A microbiological treatment system comprising three consecutive stages of packed bed bioreactors inoculated with mercury-reducing bacteria was operated in laboratory scale. The efficiency of this system for removal of mercury from the following types of industrial wastewater were determined: (1) chlor-alkali electrolysis; (2) gas scrubber solutions from the waste incineration plant TAMARA; (3) gas scrubber solutions from incineration of various types of waste from a chemical factory. The data show that all three types of wastewater could be efficiently cleaned. Factory wastewater with mercury concentrations of up to 460 mg/l had to be diluted to obtain a mercury concentration < 10 mg/l. Treatment efficiency was reduced by chloride concentrations above 39 g/l or toxic compounds, which were present in one of the wastewater batches from the chemical factory. The sand filter buffered transient changes in the bioreactor efficiency. The activated carbon filter functioned as a polishing step so that effluent concentrations below 50 mu g/l could always be maintained. The best and most stable bioreactor performance was obtained for electrolysis wastewater, which has a relatively predictable composition.

A plant for BIOlogical MERcury Remediation (BIOMER) based on mercury-resistant bacteria was operated for 3 years at a chlor-alkali factory in technical scale. Here we report on the performance of the plant and on the technical problems that had to be solved until a stable and continuous operation could be guaranteed. One basic improvement was the installation of a pre-treatment unit. Basic process characteristics were determined during long-term operation. The BIOMER plant could treat wastewater with up to 10 mg/l of mercury. The optimal operation temperature was between 25 and 35 degrees C. A salt concentration of up to 40 g/l of chloride could be tolerated by the microbes, but the fluctuations should be as small as possible. The bioreactor has to be operated at a pH of 7.0 +/- 1.0. A space velocity of up to 4 per hour could be obtained. The wastewater flow rate should be constant to avoid export of fine particles. Finally, a space time yield of 1 kg mercury per day and m(3) bed volume corresponding to 100 m(3) wastewater per day is possible.The biological system showed a high capacity for self-regeneration. Interruptions of the water inflow for up to 12 hours and of the medium supply over several days were tolerated. Toxic shock loads of high concentrations of chlorine or mercury chloride also caused only a transient reduction of the microbial activity The plant was able to quickly return to normal operation with high mercury retention efficiency after such stresses.The results from the long-term operation show that a process can be scaled up from laboratory tests to an industrial plant without any serious engineering problems. It was demonstrated that the BIOMER plant is able to work under industrial conditions at two different chlor-alkali electrolysis factories in Europe.

A multiple vector system for the intracellular high-level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N- and C-terminal fusion of a protein of interest to a Strep II and a His 6 -tag is possible. Corresponding genes are expressed under the control of a xylose-inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N- or C-terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one-step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry. Biotechnol. Bioeng. 2007;96: 525–537. © 2006 Wiley Periodicals, Inc.

Production and secretion of a 28,172 Da hydrolase from Thermobifida fusca (TFH) in Bacillus megaterium MS941 and WH323 was investigated in shake flask and pH controlled bioreactors. Successful production of heterologous TFH was achieved by adapting the original tfh gene to the optimal codon usage of B. megaterium. A codon adaption index close to one was reached. The codon optimized tfh was cloned into an open reading frame with DNA sequence for the N‐terminal signal peptide of B. megaterium lipase A and a C‐terminal His6‐tag, all under the control of a xylose inducible promoter. Successful TFH production and secretion were observed using batch reactor cultivations with complex medium. Expression of the tfh gene from the PxylA promoter and secretion of produced TFH were compared in detail to batch reactor cultivations with semi‐defined growth medium. For the first time, significant TFH secretion was achieved using a semi‐defined medium in glucose limited fed batch cultivations yielding 10‐fold higher cell densities compared to LB medium cultivation. Comparable volumetric TFH activities were obtained for both cultivation strategies. Surprisingly, measured specific TFH activities exhibited drastic discrepancies between preparations from LB and semi‐defined medium grown B. megaterium. TFH recovery by Ni‐chelate affinity chromatography resulted in higher purification factors when LB medium was used. These results indicated that secreted TFH is favorably produced by batch cultures of B. megaterium WH323 in LB medium. Biotechnol. Bioeng. 2007;96:780–794. © 2006 Wiley Periodicals, Inc.

