Michimasa Kishimoto

Oil production from algal cells of Dunaliella tertiolecta by direct thermochemical liquefaction

Algal cells of Dunaliella tertiolecta with a moisture content of 78.4 wt% were converted directly into oil by thermochemical liquefaction at around 300°C and 10 MPa. The oil yield was about 37% on an organic basis. The oil obtained at a reaction temperature of 340°C and holding time of 60 min had a viscosity of 150–330 mPas and a calorific value of 36 kJ g−1, comparable to those of fuel oil.

Effect of methanol concentration on the production of human β2-glycoprotein I domain V by a recombinant Pichia pastoris: A simple system for the control of methanol concentration using a semiconductor gas sensor

Abstract The methylotrophic yeast Pichia pastoris is one of the best hosts for the production of foreign proteins because of the presence of the strong AOX1 promoter induced by methanol. Methanol feeding during the production phase of the foreign proteins is important because methanol not only induces protein production but also provides energy source for the host cells. Excess methanol inhibits the growth of host cells, while an insufficient amount of energy source and/or methanol starvation lead to poor growth and production. We constructed a simple methanol control system consisting of a semiconductor gas sensor and a relay. Using this system, we studied the effect of methanol concentration on the production of a model foreign protein, human β2-glycoprotein I domain V. The methanol concentrations were kept constant at 1.5, 10, 17, or 31 g·l−1 (±5%) during the production phase. Although the specific rates of growth and methanol consumption decreased with increase in the methanol concentration, the specific production rates increased, indicating that the energy for the production competed with that for cell growth. Accordingly, we provided glycerol as an extra energy source during the production phase, with the result that the specific production rate increased two times. Our simple and inexpensive system will help bioengineering studies on the production of recombinant proteins in P. pastoris, the growth and production of objective proteins in which are dependent on the methanol concentration.

Amplified Gene Location in Chromosomal DNA Affected Recombinant Protein Production and Stability of Amplified Genes

Previously, we established an easy and quick construction method for obtaining a stable and highly productive gene‐amplified recombinant Chinese hamster ovary (CHO) cell line. With a gradual increase in methotrexate (MTX) concentration, gene‐amplified cell pools had high and stable specific growth and production rates. Moreover, the phenotype of gene‐amplified cells seemed to be affected by the location of the amplified gene in chromosomal DNA. We suspected that various kinds of gene‐amplified cells might appear during the long‐term selection to construct gene‐amplified cell pools. To clarify the behavior of gene‐amplified cell pools during a stepwise increase of MTX concentration, we isolated gene‐amplified clones derived from gene‐amplified cell pools. We compared the characteristics of isolated clones, such as the productivity of recombinant protein, stability of amplified genes, and the location of amplified genes. As a result, telomere‐type clones, in which the amplified gene was located near the telomeric region, were found to be more stable and productive than other types of clones. Telomere‐type clones had over 100 copies of amplified genes in the chromosomal DNA. In contrast, a large number of other types of clones had less than 10 copies of amplified genes. During long‐term cultivation in the absence of MTX, in other types of clones, amplified genes rapidly decreased in the chromosomal DNA.

CO2 fixation and oil production using micro-algae

The micro-alga Dunaliella tertiolecta ATCC 30929 could be grown under highly saline conditions (6% of NaCl aqueous solution) without the need for sterilization of the bio-reactor or the culture medium which would be greatly advantageous in a scale up of the culture system. Using CO2 as a carbon source, the micro-algae growth reached 1.0g/l under non-sterilized conditions after one week. The effect of contamination by other micro-organisms on the growth was negligible. The micro-alga contained 10% glycerol, and the yield of its conversion to oil by thermo-chemical liquefaction was 36%, indicating the possibility of effective oil production by CO2 fixation using such a micro-alga.

Effective cell harvesting of the halotolerant microalga Dunaliella tertiolecta with pH control

An effective method for cell harvesting of the halotolerant microalga, Dunaliella tertiolecta ATCC30929, was investigated. By increasing the pH of the culture broth using NaOH solution, suspended D. terfolecta cells rapidly coagulated and settled within a few minutes, which allowed simple harvesting of the concentrated cells from the culture broth. The pHs for successful coagulation were between 8.6 and 10.5 and more than 90% of cells was recovered from the culture broth. Clarified culture broth could be reused for the successive cultivation of D. tertiolecta simply by supplying fresh medium and neutralizing alkali with HCl without the requirement for an additional inoculum.

