Industrial bioconversion of renewable resources as an alternative to conventional chemistry.

There are numerous possibilities for replacing chemical techniques with biotechnological methods based on renewable resources. The potential of biotechnology (products, technologies, metabolic pathways) is for the most part well known. Often the costs are still the problem. Biotechnological advances have the best chances for replacing some fine chemicals. While the raw material costs are less of a consideration here, the environmental benefit is huge, as chemical-technical processes often produce a wide range of undesirable/harmful by-products or waste. In the case of bulk chemicals (<1 US dollar/kg) the product price is affected mainly by raw material costs. As long as fossil raw materials are still relatively inexpensive, alternatives based on renewable resources cannot establish themselves. Residues and waste, which are available even at no cost in some cases, are an exception. The introduction of new technologies for the efficient use of such raw materials is currently being promoted. The utilisation of residual wood, plant parts, waste fat, and crude glycerol, for example, provides great potential. For industrial chemicals (2-4 US dollars/kg), process and recovery costs play a greater role. Here, innovative production technologies and product recovery techniques (e.g. on-line product separation) can increase competitiveness.

Advanced nitrogen elimination by encapsulated nitrifiers.

By introducing a mixed population of nitrifiers encapsulated in gel lens beads a more selective nitrification process was found in treatment of settled sewage in lab scale at a hydraulic retention time (HRT) of about 30 to 60 minutes. The reaction rates for oxidation of soluble chemical oxygen demand (SCOD) were found to vary between 25 to 150 mg/L x h while nitrification takes place around 50 mg nitrogen per hour and litre reaction volume. However, based on this SCOD removal in the nitrification step, a consequent post-denitrification process without nitrate recycle and dosage of external carbon sources has been proven to reach substantial nitrate elimination of up to 20 mg nitrogen per litre at COD/N-ratios of approx. 6 in settled sewage. At such COD/N-ratios, suitable nitrogen elimination seems to be possible, because the bioflocs of settled sewage, produced so far by SCOD oxidation and entrapment of particulate COD, are passing through the nitrification process having a substantial contribution to the denitrification rate additionally to the remaining SCOD.

Pre-nitrification by encapsulated nitrifiers--a possibility for self-sufficient energy operation of domestic WWTPs.

The overall energy consumption of domestic wastewater treatment plants (WWTPs) increases with treatment efficiency. Approximately 30 to 45 kWh per people equivalent and year is mostly necessary for advanced nitrogen and phosphorus removal, while the aeration contains the main part of approximately 60%. A new process using encapsulated nitrifiers on gel lens beads is introduced to overcome the high energy consumption of aeration. A more selective nitrification process was found at a nitrification rate of between 50 and 60 mg nitrogen per hour and litre reaction volume corresponding to a hydraulic retention time (HRT) of about 30 to 60 minutes while the soluble Chemical Oxygen Demand (COD) removal could be less than 30% depending on operational conditions of the bio-reactor. The latter enables internal use of wastewater's COD for a post denitrification. For the new process the energy consumption as well as total volume of bio-reactor are much less (approximately 30 to 50% for both) than conventional processes due to the low sludge age for COD and nitrate removal and the avoidance of internal wastewater recycle. Therefore, self-sufficient energy operation of domestic WWTPs operating with advanced treatment efficiency could become possible, if energy recovery by anaerobic sludge digestion is included.

Biotechnological production of itaconic acid.

Itaconic acid (IA) is an unsaturated dicarbonic organic acid. It can easily be incorporated into polymers and may serve as a substitute for petrochemical-based acrylic or methacrylic acid. It is used at 1-5% as a co-monomer in resins and also in the manufacture of synthetic fibres, in coatings, adhesives, thickeners and binders. The favoured production process is fermentation of carbohydrates by fungi, with a current market volume of about 15,000 t/a. Due to the high price of about US$ 4/kg, the use of IA is restricted. At present, the production rates do not exceed 1 g l(-1) h(-1), accompanied by product concentrations of about 80 g l(-1). New biotechnology approaches, such as immobilisation techniques, screening programmes and genetic engineering, could lead to higher productivity. Also, the use of alternative substrates may reduce costs and thus open the market for new and increased applications.

Asymmetric synthesis of an (R)-cyanohydrin using enzymes entrapped in lens-shaped gels.

[structure: see text] A novel synthesis of (R)-cyanohydrins is described which is based on the use of cross-linked and subsequently poly(vinyl alcohol)-entrapped (R)-oxynitrilases. These immobilized lens-shaped biocatalysts have a well-defined macroscopic size in the mm range, show no catalyst leaching, and can be recycled efficiently. Furthermore, this immobilization method is cheap and the entrapped (R)-oxynitrilases gave similar good results compared with those of free enzymes. The (R)-cyanohydrin was obtained in good yields and with high enantioselectivities of up to >99% ee.