The transformation of extremely high concentrations of ionic mercury (up to 500 mg L−1) was investigated in a chemostat for two mercury-resistant Pseudomonas putida strains, the sediment isolate Spi3 carrying a regulated mercury resistance (mer) operon, and the genetically engineered strain KT2442∷mer73 expressing the mer operon constitutively. Both strains reduced Hg(II) with an efficiency of 99.9% even at the maximum load, but the concentration of particle bound mercury in the chemostat increased strongly. A proteome analysis using two-dimensional gel electrophoresis and mass spectrometry (2-DE/MS) showed constant expression of the MerA and MerB proteins in KT2442∷mer73 as expected, while in Spi3 expression of both proteins was strongly dependent on the Hg(II) concentration. The total cellular proteome of the two strains showed very little changes at high Hg(II) load. However, certain cellular responses of the two strains were identified, especially in membrane-related transport proteins. In Spi3, an up to 45-fold strong induction of a cation efflux transporter was observed, accompanied by a drastic downregulation (106-fold) of an outer membrane porin. In such a way, the cell complemented the highly specific mercury resistance mechanism with a general detoxification response. No indication of a higher demand on energy metabolism could be found for both strains.

A unique biotechnological method for remediation of industrial wastewater contaminated by toxic mercury, based on the enzymatic reduction of ionic mercury by live bacteria, has been developed by prof. Deckwer and co-workers at GBF (at present HZI), Germany, and implemented in a pilot-plant scale. The experience gained during operation of this installation led to the idea, that the process of bioremediation may be integrated in one bioreactor with the adsorption of mercury by immobilization of the bacteria onto the activated carbon. For this it was necessary to define several significant parameters of the activated carbon selected for the process and the adsorption process itself.The paper presents results of the equilibrium and kinetics investigations of the process of ionic mercury sorption from aqueous solutions onto 8 different types of activated carbon. The effective diffusion coefficients in the carbon particles were obtained from the transient-state experiments using a mathematical model of the process and the sorption isotherms as well as the saturation capacity of the sorbents in relation to ionic and metallic mercury were identified. From the temperature dependence of adsorption constants the values of adsorption enthalpy for both metallic and ionic mercury on activated carbon impregnated with sulfur were estimated. The obtained results enabled selection of the optimal sorbent for the fixed-bed activated-carbon bioreactor which will be applied for the modified, integrated process of biological detoxification of mercury in industrial wastewaters.

Fluxes of central carbon metabolism [glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA cycle), biomass formation] were determined for several Bacillus megaterium strains (DSM319, WH320, WH323, MS941) in C- and N-limited chemostat cultures by (13)C labelling experiments. The labelling patterns of proteinogenic amino acids were analysed by GC/MS and therefrom flux ratios at important nodes within the metabolic network could be calculated. On the basis of a stoichiometric metabolic model flux distributions were estimated for the different B. megaterium strains used at various cultivation conditions. Generally all strains exhibited similar metabolic flux distributions, however, several significant changes were found in (1) the glucose flux entering the PPP via the oxidative branch, (2) the reversibilities within the PPP, (3) the relative fluxes of pyruvate and acetyl-CoA fed to the TCA cycle, (4) the fluxes around the pyruvate node involving a futile cycle.

A sucrose-inducible promoter system (PsacB) from Bacillus megaterium was identified using a secretome approach. It was successfully employed for the extracellular production of the homologous levansucrase SacB (4252.4Ul−1) and the heterologous green fluorescent protein GFP (7.9mggCDW−1). Mutational analysis of B. megaterium PsacB allowed the identification of important promoter elements. The sucrose-inducible promoter provides a useful alternative to the established xylose-inducible promoter system (PxylA) for recombinant gene expression in B. megaterium.

