Based on an integrated approach of genetic engineering, fermentation process development, and downstream processing, a fermentative chymotrypsinogen B production process using recombinant Pichia pastoris is presented. Making use of the P. pastoris AOX1-promotor, the demand for methanol as the single carbon source as well as an inducer of protein secretion enforced the use of an optimized feeding strategy by help of on-line analysis and an advanced controller algorithm. By using an experimental system of six parallel sparged column bioreactors, proteolytic product degradation could be minimized while also optimizing starting conditions for the following downstream processing. This optimization of process conditions resulted in the production of authentic chymotrypsinogen at a final concentration level of 480 mg‚L -1 in the whole broth and a biomass concentration of 150 g‚L -1 cell dry weight, thus comprising a space-time yield of 5.2 mg‚L -1 ‚h -1 . Alternatively to the high cell density fermentation approach, a continuous fermentation process was developed to study the effects of reduced cell density toward oxygen demand, cooling energy, and biomass separation. This development led to a process with a highly increased spacetime yield of 25 mg‚L -1 ‚h -1 while reducing the cell dry weight concentration from 150 g‚L -1 in fed-batch to 65 g‚L -1 in continuous cultivation.

BackgroundConsolidated bioprocessing (CBP) of lignocellulosic biomass to ethanol using thermophilic bacteria provides a promising solution for efficient lignocellulose conversion without the need for additional cellulolytic enzymes. Most studies on the thermophilic CBP concentrate on co-cultivation of the thermophilic cellulolytic bacterium Clostridium thermocellum with non-cellulolytic thermophilic anaerobes at temperatures of 55°C-60°C.ResultsWe have specifically screened for cellulolytic bacteria growing at temperatures >70°C to enable direct conversion of lignocellulosic materials into ethanol. Seven new strains of extremely thermophilic anaerobic cellulolytic bacteria of the genus Caldicellulosiruptor and eight new strains of extremely thermophilic xylanolytic/saccharolytic bacteria of the genus Thermoanaerobacter isolated from environmental samples exhibited fast growth at 72°C, extensive lignocellulose degradation and high yield ethanol production on cellulose and pretreated lignocellulosic biomass. Monocultures of Caldicellulosiruptor strains degraded up to 89-97% of the cellulose and hemicellulose polymers in pretreated biomass and produced up to 72 mM ethanol on cellulose without addition of exogenous enzymes. In dual co-cultures of Caldicellulosiruptor strains with Thermoanaerobacter strains the ethanol concentrations rose 2- to 8.2-fold compared to cellulolytic monocultures. A co-culture of Caldicellulosiruptor DIB 087C and Thermoanaerobacter DIB 097X was particularly effective in the conversion of cellulose to ethanol, ethanol comprising 34.8 mol% of the total organic products. In contrast, a co-culture of Caldicellulosiruptor saccharolyticus DSM 8903 and Thermoanaerobacter mathranii subsp. mathranii DSM 11426 produced only low amounts of ethanol.ConclusionsThe newly discovered Caldicellulosiruptor sp. strain DIB 004C was capable of producing unexpectedly large amounts of ethanol from lignocellulose in fermentors. The established co-cultures of new Caldicellulosiruptor strains with new Thermoanaerobacter strains underline the importance of using specific strain combinations for high ethanol yields. These co-cultures provide an efficient CBP pathway for ethanol production and represent an ideal starting point for development of a highly integrated commercial ethanol production process.

Continuous fermentation was applied to the production of recombinant human chymotrypsinogen B (hCTRB) by the methylotrophic yeast Pichia pastoris as a tool for the kinetic analysis of growth and product formation. Using methanol as the sole source of carbon, energy, and induction, cell growth could be described by a non‐competitive Monod approach. Maximum growth rate μmax was determined to 0.084 h‐‐1 and the KM‐value for methanol to 0.22 g·L‐‐1, respectively. With respect to product formation, a similar model was established exhibiting a methanol concentration of 0.13 g·L‐‐1 as the KM‐value and a maximum biomass‐specific product‐formation rate of πmax = 0.23 mg·g‐‐1·h‐‐1. The production of hCTRB was strictly growth‐coupled. The data provided covers the range of methanol concentrations between 0 and 4 g·L‐‐1. Substrate concentrations exceeding this upper value led to a complete collapse of product formation. This change in phenotype turned out to be irreversible indicating a genetic instability of transformed Pichia pastoris caused by excess methanol.

The purification of human chymotrypsinogen B (hCTRB) after expression and secretion by the yeast Pichia pastoris is described based on two different approaches using integrated initial recovery. Extraction employing aqueous two-phase systems (ATPS) from poly(ethylene glycol) and sodium sulfate allows direct processing of cell containing yeast suspensions of 50% wet weight. The target protein is obtained partially purified in the top phase while cells and cell debris are partitioned to the bottom phase of the system. hCTRB is further purified by adsorption from the top phase to the cation exchanger SP Sepharose Big Beads and elution in a salt step. The single step isolation of hCTRB is possible by expanded bed adsorption (EBA) using a fluidized cation exchanger (Streamline SP XL). A design strategy is shown taking both target protein binding and stable fluidization of the stationary phase in cell containing suspensions into consideration. For the example of hCTRB isolation from cell containing P. pastoris suspensions, a successful use of this strategy is demonstrated. Both initial recovery strategies deliver a product that can be further purified and formulated by ultrafiltration/diafiltration followed by lyophilization, resulting in a homogeneous product. Scale-up to 30-90 L of culture suspension was shown for both methods, resulting in a product of similar quality. Comparing both strategies reveals that the two-step ATPS route is better suited for high cell density cultures, while the single step EBA method is preferred for cultures of moderate cell density. This is due to the fact that application of EBA is restricted to suspensions of 10-12.5% wet weight cell concentration, thus necessitating dilution of the original broth prior to sample application. The data presented show that integrated recovery operations are a valuable alternative to traditional processing for systems that are problematic during initial solid-liquid separation.

Formation of stable emulsions with the organic solvent is a general complication in the extractive workup of aqueous wholecell biotransformations. This hold-up has been overcome by biocatalytic lysis of emulsifying agents present in the medium through addition of living microorganisms. Of these, Bacillus subtilis and Rhodococcus erythropolis exhibited the most powerful de-emulsifying activity. As exemplified by microbial treatment of cell-free biotransformation media of Saccharomyces cereWisiae and Lactobacillus kefiri, phase separation time (t p ) was drastically reduced from one week to 20 s without significantly affecting product integrity. This practicable readyto-use method is appropriate to both fungal and bacterial biocatalysts. The highly efficient de-emulsification power and the considerably short phase separation time of this technique allow for cost-effective continuous extractions on a large-scale, for example with mixer-settler units.