A novel encapsulation technique for the production of artificial seeds.

A novel technique for the encapsulation of plant material in calcium alginate hollow beads was tested. The technique involves suspending plant material (i.e. plant cells, tissues, organs, shoot tips, somatic embryos) in a solution containing carboxymethylcellulose and calcium chloride and then dripping it into a stirred sodium alginate solution. In initial experiments with Daucus carota (carrot), it was found that after 14 days of cultivation, 100 % of seeds encapsulated in calcium alginate hollow beads would germinate in the liquid core and that 13% would burst the capsules. Embryogenic calli developed inside hollow beads and formed somatic embryos while calli in conventional calcium alginate beads became detached from the beads early in development, and no somatic embryogenesis occurred. With Solanum tuberosum (potato), development of calli was observed in 50% of hollow beads. Eighty-one percent of shoot tips encapsulated in hollow beads sprouted and grew out of the capsules.

A nitrite sensor based on a highly sensitive nitrite reductase mediator-coupled amperometric detection.

Highly sensitive nitrite sensors have been developed for the first time based on mediator-modified electrodes. Tetraheme cytochrome c nitrite reductase from Sulfurospirillum deleyianum and cytochrome cd(1) nitrite reductase from Paracoccus denitrificans are able to accept electrons from artificial electron donors, which simultaneously act as electron mediators between the enzyme and an amperometric electrode. In addition to methyl viologen, redox-active compounds such as phenazines (phenosafranin, safranin T, N-methylphenazinium, 1-methoxy-N-methylphenazinium) and triarylmethane redox dyes (bromphenol blue and red) were selected from a range of redox compounds exhibiting the most efficient performance for nitrite detection. After precipitation, the electron mediators were incorporated in a graphite electrode material. Enzyme immobilization is performed by entrapment in a poly(carbamoyl sulfonate) (PCS) hydrogel. Diffusion coefficients and apparent heterogeneous rate constants of the mediators as well as homogeneous rate constants of nitrite sensors were determined by chronoamperometry and cyclic voltammetry. The phenosafranin-modified electrode layered with the PCS hydrogel immobilization of tetraheme cytochrome c nitrite reductase yielded linear current responses up to 250 µM nitrite with a sensitivity of 446.5 mA M(-)(1) cm(-)(2). The detection limit of the enzymatic nitrite sensor was found to be 1 µM nitrite.

Preparation and characterization of pancreatic lipase immobilized in Eudragit-matrix.

Pancreatic lipase (EC 3.1.1.3) was immobilized by entrapping in a commercial preparation of acrylic/methacrylic acid ester-based copolymer (Eudragit E 30 D). The activity of the immobilized lipase beads with a diameter of 1.5-2.0 mm was found to be lower than that of the free lipase. The optimum pH was shifted to the alkaline region and the thermal stability increased, whereas the optimum temperature level remained unchanged. The most important reason for the decreased activity was diffusion limitations. The diffusion of the substrate and products became more pronounced, and lipolytic activity increased upon addition of n-hexane into the reaction medium. The storage and operational stabilities of the immobilized lipase were investigated, and both characteristics were found to be increased when compared to the free enzyme. Furthermore, mechanical or magnetic stirring during the operation were found to have no influence on the carrier-matrix as determined by nephelometric measurements.

Immobilization of anaerobic thermophilic bacteria for the production of cell-free thermostable alpha-amylases and pullulanases.

For the production of cell-free thermostable alpha-amylases and pullulanases various anaerobic thermophilic bacteria that belong to the genera Clostridium and Thermoanaerobacter were immobilized in calcium alginate gel beads. The entrapment of bacteria was performed in full as well as in hollow spheres. An optimal limited medium, which avoided bacterial outgrowth, was developed for the cultivation of immobilized organisms at 60 degrees C using 0.4% starch as substrate. Compared to non-immobilized cells these techniques allowed a significant increase (up to 5.6-fold) in the specific activities of the extracellular enzymes formed. An increase in the productivity of extracellular enzymes was observed after immobilization of bacteria in full spheres. In the case of C. thermosaccharolyticum, for instance, the productivity was raised from 90 units (U)/10(12) cells up to 700 U/10(12) cells. Electrophoretic analysis of the secreted proteins showed that in all cases most of the amylolytic enzymes formed were released into the culture medium. Proteins that had a molecular mass of less than 450,000 daltons could easily diffuse through the gel matrix. Cultivation of immobilized bacteria in semi-continuous and fed-batch cultures was also accompanied by an elevation in the concentration of cell-free enzymes.