Bioinformatic tools can be applied to reconstruct the potential metabolic network of a microorganism from the genome alone. Networks are the rational basis of systems biology. For a systems biology focusing on processes and products, it is a must to functionalize the potential networks. This particularly requires that the phenotype which yields optimal processes and productivities be found out. The functionality of the gene regulatory and the metabolic networks as well can be achieved by the 'omics' methods. As the functionality depends on the environome, it is essential to apply these techniques coordinatively and possibly simultaneously. Presently, the fluxome (flux distribution of the central metabolic pathways), which has to be determined under variation of genetic properties (metabolic engineering) as well as alterations of the environment, is of particular interest. A process-oriented systems biology offers manifold tasks for the biochemical engineer, which can only be solved in close cooperation with molecular biologists and bioinformaticians. Among others, engineering is not only responsible for developing defined and reproducible cultivation strategies in highly instrumented bioreactors but also for reliably determining kinetic relationships in dependence on genomic and environmental properties and efficiently performing network simulations. The recognition of influences of engineering parameters on network functionality is only at the beginning.

AbstractBei biotechnologischen Verfahren werden die eigentlichen wertschöpfenden chemischen Reaktionen durch Proteine durchgeführt. Diese Biokatalysatoren wandeln ihre Substrate direkt in Produkte um, die sich durch klassische chemische Synthese gar nicht oder nur unter großem Aufwand darstellen lassen. Die Produktion von Biokatalysatoren erfolgt im Allgemeinen in wenigen, gut charakterisierten Mikroorganismen. Es wird gezeigt, dass Bacillus megaterium als Produktionssystem für rekombinante Proteine eine gute Alternative zu bereits etablierten Systemen darstellt. Probleme mit der Gewinnung sekretierter Proteinmengen sind auf Schwierigkeiten der Adaption der Prozesse für Hochzelldichtekultivierungen zurückzuführen.

AbstractMit Hilfe der Bioinformatik kann aus dem Genom eines Mikroorganismus das potenzielle metabolische Netzwerk rekonstruiert werden. Netzwerke stellen die eigentliche Basis der Systembiologie bzw. Systembiotechnologie dar. Für eine Systembiotechnologie, die auf Prozesse und Produkte fokussiert ist, müssen die potenziellen Netzwerke in geeigneter Weise funktionalisiert, d. h. der Phänotyp ermittelt werden, der optimale Prozesse bzw. Produkte und Produktivitäten liefert. Die Funktionalität der genregulatorischen und metabolischen Netzwerke wird durch die „Omics”︁‐Techniken abgefragt. Da die Funktionalität vom Environom abhängt, ist es erforderlich, diese Techniken in koordinierter Weise möglichst simultan anzuwenden. Das Fluxom (Flussverteilung der zentralen Stoffwechselwege) erfordert derzeit besonderes Interesse, das sowohl unter Variation genetischer Eigenschaften (Metabolic Engineering) als auch Änderungen des Environoms zu ermitteln ist. Im Rahmen einer im SFB 578 „Vom Gen zum Produkt”︁ angestrebten prozessorientierten Systembiotechnologie stellen sich für den Verfahrenstechniker vielfache Aufgaben, die nur in Kooperation mit Molekularbiologen und Bioinformatikern zu lösen sind. Dazu gehört neben der Bereitstellung definierter, reproduzierbarer Kultivierungsverfahren in hochinstrumentierten Bioreaktoren die Erfassung kinetischer Zusammenhänge in Abhängigkeit vom Environom sowie von Netzwerksimulationen. Die Erkennung des Effekts ingenieurtypischer Parameter auf die Funktionalität der Netzwerke steht jedoch erst am Anfang.

