Baculovirus production for gene therapy: the role of cell density,multiplicity of infection and medium exchange

One of the major concerns regarding the use of insect cells and baculovirus expression vectors for the production of recombinant proteins is the drop in production observed when infecting cultures at high cell densities; this work attempts to understand this so-called cell density effect in the scope of baculovirus production for gene therapy purposes. A Spodoptera frugiperda insect cell line (Sf-9) was cultured and infected in serum-free medium, and the patterns of production of a recombinant baculovirus expressing the green fluorescent protein (GFP) were analyzed at different cell concentrations at infection (CCIs) and multiplicities of infection (MOIs). The results confirm that a cell density effect on productivity occurs which is dependent on the MOI used, with a high MOI “delaying” the drop in production to higher cell densities. Medium replacement at the time of infection using a high MOI considerably improved baculovirus production, with the different production indicators, namely the titer, specific yield, amplification factor, and time of harvesting, increasing with cell concentration for the CCI range tested. Virus titers as high as 2.6 × 10¹⁰ IP.mL⁻¹ were obtained in cultures infected at 3.5 × 10⁶ cells.mL⁻¹, while the amplification factor was roughly 19 times higher than the highest value obtained without medium exchange.     

Growth, metabolism and baculovirus production in suspension cultures of an Anticarsia gemmatalis cell line

The UFL-AG-286 cell line, established from embryonic tissue of the lepidopteran insect Anticarsia gemmatalis, has been identified as a good candidate to be used as a cellular substrate in the development of a process for in vitro production of the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, a baculovirus widely used as bioinsecticide. In order to characterize the technological properties of this cell line and evaluate its feasibility to use it for the large-scale production of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, UFL-AG-286 cells were adapted to grow as agitated suspension cultures in spinner-flasks. Batch suspension cultures of adapted cells in serum-supplemented TC-100 medium grew with a doubling time of about 29 h and reached a maximum cell density higher than 3.5 × 10⁶ viable cells ml⁻¹. At the end of the growth period glucose was completely depleted from the culture medium, but l-lactate was not produced. Amino acids, with the exception of glutamine, were only negligibly consumed or produced. In contrast to other insect cell lines, UFL-AG-286 cells appeared to be unable to synthesize alanine as a metabolic way to dispose the by-product ammonia. The synchronous infection of suspension cultures with Anticarsia gemmatalis multicapsid nucleopolyhedrovirus in the early to medium exponential growth phase yielded high amounts of both viral progenies per cell and reduced the specific demands of UFL-AG-286 cells for the main nutrients.     

Influence of culture conditions on production and freeze-drying tolerance of Paecilomyces fumosoroseus blastospores

With the goal of developing a defined medium for the production of desiccation-tolerant blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus, we evaluated the impact of various media components such as amino acids, carbohydrates, trace metals and vitamins on hyphal growth and sporulation of P. fumosoroseus cultures and on the freeze-drying tolerance of blastospores produced under these conditions. A comparison of 13 amino acids as sole nitrogen sources showed that glutamate, aspartate, glycine and arginine supported biomass accumulations (12–16 mg ml⁻¹) and blastospore yields (6–11 × 10⁸ blastospores ml⁻¹) comparable to our standard production medium which contains casamino acids as the nitrogen source. Using glutamate as the sole nitrogen source, tests with various carbohydrates showed that P. fumosoroseus grew best on glucose (18.8 mg biomass ml⁻¹) but produced similar blastospore concentrations (7.3–11.0 × 10⁸) when grown with glucose, glycerol, fructose or sucrose. P. fumosoroseus cultures grown in media with sodium citrate or galactose as the sole carbohydrate produced lower blastospore concentrations but more-desiccation-tolerant spores. Zinc was the only trace metal tested that was required for optimal growth and sporulation. In a defined medium with glutamate as the nitrogen source, vitamins were unnecessary for P. fumosoroseus growth or sporulation. When blastospores were freeze-dried in the absence of a suspension medium, residual glucose (>2.5% w/v) was required for enhanced spore survival. Thus, a defined medium containing basal salts, glucose, glutamate and zinc can be used to produce optimal concentrations of desiccation-tolerant blastospores of P. fumosoroseus. Received 27 October 1998/ Accepted in revised form 06 May 1999     

Immobilization of 293 cells using porous support particles for adenovirus vector production