Computer control of glutamic acid production based on fuzzy clusterization of culture phases

The reaction mechanism in a microorganism is much more complicated than that of an ordinary chemical reactor. The mechanism is fundamentally programmed by the DNA sequences of the organism involved, which can change from their original situation with time. It is difficult to keep all of the characteristics of a microorganism constant for a long period, and the improvement of strains by genetic engineering and/or screening techniques is often tried in order to improve industrial fermentation processes. As a result, models of a real fermentation process usually work only for a limited period, and it is virtually impossible to construct a comprehensive and robust model. Furthermore, only a few kinds of sensors are available for monitoring the culture states in fermentation processes, and we cannot measure the state inside cells directly. Therefore, a deterministic model for the application to the control or the simulation of fermentation processes cannot easily be constructed.

Evaluation of stable and highly productive gene amplified CHO cell line based on the location of amplified genes

In order to establish an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, variouskinds of stepwise methotrexate (MTX) selection were carriedout. The specific growth and production rates of the cell were compared with each other, and the distribution of the amplified gene location was determined using fluorescence in situ hybridization (FISH). The specific growth andproduction rates of the cell pool reached the highest levels under the selection condition in which the stepwise increase in the MTX concentration was most gradual; about 82% of amplified genes were observed near the telomeric region. During long-term cultivation without MTX, the percentage ofamplified genes near the telomeric region hardly changed, butthat of amplified genes at other regions decreased. Based on these results, stable and highly productive cell pools could be easily and quickly constructed and amplified and gradual stepwise increase of the MTX concentration. In addition, the FISH technique was powerful tool to evaluate highly productiveand stable gene-amplified cells based on the chromosomal location of the amplified gene.

Application of fuzzy control to industrial bioprocesses in Japan

Applications of fuzzy control to industrial biological processes in Japan were summarized, compared and discussed in terms of the system features, control purpose, input and output variables, development of fuzzy rules and its effectiveness. Fuzzy control was mainly applied to the fed-batch cultures of microorganisms and used for the on-line control of feeding rate of substrate. Most of the control systems were developed based on the knowledge of experienced. Fuzzy control is regarded as a promising method for automating the bioprocesses where the experienced operators play significant roles for their successful operation.

Dewatering characteristics of activated sludges and effect of extracellular polymer

Shiratos expression theory, which is based on Ruths filtration theory and Terzaghis consolidation theory, was applied to an analysis of the dewatering characteristics in activated sludges. It was ascertained that the process could be divided into periods of filtration and consolidation. It was further confirmed that Ruths average specific resistance (αav) was increased and Shiratos modified consolidation coefficient (Ce) decreased by adding the extracellular polymer substance extracted from the sludges. That is to say, the extracellular polymer was found to have a bad effect on the dewatering process through the two periods. From the results of compression-permeability testing, the addition of extracellular polymer was found not to affect the local porosity (ϵ), but it did have a great influence on the local specific filtration resistance (α).

Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag)

Single-chain Fv antibodies (scFv) genetically fused with polystyrene-binding peptides (PS-tags, (PS19-1; RAFIASRRIRRP, PS19-6; RIIIRRIRR)) were generated by recombinant Escherichia coli for direct and site-specific immobilization of scFv on polystyrene supports with high antigen-binding activity. PS-tag-fused scFvs (scFv-PS-tags) specific for human C-reactive protein (CRP) were successfully over-expressed as an inclusion body and were refolded using the batch-dilution method. When scFv-PS-tags were immobilized on a hydrophilic PS (phi-PS) plate in the presence of Tween 20, they showed high antigen-binding activity comparable to, or greater than, that of a whole monoclonal antibody (mAb) on a hydrophobic PS (pho-PS) plate, which has been the exclusive method for enzyme-linked immunosorbent assay (ELISA). Furthermore, when a scFv-PS-tag was used as a ligand antibody in one- and two-step ELISA, the assay time was reduced without loss of sensitivity. These results indicate that strong and specific attachment of PS-tags onto the phi-PS surface prevented scFv conformational changes and consequently, the high antigen-binding activities of scFvs were preserved. Nearly identical results were obtained by use of PS-tag-fused scFvs with different VH/VL pairs. Therefore, a variety of scFvs could be functionalized onto phi-PS plates by genetic fusion of PS-tags. ScFv-PS-tags, which possess high antigen-binding activity on the phi-PS plate, are more useful ligand antibodies than whole mAbs. Thus, scFv-PS-tags are applicable in both clinical diagnosis and proteomic research.