A hydrocyclone with a volume of 2.56 cm(3) was studied as a potential cell retention device for mammalian cell cultures (6 L volume). For the feasible operation range (0.9 to 1.6 L/min flow corresponding to pressure drops of 0.4 to 1.3 bar) the hydrocyclone was characterized with regard to flow split (underflow-to-overflow ratio) and flow ratio (underflow to supply). Cultures of BHK and HeLa cells (with low cell concentrations) were applied to measure separation efficiency and cell viability for a hydrocyclone operation period of 3 min corresponding to a cell suspension throughput of 2.7 to 4.8 L. Cell separation efficiencies ranged from 0.77 to 0.97 and cell viability was not affected except for BHK cells in the overflow at the highest pressure drop (1.3 bar). As the overflow is commonly used for product harvest and cells are discarded, the application of the hydrocyclone has no detrimental effect on the reactor perfusion system. The results indicate that only cells passing from the primary vortex downwards into the inner secondary vortex and from there upwards could be damaged. Evidence for this hypothesis is obtained from operating the hydrocyclone with closed overflow (only centrifugal forces acting) for a period of 3 h. In these studies no significant effect on cell viability could be detected for HeLa and CHO cells. Hence, the results indicate that the hydrocyclone can be appropriately used for cell retention and separation in perfusion cultures. Application at higher pressures is recommended whereby separation efficiencies of 0.97 without any loss in viability can be achieved.

Intracellular and extracellular proteome analysis was carried out by combined two-dimensional gel electrophoresis and mass spectrometric analysis (2DE/MS) for high cell density fed-batch culture of recombinant Bacillus megaterium strains. In the early feeding phase with a constant growth rate of 0.12 h−1 under glucose limitation, high expression and secretion of a metalloprotease (referred as Bmg1465) was detected. The transient appearance of this metalloprotease was found both as cell-associated and as secreted into the culture medium. Searching homologous proteins for functional assignment led to an unambiguous identification of Bmg1465 as a zinc-binding metalloprotease of the type immune inhibitor A (InhA). Metalloproteases of this type are currently considered as typical virulence factors associated with pathogenic Bacillus species. The result raises questions concerning the intrinsic function(s) of Bmg1465 in B. megaterium, which has the GRAS status, with respect to its stress response in high cell density culture.

Neuere Untersuchungen haben gezeigt, dass Bacillus megaterium auch als Produktionsstamm bei der Expression von Fremd-Genen vorteilhaft eingesetzt werden kann. Gegenstand dieses Beitrags sind Ergebnisse aus dynamischen Untersuchungen des Aminosaure-Stoffwechsels von Bacillus megaterium. Vorgestellt wird eine dynamische Methode zur Markierung mit stabilen Isotopen (13C). Damit ist es moglich, kinetische Informationen uber die Biosynthese von proteinogenen Aminosauren zu erhalten und diese als Grundlage zum verbesserten Verstandnis der fur die Proteinproduktion wichtigen Stoffwechselwege zu nutzen.