Adenovirus vector production by anchorage-independent 293 cells immobilized using porous biomass support particles (BSPs) was investigated in static and shake-flask cultures for efficient large-scale production of adenovirus vectors for gene therapy applications. The density of cells immobilized within BSPs was evaluated by measuring their WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) reduction activity. In shake-flask culture, 293-F cells, which were adapted to serum-free suspension culture, were not successfully retained within reticulated polyvinyl formal (PVF) resin BSPs (2 × 2 × 2 mm cubes) with matrices of relatively small pores (pore diameter 60 μm). When the BSPs were coated with a cationic polymer polyethyleneimine, a high cell density of more than 10⁷ cells cm⁻³-BSP was achieved in both static and shake-flask cultures with regular replacement of the culture medium. After infection with an adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP), the specific Ad EGFP productivity of the immobilized cells was comparable to the maximal productivity of non-immobilized 293-F cells by maintaining favorable conditions in the culture environment.     

Production of adenoviral vectors and its recovery

The current demands for adenoviral vectors are increasing to satisfy pre-clinical and clinical gene therapy protocols. Consequently, there is a necessity of methodologies to improve production and recovery of intact particles with the minimum effect upon bioactivity. The production of adenoviral vectors in HEK 293 cells and the potential of an alternative aqueous two-phase system (ATPS) composed of PEG 300-phosphate in recovery of adenoviral vectors were investigated. The production of adenoviral vectors was carried out using a 2 L bioreactor equipped with two Rushton impellers. Different parameters including initial cell density, harvesting time and the addition of a buffer (HEPES) were studied in order to improve the production of adenoviral vectors in HEK 293 cells. A yield of 8 × 10¹¹ infective particles was achieved under the conditions characterized by the addition of Pluronic F-68, inoculation at an initial cell density of 3.5 × 10⁵ cells/mL and harvest of infected cells at 48 h post infection (hpi). This material was used for the evaluation of the ATPS recovery processes. It was demonstrated that the chemical components of the ATPS did not have a significant effect upon the infectivity of the adenoviral vectors and a total recovery of approximately 90% was obtained. These findings contribute to the process development for the manufacture of adenoviral vectors and other nanoparticulate bioproducts.     

Dynamics of bacterial communities during solid-state fermentation using agro-industrial wastes to produce poly-γ-glutamic acid,revealed by real-time PCR and denaturing gradient gel electrophoresis (DGGE)

The dynamics of bacterial communities play an important role in solid-state fermentation (SSF). Poly-γ-glutamic acid (γ-PGA) was produced by Bacillus amyloliquefaciens C1 in SSF using dairy manure compost and monosodium glutamate production residuals as basic substrates. The production of γ-PGA reached a maximum of 0.6% after 20 days fermentation. Real-time polymerase chain reaction showed the amount of total bacteria reached 3.95 × 10⁹ 16S rDNA copies/g sample after 30 days, which was in good accordance with the 4.80 × 10⁹ CFU/g obtained by plate counting. Denaturing gradient gel electrophoresis profile showed a reduction of microbial diversity during fermentation, while the inoculum, B. amyloliquefaciens C1, was detected as the dominant organism through the whole process. In the mesophilic phase of SSF, Proteobacteria was the dominant microbial, which was replaced by Firmicutes and Actinobacteria in the thermophilic phase. The molecular analysis of the bacterial diversity has significant potential for instructing the maturing process of SSF to produce γ-PGA at a large-scale level, which could be a benefit in the production of high quality and stable SSF products.     

Elicitation with methyl-jasmonate stimulates peruvoside production in cell suspension cultures of <Emphasis Type="Italic">Thevetia peruviana</Emphasis>

Thevetia peruviana is a small tree that produces several compounds with pharmaceutical application, among which peruvoside could be highlighted. However, these compounds are produced in low concentration in the plant, making it important to develop strategies such as plant cell culture and elicitation to obtain higher quantities of the desired product. In this work, cell suspension cultures of T. peruviana were established in four different culture media: Murashige–Skoog (MS), half Murashige–Skoog (half MS), Schenk–Hildebrandt (SH), and Gamborg (B5) to study their effect on cell growth. Cell growth kinetics were studied in SH medium, and the extracellular peruvoside production during the culture time was determined. The best culture medium for the establishment of cell suspension cultures was MS with a growth index of 3.17 ± 0.2 g g⁻¹ inoculum. The cell growth kinetics showed the four characteristic growth phases of a cell culture (lag, exponential, stationary, and death), and during none of these phases was it possible to observe peruvoside production. The elicitor effect of methyl-jasmonate (MeJ) was studied in cell suspension cultures established in SH medium. The effect of MeJ concentration and the time in which it should be applied were determined. The best results were obtained at a concentration of 100 mg l⁻¹ of MeJ applied at the beginning of the culture, which induced a peruvoside production of 8.93 mg l⁻¹ medium. The current results are the first report of an in vitro peruvoside production system.     

Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells

Human 293S cells, a cell line adapted to suspension culture, were grown to 5×10⁶ cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×10⁵ cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×10⁶ cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.Abbreviations CFDMEM calcium-free DMEM - CS bovine calf serum - hpi hours post-infection - J+ enriched Joklik medium - MLP major late promoter - MOI multiplicity of infection (# of infectious viral particle/cell) - q specific consumption rate (mole/cell.h) - pfu plaque forming unit (# of infectious viral particle) - Y yield (g/E6 cells or mole/cell)     

Production of transgenic gentian plants by particle bombardment of suspension-culture cells

 Cell suspension cultures were established from leaf explants of gentian (Gentiana triflora×G. scabra) for the generation of transgenic plants by particle bombardment. The parameters for the bombardment of suspension culture cells with a particle gun were examined by monitoring the transient expression of a gene for β-glucuronidase driven by the cauliflower mosaic virus (CaMV) 35S promoter. We found that prior culture of suspension culture cells for 5 days on solid medium was optimum for successful particle bombardment. Putative transformed calli were obtained from bombarded cells after a two-step selection procedure. Cells were cultured first with 30 mg l^–1 hygromycin in liquid MS medium that contained 10 mg l^–1 N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, 1 mg/l 1-naphthaleneacetic acid and 30 g l^–1 sucrose and then on solid medium prepared from the same liquid medium plus 2 g l^–1 gellan gum. After 12 weeks of selection on solid medium that contained 30 mg l^–1 hygromycin, two transgenic gentian plants were regenerated from each selected callus. Analysis by the polymerase chain reaction and Southern blotting revealed the stable integration of transferred DNA. Received: 3 June 1999 / Revision received: 21 September 1999 / Accepted: 20 September 1999     

腺相关病毒载体工程毒株精细化纯化

[背景] 大量制备高纯度的重组腺相关病毒（Recombinant Adeno-Associated Virus,rAAV）,是以腺相关病毒（AAV）为投递载体的基因靶向治疗或基因工程药物研发过程中需要解决的重要问题。[目的] 利用层析柱法大量纯化质粒和rAAV细胞培养液,以获得高纯度的rAAV颗粒。[方法] 对组装rAAV的3种质粒用HiPrep^TM10 Sepharose 6FF和HiTrap PlasmidSelect Xtra凝胶层析柱纯化,目的是得到组装AAV的超螺旋质粒DNA,然后对组装rAAV过程中的AAV-293细胞培养方法进行优化,组装成功病毒后,再对收集到的细胞提取液用HiLoad^TM10Q和HiLoad^TM10SP阴阳离子交换层析柱纯化,最后用纯化浓缩后的rAAV感染HT1080细胞,通过流式细胞仪检测荧光表达细胞数,计算浓缩后活病毒感染滴度。[结果] HiPrep和HiTrap层析柱纯化后得到大量高纯度的超螺旋质粒DNA,组装病毒后,用HiLoad柱纯化获得大量高纯度的rAAV颗粒,通过测定,rAAV基因组滴度达到（2.46±0.37）×10¹² TCID₅₀/mL （MOI=1.0×10⁴）。[结论] 通过一系列精细化组装纯化制备出高纯度、高滴度rAAV病毒颗粒。     

Development of a measles vaccine production process in MRC-5 cells grown on Cytodex1 microcarriers and in a stirred bioreactor

