Mauricio Moura da Silveira

The biotechnological production of sorbitol

Abstract. Sorbitol, a polyol found in many fruits, is of increasing industrial interest as a sweetener, humectant, texturizer and softener. At present, it is produced chemically. The bacterium Zymomonas mobilis is able to produce sorbitol and gluconic acid from fructose and glucose, respectively. This is possible in a one-step reaction via a glucose-fructose oxidoreductase so far only known from Z. mobilis. The possibilities for the industrial production of sorbitol by Z. mobilis are discussed, and compared with the current chemical production method as well as other microbiological processes.

Influence of medium composition and pH on the production of polygalacturonases by Aspergillus oryzae

ABSTRACT A liquid medium containing wheat bran, salts and a source of inducer (pectin) was found to be suitable for the production of exo- and endo-polygalacturonases by Aspergillus oryzae CCT3940. Induction of polygalacturonases by purified pectin was significantly higher than when rinds of citrus fruits were used as inducer. A. oryzae growth was favoured by pH close to 4, although a drop of pH to around 3 was needed for enzymes production. Afterwards, decreasing activities were observed with the normal increase in pH to near neutrality. The highest activities were achieved with an initial pH of 4 and controlled when it decreased to a value slightly below 3 (159 units endo-PG.mL -1 at 83 h and 45 units exo-PG.mL -1 at 64 h), being the loss in polygalacturonases activities strongly reduced at this condition. The best values of pH and temperature for the action of exo-PG (4.5/57oC) and endo-PG (4.3/40oC) were assessed. Key words: Pectinase, polygalacturonase, submerged process, production medium, pH

Comparison of stirred tank and airlift bioreactors in the production of polygalacturonases by Aspergillus oryzae

The production of endo and exo-polygalacturonase (PG) by Aspergillus oryzae IPT 301 was studied in a stirred tank bioreactor (STR) and an internal circulation airlift bioreactor. Using a factorial experimental design, a soluble culture medium was defined which allowed the production of exo- and endo-PG comparable to that obtained in a medium containing suspended wheat bran. The soluble medium was used in tests to compare the production of these enzymes in the STR and airlift bioreactor. In these tests, after 96 h, maximum enzymatic activity values achieved for exo- and endo-PG were 65.2 units (U) per mL and 91.3 U mL(-1), in the STR, with similar activity values of 60.6 U mL(-1) and 86.2 U mL(-1), respectively, being achieved in the airlift bioreactor. The airlift bioreactor also showed satisfactory results regarding the oxygen transfer rate in this process, indicating its potential to be used in an eventual larger scale production of exo- and endo-PG, with lower costs for both installation and operation.

Bioconversion of glucose and fructose to sorbitol and gluconic acid by untreated cells of Zymomonas mobilis

The bioconversion of glucose and fructose to gluconic acid and sorbitol, respectively, by the enzymes glucose-fructose oxidoreductase (GFOR) and glucono-delta-lactonase (GL), contained in untreated cells of Zymomonas mobilis ATCC 29191, was investigated in batch runs with glucose plus fructose concentrations (S0) varying from 100 to 750 g l-1 in equimolar ratio. When S0 was increased to 650 g l-1, the yields were improved, reaching a maximum of 91% for both products, with productivities of 1.6 and 1.5 g g-1 cell h-1 for gluconic acid and sorbitol, respectively. Above this level (S0 = 750 g l-1), no further improvement in yields was observed and productivities decreased due to the longer process time. The high yields of bioconversion runs with S0 > or = 650 g l-1 are a consequence of the sequential inhibition of the normal metabolism of Z. mobilis by substrates and products, resulting in preferential utilization of substrates via the GFOR/GL system.

Sorbitol Can Be Produced Not Only Chemically But Also Biotechnologically

Sorbitol, a polyol found in many fruits, is attracting increasing industrial interest as a sweetener, humectant, texturizer, and softener. It is principally produced by chemical means. The bacterium Zymomonas mobilis is able to produce sorbitol together with gluconic acid from fructose and glucose, respectively. This is possible in a one-step reaction via the enzyme glucose-fructose oxidoreductase, so far only known from Z. mobilis. The possibilities for the production of sorbitol by Z. mobilis are discussed also under the aspect of an industrial process and compared with the current chemical as well as other microbiologic processes. The production process by Z. mobilis shows economic possibilities for certain countries, such as Brazil, considering only the products sorbitol and ethanol as an important byproduct. For the other byproduct, gluconic acid, further studies for its partial substitution must be conducted.

Production of polygalacturonases by Aspergillus oryzae in stirred tank and internal- and external-loop airlift reactors

The production of endo- and exo-polygalacturonase (PG) by Aspergillus oryzae was assessed in stirred tank reactors (STRs), internal-loop airlift reactors (ILARs) and external-loop airlift reactors (ELARs). For STR production, we compared culture media formulated with either pectin (WBE) or partially hydrolyzed pectin. The highest enzyme activities were obtained in medium that contained 50% pectin in hydrolyzed form (WBE5). PG production in the three reactor types was compared for WBE5 and low salt WBE medium, with additional salts added at 48, 60 and 72h (WBES). The ELARs performed better than the ILARs in WBES medium where the exo-PG was the same concentration as for STRs and the endo-PG was 20% lower. These results indicate that PG production is higher under experimental conditions that result in higher cell growth with minimum pH values less than 3.0.

