugation occurred. The efficiency of conjugation was 10% of recipient strain, which is considered a very efficient process. When the plasmid pHT73 was introduced by transformation, erythromycin-resistant cells contained the plasmid as an episome with no arrangements, as assayed by Southern blot analysis. In contrast, conjugated-resistant cells harbor the plasmid integrated into the chromosome. These data suggest a common mechanism of cell communication between nonrelated bacterial species with similar ecological habitats, and also that both electroporation and conjugation can be used to transform C. flavigena efficiently.     

Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa

Pectin administered to Uncaria tomentosa cell suspension cultures, was found to increase the production of triterpene acids (ursolic and oleanolic acid), however, neither growth nor sterol accumulation were affected. Cell cultures showed that pectin treatment caused a rapid threefold increase in the activities of enzymes involved in the biosynthesis of C(5) and C(30 )isoprenoid, such as isopentenyl diphosphate isomerase and squalene synthase. The activity of a farnesyl diphosphatase, which could divert the flux of farnesyl diphosphate to farnesol, was two times lower in elicited than in control cells. Elicited cells also transformed more rapidly a higher percentage of [5-(3)H]mevalonic acid into triterpene acids. Interestingly, addition of terbinafine, an inhibitor of squalene epoxidase, to elicited cell cultures inhibited sterol accumulation while triterpene production was not inhibited. These results suggest that in U. tomentosa cells, both the previously mentioned enzymes and those involved in squalene 2,3-oxide formation play an important regulatory role in the biosynthesis of sterols and triterpenes.     

Comparison of two continuous fungal bioreactors for posttreatment of anaerobically pretreated weak black liquor from kraft pulp mills

The purpose of this work was to evaluate and compare two continuous systems of posttreatment of anaerobically pretreated weak black liquor (WBL). The first system consisted of a packed bed reactor (PBR) with Trametes versicolor (Tv) immobilized on wood cubes of holm oak (biocubes). The second system was a fluidized bed reactor (FBR) with Lentinus edodes (Le) immobilized on wood cubes of holm oak. The reactors operated for 65 days at a hydraulic retention time (HRT) of 5 days, at 28 degrees C, with continuous aeration. Response variables monitored were conventional and specific, unit, net removal efficiency (eta and eta(sun), respectively) of chemical oxygen demand (COD), color, and ligninoids, and enzymatic activities of manganese peroxidase (MnP), lignin peroxidase (LiP), laccase (Lac) and proteases. The PBR showed an average color eta superior to that of the FBR (52.42 +/- 21.78% and 25.34 +/- 14.38% for PBR and FBR, respectively); removals of COD and ligninoids presented a similar pattern to that of color. Lac activity was significantly larger in PBR than in FBR. Activity of MnP in PBR was higher than that of the FBR (0.004 and 0.002 U MnP/mL, respectively). This difference could be ascribed to the different fungi present in each bioreactor. LiP activity was very low in both reactors. Average value of proteases was almost double in the FBR as compared with PBR (0.472 and 0.209 U Proteases/mL, respectively). During the last 2 weeks of operation, biocubes in the FBR experienced a significant loss of the attached Le biomass, probably by attrition. This and higher protease activity in the FBR could explain the lower pollutant removals achieved in the FBR. Overall, PBR with immobilized Tv showed a better performance than the FBR with Le for the posttreatment of the recalcitrant anaerobic effluent. Extended and sustained pollutant removal (65 days) was achieved in the PBR, although more research is needed to evaluate bioreactor performance at shorter hydraulic retention times.     

Induction of xylanases by sugar cane bagasse at different cell densities of Cellulomonas flavigena

The effect of cell density on xylanolytic activity and productivity of Cellulomonas flavigena was evaluated under two different culturing conditions: fed-batch culture with discontinuous feed of sugar cane bagasse (SCB; condition 1) and glycerol fed-batch culture followed by addition of SBC as xylanases inducer (condition 2). The enzymatic profile of xylanases was similar in both systems, regardless of the initial cell density at time of induction. However, the xylanolytic activity changed with initial cell density at the time of induction (condition 2). The maximum volumetric xylanase activity increased with increased initial cell density from 4 to 34 g l⁻¹ but decreased above this value. The largest total volumetric xylanase productivity under condition 2 (1.3 IU ml⁻¹ h⁻¹) was significantly greater compared to condition 1 (maximum 0.6 IU ml⁻¹ h⁻¹). Consequently, induction of xylanase activity by SCB after growing of C. flavigena on glycerol at intermediate cell density can be a feasible alternative to improve activity and productivity of xylanolytic enzymes.     

Multiple steady states in a continuous stirred tank reactor: an experimental case study for hydrolysis of sucrose by invertase

The purpose of this work was to validate experimentally that multiple steady states may be achieved in a continuous stirred tank reactor (CSTR) during hydrolysis of sucrose by invertase. Experiments were done with four initial sucrose concentrations (0.1, 0.175, 0.584 and 1 M) to study their effect on residual sucrose and reaction rate at steady state. Two different steady states (S=0.7 M, r=9×10⁻⁴ mol/l min and S=0.135 M, r=1.54×10⁻³ mol/l min) were found depending on initial concentration of sucrose in the reactor. Two stable steady states were possible in a CSTR using invertase for the hydrolysis of sucrose. A third possible steady state can be derived theoretically, but it should be a metastable condition because any small disturbance in the system will result in transitory states stabilizing at sugar concentrations of either 0.135 or 0.7 M.     