Measles vaccination remains the most efficient way to control the spread of the virus. This work focuses on the production of a measles vaccine using stirred conditions as an advanced option for process scale up. Non-porous Cytodex 1 microcarriers were used to support MRC-5 cell growth in suspension cultures. Virus replication was first optimized in spinner flasks, and the effects of various operational parameters were investigated. Cell infection with AIK-C measles strain at an MOI (multiplicity of infection) of 0.005, without glucose regulation and in M199 medium, resulted in a virus titer of 10^6.25 TCID₅₀ (median tissue culture infective dose)/ml. To optimize the production process in a 7-l bioreactor, we carried out various perfused cultures using minimum essential medium (MEM) + 5% FCS diluted with phosphate-buffered saline (PBS). We achieved a high cell density level (4.1 × 10⁶ cells/ml) with an efficient use of the medium when MEM + 5% FCS diluted with PBS at 25% was used during the cell amplification step. Optimization of measles production in MRC-5 cells grown on Cytodex 1 beads in a 7-l bioreactor showed that perfusion was the most efficient when compared to repeated-batch culture. Perfusion at a rate of 0.25 V (reactor volume)/day showed the highest specific productivity (1.6 IVP [infectious virus particle] cell⁻¹ day⁻¹). Testing of several stabilizers containing pharmaceutically improved components such as sugars, amino acids, and charged ions showed that the formulation composed of sucrose and MgCl₂, led to the maintenance of the infectivity of the AIK-C measles virus strain to a significant level, when stored at +28 °C, +4 °C and −60 °C.     

Effect of ammonia on the anaerobic degradation of protein by a mesophilic and thermophilic biowaste population

The influence of ammonia on the anaerobic degradation of peptone by mesophilic and thermophilic populations of biowaste was investigated. For peptone concentrations from 5 g l⁻¹ to 20 g l⁻¹ the mesophilic population revealed a higher rate of deamination than the thermophilic population, e.g. 552 mg l⁻¹ day⁻¹ compared to 320 mg l⁻¹ day⁻¹ at 10 g l⁻¹ peptone. The final degree of deamination of the thermophilic population was, however, higher: 102 compared to 87 mg NH₃/g peptone in the mesophilic cultures. If 0.5–6.5 g l⁻¹ ammonia was added to the mesophilic biowaste cultures, deamination of peptone, degradation of its chemical oxygen demand (COD) and formation of biogas were increasingly inhibited, but no hydrogen was formed. The thermophilic biowaste cultures were most active if around 1 g ammonia l⁻¹ was present. Deamination, COD degradation and biogas production decreased at lower and higher ammonia concentrations and hydrogen was formed in addition to methane. Studies of the inhibition by ammonia of peptone deamination, COD degradation and methane formation revealed a K _i (50%) for NH₃ of 92, 95 and 88 mg l⁻¹ at 37 °C and 251, 274 and 297 mg l⁻¹ at 55 °C respectively. This indicated that the thermophilic flora tolerated significantly more NH₃ than the mesophilic flora. In the mesophilic reactor effluent 4.6 × 10⁸ peptone-degrading colony-forming units (cfu)/ml were culturable, whereas in the thermophilic reactor effluent growth of only 5.6 × 10⁷ cfu/ml was observed. Received: 24 April 1998 / Received revision: 26 June 1998 / Accepted: 27 June 1998     

A novel animal-component-free medium for rabies virus production in Vero cells grown on Cytodex 1 microcarriers in a stirred bioreactor

Vero cells growth and rabies production in IPT-AF medium, a property animal-component-free medium are described in this work. Kinetics of cell growth and rabies virus (strain LP 2061) production were first conducted in spinner flasks. Over eight independent experiments, Vero cell growth in IPT-AF medium, on 2 g/l Cytodex 1 was consistent. An average Cd (cell division number) of 3.3 ± 0.4 and a specific growth rate μ of 0.017 ± 0.006 h⁻¹ were achieved. Such performances were comparable to those obtained in serum-containing medium (MEM + 10% FCS). Rabies virus production on Vero cells in IPT-AF medium was also optimised in spinner flasks. The effects of multiplicity of infection (MOI), regulation of glucose level at 1 g/l and cell washing step, were investigated. The highest virus titer was achieved when the cells were infected at an MOI of 0.1; this level was equal to 10⁷ FFU/ml. The step of medium exchange before cell infection can be omitted; nevertheless in this case glucose level should be maintained at 1 g/l to avoid a decrease of specific virus productivity. Process optimisation in a 2-l stirred bioreactor pointed out that the aeration mode was the prominent parameter that affected cell growth in IPT-AF medium and on Cytodex 1 microcarriers. An acceptable level of cell density (cell density level of 1.5 × 10⁶ cells/ml) was achieved when cells were grown in batch mode and using headspace aeration. Nevertheless, this aeration mode is not optimal for large-scale culture. The addition of Pluronic F68 at 0.1% at 24 h post inoculation as well as the switch from surface aeration mode to the sparged mode, 2 days after the start of the culture, had markedly improved cell growth performance. A cell density level of 5.5 × 10⁶ cells/ml was reached when cells were grown in a 2-l bioreactor, on 3 g/l Cytodex 1 in IPT-AF medium and using the recirculation culture mode. Cell infection at an MOI of 0.1 and using perfused culture, resulted in a maximal virus titer of 3.5 × 10⁷ FFU/ml. The activity of the pooled inactivated rabies virus harvests showed a protective activity that meets WHO requirements.     