High cell density cultivations were performed under identical conditions for two Bacillus megaterium strains (MS941 and WH320), both carrying a heterologous dextransucrase (dsrS) gene under the control of the xylA promoter. At characteristic points of the cultivations (end of batch, initial feeding, before and after induction) the proteome was analyzed based on two dimensional gel electrophoresis and mass spectrometric protein identification using the protein database "bmegMEC.v2" recently made available. High expression but no secretion of DsrS was found for the chemical mutant WH320 whereas for MS 941, a defined protease deficient mutant of the same parent strain (DSM319), not even expression of DsrS could be detected. The proteomic analysis resulted in the identification of proteins involved in different cellular pathways such as in central carbon and overflow metabolism, in protein synthesis, protein secretion and degradation, in cell wall metabolism, in cell division and sporulation, in membrane transport and in stress responses. The two strains exhibited considerable variations in expression levels of specific proteins during the different phases of the cultivation process, whereas induction of DsrS production had, in general, little effect. The largely differing behaviour of the two strains with regard to DsrS expression can be attributed, at least in part, to changes observed in the proteome which predominantly concern biosynthetic enzymes and proteins belonging to the membrane translocation system, which were strongly down-regulated at high cell densities in MS941 compared with WH320. At the same time a cell envelope-associated quality control protease and two peptidoglycan-binding proteins related to cell wall turnover were strongly expressed in MS941 but not found in WH320. However, to further explain the very different physiological responses of the two strains to the same cultivation conditions, it is necessary to identify the mutated genes in WH320 in addition to the known lacZ. In view of the results of this proteomic study it seems that at high cell density conditions and hence low growth rates MS941, in contrast to WH320, does not maintain a vegetative growth which is essential for the expression of the foreign dsrS gene by using the xylA promoter. It is conceivable that applications of a promoter which is highly active under nutrient-limited cultivation conditions is necessary, at least for MS941, for the overexpression of recombinant genes in such B. megaterium fed-batch cultivation process. However to obtain a heterologous protein in secreted and properly folded form stills remains a big challenge.

Peptide mass fingerprint (PMF) matching is a high-throughput method used for protein spot identification in connection with two-dimensional gel electrophoresis (2DE). However, the success of PMF matching largely depends on whether the proteins to be identified exist in the database searched. Consequently, it is often necessary to apply other more sophisticated but also time-consuming technologies to generate sequence-tags for definitive protein identification. On the other hand, modern sequencing technologies are generating a large quantity of DNA sequences, first in unfinished form or with low genome coverage due to the time-consuming and thus limiting steps of finishing and annotation. We recently started to sequence the genome of Bacillus megaterium DSM 319, a bacterium of industrial interest. In this study, we demonstrate that a protein database generated from merely three-fold coverage, unfinished genomic sequences of this bacterium allows a fast and reliable protein spot identification solely based on PMF from high-throughput MALDI-TOF MS analysis. We further show that the strain-specific protein database from low coverage genomic sequence greatly outperforms the commonly used cross-species databases constructed from 13 completely sequenced Bacillus strains for protein spot identification via PMF.

Mercury‐resistant microorganisms are widespread in natural environments and can effectively be used to demercurize Hg(II)‐contaminated wastewaters as was already demonstrated on an industrial scale. The aim of this paper is to find the performance limits with regard to Hg(II) loadings D cHg,in (dilution rate × Hg(II) inlet concentration) and residual Hg(II) at the reactor outlet and to provide a reasonable basis for an optimal and safe process design. To this end, comprehensive studies were carried out with different single microbes (natural isolates and a genetically engineered strain) as well as microbial consortia in batch and continuous stirred reactors and fixed beds with microorganisms immobilized as films. The rate of the biotransformation (reduction of inorganically and organically bound Hg(II) to elemental Hg(0)) was found to follow a uniform mechanism with inhibition kinetics (Haldane type). Both reactor types are able to cope with high Hg(II) loadings and yield conversions up to 98 %. The stirred vessel is particularly suited for high cHg,in but restricted to low D (D < μmax), while the fixed bed can be operated at high D, say 10 h–1, but can only deal with cHg,in < 10 mg/L due to the limited Hg(II) tolerance of microorganisms. The loading limitations can be removed by appropriate recycle flows for both reactor types. However, irrespective of reactor type used, the residual Hg at the outlet cannot be reduced below the legal discharge limit (50 μg/L) mainly owing to the adsorption of Hg(II) on biomass. Therefore, a separation step following the reactor is required (sand bed, activated carbon filter). Comparing the reactor types exhibits the superiority of the fixed bed system due to its simpler construction, easier operation and higher cost effectiveness. Furthermore, the fixed bed shows better flexibility and robustness to extreme loadings. This justifies a posteriori the choice of a fixed bed reactor applied in the technical process.