Evaluation of Filamentous Fungi and Inducers for the Production of Endo-Polygalacturonase by Solid State Fermentation

Five strains of filamentous fungi (Aspergillus niger strains NRRL 3122 and T0005007-2, Aspergillus oryzae CCT 3940, Aspergillus awamori NRRL 3112 and a Trichoderma sp.) were compared for their capacity to produce endo-polygalacturonase (endo-PG) in solid state fermentation. Maximum pectinolytic activity was reached in 72 h of growth, the best two fungal strains being A. niger T0005007-2 and A. oryzae CCT 3940. Three types of commercial purified pectin and four of unprocessed pectin (tangerine, orange, Tahiti lime and sweet lime rind) were used to assess the effect of pectin on the production of endo-PG by A. niger T0005007-2. Maximum pectinolytic activity was achieved using 6 and 10% (w/w) of purified pectin as inducer. Depending on the origin of the commercial pectin used as inducer, maximum endo-PG levels varied from 223 to 876 units per gram of dry medium (one endo-PG unit (U) was defined as the quantity of enzyme which caused a reduction in viscosity of 50% in a 1% w/v solution of pectin in 30 min), indicating that care should be taken when choosing this component of the medium. When the crude pectins were used as inducers at the same concentration as purified pectin, maximum endo-PG activities were 250-300 units/g. However, by increasing the amount of Tahiti lime rind to 50% (w/w) maximum endo-PG was 919 U/g, thus opening up the possibility of a low cost medium for endo-PG production.

Cellulase and Xylanase Production by Penicillium echinulatum in Submerged Media Containing Cellulose Amended with Sorbitol

The present work investigated the use of sorbitol as a soluble carbon source, in association with cellulose, to produce cellulases and xylanases in submerged cultures of Penicillium echinulatum 9A02S1. Because cellulose is an insoluble carbon source, in cellulase production, there are some problems with rheology and oxygen transfer. The submerged fermentations containing media composed of 0, 0.25, 0.5, 0.75, and 1% (w/v) sorbitol and cellulose that were added at different times during the cultivation; 0.2% (w/v) soy bran; 0.1% (w/v) wheat bran; and a solution of salts. The highest filter paper activity (FPA) (1.95  ±  0.04 IU·mL−1) was obtained on the seventh day in the medium containing 0.5% (w/v) sorbitol and 0.5% (w/v) cellulose added 24 h after the start of cultivation. However, the CMCases showed an activity peak on the sixth day (9.99 ± 0.75 IU·mL−1) in the medium containing 0.75% (w/v) sorbitol and 0.75% (w/v) cellulose added after 12 h of cultivation. The xylanases showed the highest activity in the medium with 0.75% (w/v) sorbitol and 0.25% (w/v) cellulose added 36 h after the start of cultivation. This strategy enables the reduction of the cellulose concentration, which in high concentrations can cause rheological and oxygen transfer problems.

Ammonium phosphate as a sole nutritional supplement for the fermentative production of 2,3-butanediol from sugar cane juice.

Abstract The production of 2,3-butanediol by Klebsiella pneumoniae from sugar cane juice supplemented with different salts was studied. This microorganism is able to degrade sucrose present in sugar cane juice containing ammonium phosphate as the sole nutritional supplement. With a sugar cane juice-based medium containing ~180 g sucrose /1 and 8.0 g (NH4)2HPO4, / 1, over 70 g 2,3-butanediol plus acetoin / 1 were formed. This result is comparable to that achieved with a sugar cane juice-based medium containing several nutrients, although the kinetic profiles of these runs presented significant differences. With the ammonium phosphate- enriched medium, cell growth was initially favoured by both the strong oxygen supply and the higher water activity due to the lower concentration of nutrients. After 14 h, the limitation in some nutrients led to the interruption of cell growth, and decreasing rates for product formation and substrate consumption were observed. During the stationary phase of this run, sucrose was preferentially converted to product, and the substrate was completely depleted after 35 h of the process. With the complete medium, the substrate was totally consumed after 36 h of run. In this case, the higher initial concentration of nutrients reduced the overall process rate but sustained the cell growth for 27 h. Conversion yields of 0.40 g product / g sucrose and productivities close to 2.0 g/ lxh were obtained under both conditions.

Production and characterization of endo-polygalacturonase from Aspergillus niger in solid-state fermentation in double-surface bioreactor

Endo-polygalacturonase (endo-PG) production by Aspergillus niger T0005/007-2 in solid medium with 170 mm of height was evaluated in a cylindrical double surface bioreactor in 96-h experiments. Cell concentration close to 92 mg.g -1 dm (mg per g of dry medium) in the standard condition (static) was achieved, whereas in tests under forced aeration of 1.4 and 2.8 L.min-1. Kg-1 mm (L of air per minute per Kg of moist medium) and with the central shaft fungal biomass attained approximately 100 mg.g-1 dm. Superior endo-PG activity was obtained with the central-shaft system, 78 U.g-1 dm (units per g of dry medium). Forced aeration and pressure pulse showed no positive effect on the production of endo-PG, 45 U.g-1 dm and 28 U.g-1 dm, respectively. None of the conditions evaluated was efficient for medium temperature control. Endo-PG was stable up to 40oC. The activity decreased in 50% after 120 minutes at 50oC, which is a temperature normally found during this process.