Changes in Morphology and Elemental Composition of <Emphasis Type="Italic">Vibrio splendidus</Emphasis> Along a Gradient from Carbon-limited to Phosphate-limited Growth

We examined morphology, elemental composition (C, N, P), and orthophosphate-uptake efficiency in the marine heterotrophic bacterium Vibrio splendidus grown in continuous cultures. Eight chemostats were arranged along a gradient of increasing glucose concentrations in the reservoirs, shifting the limiting factor from glucose to phosphate. The content of carbon, nitrogen, and phosphorus was measured in individual cells by x-ray microanalysis using a transmission electron microscope (TEM). Cell volumes (V) were estimated from length and width measurements of unfixed, air-dried cells in TEM. There was a transition from coccoid cells in C-limited cultures toward rod-shaped cells in P-limited cultures. Cells in P-limited cultures with free glucose in the media were significantly larger than cells in glucose-depleted cultures (P < 0.0001). We found functional allometry between cellular C-, N-, and P content (in femtograms) and V (in cubic micrometers) in V. splendidus (C = 224 × V ^0.89, N = 52.5 × V ^0.80, P = 2 × V ^0.65); i.e., larger bacteria had less elemental C, N, and P per V than smaller cells, and also less P relative to C. Biomass-specific affinity for orthophosphate uptake in large P-limited V. splendidus approached theoretical maxima predicted for uptake limited by molecular diffusion toward the cells. Comparing these theoretical values to respective values for the smaller, coccoid, C-limited V. splendidus indicated, contrary to the traditional view, that large size did not represent a trade-off when competing for the non-C-limiting nutrients.     

Influence of inoculum density on population dynamics and dauer juvenile yields in liquid culture of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida)

For improvement of mass production of the rhabditid biocontrol nematodes Steinernema carpocapsae and Steinernema feltiae in monoxenic liquid culture with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, the effect of the initial nematode inoculum density on population development and final concentration of dauer juveniles (DJs) was investigated. Symbiotic bacterial cultures are pre-incubated for 1 day prior to inoculation of DJs. DJs are developmentally arrested and recover development as a reaction to food signals provided by their symbionts. After development to adults, the nematodes produce DJ offspring. Inoculum density ranged from 1 to 10 × 10³ DJ per milliliter for S. carpocapsae and 1 to 8 × 10³ DJs per milliliter for S. feltiae. No significant influence of the inoculum density on the final DJ yields in both nematode species was recorded, except for S. carpocapsae cultures with a parental female density <2 × 10³ DJs per milliliter, in which the yields increased with increasing inoculation density. A strong negative response of the parental female fecundity to increasing DJ inoculum densities was recorded for both species with a maximum offspring number per female of >300 for S. carpocapsae and almost 200 for S. feltiae. The compensative adaptation of fecundity to nematode population density is responsible for the lack of an inoculum (or parental female) density effect on DJ yields. At optimal inoculation density of S. carpocapsae, offspring were produced by the parental female population, whereas S. feltiae always developed a F1 female population, which contributed to the DJ yields and was the reason for a more scattered distribution of the yields. The F1 female generation was accompanied by a second peak in X. bovienii density. The optimal DJ inoculum density for S. carpocapsae is 3–6 × 10³ DJs per milliliter in order to obtain >10³ parental females per milliliter. Density-dependent effects were neither observed on the DJ recovery nor on the sex ratio in the parental adult generation. As recovery varied between different batches, assessment of the recovery of inoculum DJ batches is recommended. S. feltiae was less variable in DJ recovery usually reaching >90%. The recommended DJ inoculum density is >5 × 10³ DJs per milliliter to reach >2 × 10³ parental females per milliliter. The mean yield recorded for S. carpocapsae was 135 × 10³ and 105 × 10³ per mililiter for S. feltiae.     