Expression,purification and immobilization of the intracellular invertase INVA,from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on crystalline cellulose and Nylon-6

This paper presents two immobilization methods for the intracellular invertase (INVA), from Zymomonas mobilis. In the first method, a chimeric protein containing the invertase INVA, fused through its C-terminus to CBD _Cex from Cellulomonas fimi was expressed in Escherichia coli strain BL21 (DE3). INVA was purified and immobilized on crystalline cellulose (Avicel) by means of affinity, in a single step. No changes were detected in optimal pH and temperature when INVA-CBD was immobilized on Avicel, where values of 5.5 and 30 °C, respectively, were registered. The kinetic parameters of the INVA-CBD fusion protein were determined in both its free form and when immobilized on Avicel. K _m and V _max were affected with immobilization, since both showed an increase of up to threefold. Additionally, we found that subsequent to immobilization, the INVA-CBD fusion protein was 39% more susceptible to substrate inhibition than INVA-CBD in its free form. The second method of immobilization was achieved by the expression of a 6xHis-tagged invertase purified on Ni-NTA resin, which was then immobilized on Nylon-6 by covalent binding. An optimal pH of 5.5 and a temperature of 30 °C were maintained, subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating to V _max increased up to 5.7-fold, following immobilization, whereas K _m increased up to 1.7-fold. The two methods were compared showing that when invertase was immobilized on Nylon-6, its activity was 1.9 times that when immobilized on cellulose for substrate concentrations ranging from 30 to 390 mM of sucrose.     

Purification and characterization of two sugarcane bagasse-absorbable thermophilic xylanases from the mesophilic <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis>

We report the purification and characterization of two thermophilic xylanases from the mesophilic bacteria Cellulomonas flavigena grown on sugarcane bagasse (SCB) as the only carbon source. Extracellular xylanase activity produced by C. flavigena was found both free in the culture supernatant and associated with residual SCB. To identify some of the molecules responsible for the xylanase activity in the substrate-bound fraction, residual SCB was treated with 3 M guanidine hydrochloride and then with 6 M urea. Further analysis of the eluted material led to the identification of two xylanases Xyl36 (36 kDa) and Xyl53 (53 kDa). The pI for Xyl36 was 5.0, while the pI for Xyl53 was 4.5. Xyl36 had a K _m value of 1.95 mg/ml, while Xyl53 had a K _m value of 0.78 mg/ml. In addition to SCB, Xyl36 and Xyl53 were also able to bind to insoluble oat spelt xylan and Avicel, as shown by substrate-binding assays. Xyl36 and Xyl53 showed optimal activity at pH 6.5, and at optimal temperature 65 and 55°C, respectively. Xyl36 and Xyl53 retained 24 and 35%, respectively, of their original activity after 8 h of incubation at their optimal temperature. As far as we know, this is the first study on the thermostability properties of purified xylanases from microorganisms belonging to the genus Cellulomonas.     

Draw-fill batch culture mode for production of xylanases by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> on sugar cane bagasse

Draw-fill culture was evaluated as a method for xylanase production by Cellulomonas flavigena on sugar cane bagasse. Specific xylanase activity and volumetric xylanase activities were measured by harvesting 50%, 55%, 60% and 70% of fermented broth at the end of each subculture. Maximum specific (64 IU mg(-1) protein) and volumetric (166 IU ml(-1)) xylanase activities were obtained by harvesting 50-55% of broth. Values were 3.4 and 3.8 times greater than those obtained in batch cultures carried out under the same conditions. Enzyme productivity of 4.2 IU ml(-1) h(-1) was significantly greater than that obtained in continuous cultures (2.4 IU ml(-1) h(-1)) (P<0.05).     

β-Methyl-xyloside: positive effect on xylanase induction in Cellulomonas flavigena

Synthesis of extracellular xylanase in Cellulomonas flavigena is induced in the presence of xylan and sugarcane bagasse as substrates. The essential factors for efficient production of xylanase are the appropriate medium composition and an inducing substrate. The increase in xylanase production levels in C. flavigena were tested with a number of carbon sources and different culture conditions. Xylose, arabinose, glycerol and glucose did not induce xylanase production in this microorganism. β-Methyl-xyloside (β-mx), a structural analog of xylobiose, also did not induce xylanase when used as the sole carbon source, but when xylan or sugar cane bagasse was supplemented with β-mx, extracellular xylanase production increased by 25 or 46%, respectively. The response of C. flavigena to xylan plus β-mx was accompanied by a significant accumulation of reducing sugar, an effect not observed with the combination sugarcane bagasse plus β-mx as substrate. To our knowledge, this is the first report on the effect of β-mx on the induction of xylanase in C. flavigena.     

Haematococcus pluvialis cultivation in split-cylinder internal-loop airlift photobioreactor under aeration conditions avoiding cell damage

The effects of superficial gas velocity in the riser (U_Gr) and gas entrance velocity (v) on the growth of Haematococcus pluvialis cultivated in a split-cylinder internal-loop airlift photobioreactor were investigated. Cell growth decreased when U_Gr and v were increased above 12 mm s^–1 and 22.8 m s^–1, respectively. The maximum cell density of H. pluvialis was 110×10⁴ vegetative cells ml^–1 and the chlorophyll-a titer was 7 mg l^–1. The cell damage in the photobioreactor was greater when v was increased by an increase in U_Gr rather than by a decrease in sparger internal diameter. The overall volumetric mass transfer coefficient (k_La) of the photobioreactor was measured at the same U_Gr (6–24 mm s^–1) and v (12–80 m s^–1). The k_La values reached in the airlift photobioreactor were between 10 h^–1 and 32 h^–1.