Towards a scalable bioprocess for rAAV production using a HeLa stable cell line

The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 10¹¹ vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 10¹¹ vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.     

Insect cell density in bioreactor cultures can be estimated from on-line measurements of optical density

Total cell density in growing insect cell suspension cultures was accurately estimated from both off-line and on-line measurements of optical density (OD). The latter measurements were done with an in-situ autoclavable OD sensor. The ability to continuously monitor cell density in insect cell cultures may be useful for the development of a large-scale process for recombinant protein production using baculovirus expression vectors.     

Enhancement of polysaccharides production in <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> by the addition of ethyl acetate extracts from <Emphasis Type="Italic">Eupolyphaga sinensis</Emphasis> and <Emphasis Type="Italic">Catharsius molossus</Emphasis>

To screen stimulators from Chinese medicinal insects for mycelial growth and polysaccharides production of Ganoderma lucidum, G. lucidum was inoculated into the media with and without supplementation of medicinal insect extracts. The ethyl acetate extract of Eupolyphaga sinensis at 55 mg l⁻¹ lead to significant increase in both biomass and intracellular polysaccharides (IPS) concentration from 8.53 ± 0.41 to 14.16 ± 0.43 and 1.28 ± 0.09 to 2.13 ± 0.11 g l⁻¹, respectively. In addition, the ethyl acetate extract of Catharsius molossus at 55 mg l⁻¹ significantly enhanced extracellular polysaccharides (EPS) production; the EPS yield increased from 350.9 ± 14.1 to 475.1 ± 15.3 mg l⁻¹. There were no new components in the two types of polysaccharides obtained by the addition of the insect extracts.     

The adverse effect of nitrogen limitation and excess-cellobiose on Fibrobacter succinogenes S85

Fibrobacter succinogenes S85 cultures that were cellobiose-limited converted cellobiose to succinate and acetate, produced little glucose or cellotriose, maintained an intracellular ATP concentration of 4.1 mM and a membrane potential of 140 mV for 24 h, did not lyse at a rapid rate once they had reached stationary phase, and had a most probable number of viable cells that was greater than 10⁶/ml. When the cellobiose concentration was increased 6-fold (5 mM to 30 mM), ammonia was depleted and the cultures left 10 mM cellobiose. Cultures provided with excess cellobiose produced succinate and acetate while they were growing, but there was little increase in fermentation acids after the ammonia was depleted and growth ceased. The stationary-phase, cellobiose-excess cultures had a lysis rate that was 7-fold faster than that of the cellobiose-limited cultures, and the most probable number was only 3.3 × 10³ cells/ml. The stationary-phase, cellobiose-excess cultures had 2.5 times as much cellular polysaccharide as the cellobiose-limited cultures, but the intracellular ATP and membrane potential were very low (0.1 mM and 40 mV respectively). Methylglyoxal, a potentially toxic end-product of carbohydrate fermentation, could not be detected, and fresh inocula grew rapidly in spent medium that was supplemented with additional ammonia. Stationary-phase, cellobiose-excess cultures converted cellobiose to glucose and cellotriose, but the apparent K _m of cellotriose formation was 15-fold lower than the K _m of glucose production (0.7 mM compared to 10 mM). Received: 26 June 1997 / Received revision: 12 August 1997 / Accepted: 29 August 1997     

Enhancement of tessaric acid production in Tessaria absinthioides cell suspension cultures

The total production of the sesquiterpene tessaric acid (TA) by cell cultures of Tessaria absinthioides at day 25 of the culture period reached 0.086 mg g⁻¹ DW, with intracellular accumulation accounting for 0.059 mg g⁻¹ DW. Dimethylsulfoxide-induced permeabilization of the cells effected both total production and extracellular accumulation of the sesquiterpene to reach levels of 148% and 271%, respectively. Cultures treated with elicitor preparations of Verticillum sp., Monodyctis cataneae, Acremonium sp., and Aspergillus niger produced TA at levels of 281%, 197%, 149%, and 139%, respectively. Treatment of cell suspension cultures with cis-(-)-jasmonic acid (5 μM) increased production to 267%, whereas jasmonic acid pretreatment and subsequent elicitation raised external tessaric acid to 702%. Received: 16 December 1998 / Revision received: 02 November 1999 / Accepted: 19 November 1999