Investigation of acid proteinase biosynthesis by the fungus Humicola lutea 120-5 in an airlift bioreactor

Acid proteinase production using filamentous fungus Humicola lutea 120-5 was studied under batch and continuous fermentation conditions in an airlift bioreactor. A comparison with proteinase production by fungal cells, cultivated in stirred tank bioreactor was made. The process performance in both fermentation devices was similar with respect to substrate utilization, biomass, and enzyme concentration. Continuous acid proteinase production was achieved for 14 days at an optimal dilution rate of 0.05/h with maximum specific activity of 90 U/mg DW of mycelia and yield of 38 U/mg glucose. The volumetric productivity (50 U/ml. h) was approximately 3 times higher than this of the batch system. All continuous experiments were carried out without any bacterial contamination, due to the low pH (3.0-3.5) during the process. The "pellet" type growth of the fungus in the airlift reactor prevented the system from plugging with filaments.     

Impact of ammonia concentration on Spirulina platensis growth in an airlift photobioreactor

Spirulina platensis was cultivated in a bench-scale airlift photobioreactor using synthetic wastewater (total nitrogen 412 mg L(-1), total phosphorous 90 mg L(-1), pH 9-10) with varying ammonia/total nitrogen ratios (50-100% ammonia with balance nitrate) and hydraulic residence times (15-25 d). High average biomass density (3500-3800 mg L(-1)) and productivity (5.1 g m(-2) d(-1)) were achieved when ammonia was maintained at 50% of the total nitrogen. Both high ammonia concentrations and mutual self-shading, which resulted from the high biomass density in the airlift reactor, were found to partially inhibit the growth of S. platensis. The performance of the airlift bioreactor used in this study compared favorably with other published studies. The system has good potential for treatment of high strength wastewater combined with production of algae for biofuels or other products, such as human and animal food, food supplements or pharmaceuticals.     

Production of glomus intraradices propagules, an arbuscular mycorrhizal fungus, in an airlift bioreactor

This work addresses the symbiotic culture of the arbuscular mycorrhizal (AM) fungus Glomus intraradices with Daucus carota hairy roots transformed by Agrobacterium rhizogenes, in two submerged culture systems: Petri dish and airlift bioreactor. AM fungi play an active role in plant nutrition and protection against plant pathogens. These fungi are obligate biotrophs as they depend on a host plant for their needs in carbohydrates. The effect of the mycorrhizal roots inoculum-to-medium volume ratio on the growth of both symbionts was studied. A critical inoculating condition was observed at approximately 0.6 g dry biomass (DW). L-1 medium, above which root growth was significantly reduced when using a low-salt minimal (M) liquid medium previously developed for hairy root-AM fungi co-culture. Below critical inoculum conditions the maximum specific root growth and specific G. intraradices spore production rates of 0.021 and 0.035 d-1, respectively, were observed for Petri dish cultures. Maximum spore production in the airlift bioreactor was ten times lower than that of Petri dish cultures and obtained with the lowest inoculum assessed (0.13 g DW. L-1 medium) with 1.82 x 10(5) +/- 4.05 x 10(4) (SEM) spores (g DW inoculum)-1 (L medium)-1 in 107 d. This work proposes a second-generation bioprocess for AM fungi propagule production in bioreactors. Copyright 1999 John Wiley & Sons, Inc.     

From field sampling to pneumatic bioreactor mycelia production of the ectomycorrhizal mushroom Laccaria trichodermophora

In order to increase survival rates of greenhouse seedlings destined for restoration and conservation programs, successful mycorrhization of the seedlings is necessary. To reforest forest ecosystems, host trees must be inoculated with ectomycorrhizal fungi and, in order to guarantee a sufficient supply of ectomycorrhizal inoculum, it is necessary to develop technologies for the mass production of ectomycorrhizal fungi mycelia. We selected the ectomycorrhizal fungus Laccaria trichodermophora, due to its ecological traits and feasible mycelia production in asymbiotic conditions. Here, we report the field sampling of genetic resources, as well as the highly productive nutritional media and cultivation parameters in solid cultures. Furthermore, in order to achieve high mycelial production, we used strain screening and evaluated pH, carbon source concentration, and culture conditions of submerged cultures in normal and baffled shake flasks. The higher productivity culture conditions in shake flasks were selected for evaluation in a pneumatic bioreactor, using modified BAF media with a 10 g/L glucose, pH 5.5, 25 °C, and a volumetric oxygen transfer coefficient (K_La) of 36 h⁻¹. Under those conditions less biomass (12–37 %) was produced in the pneumatic bioreactor compared with the baffled shake flasks. This approach shows that L. trichodermophora can generate a large biomass concentration and constitute the biotechnological foundation of its mycelia mass production.     

Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis

The optimization of submerged culture conditions for mycelial growth and exopolysaccharide (EPS) production in an edible mushroom Tremella fuciformis was studied in shake flasks and bioreactors. The temperature of 28 degrees C and pH 8 in the beginning of fermentation in agitated flasks was the most efficient condition to obtain maximum mycelial biomass and EPS. The optimal medium constituents were as follows (gL(-1)): glucose 20, tryptone 2, KH(2)PO(4) 0.46, K(2)HPO(4) 1 and MgSO(4).7H(2)O 0.5. The fungus was cultivated under various agitation and aeration conditions in a 5L stirred-tank bioreactor. The maximum cell mass and EPS production were obtained at a relatively high agitation speed of 200 rpm and at an aeration rate of 2 vvm. The flow behavior of the fermentation broth was Newtonian and the maximum apparent viscosity (35 cP) was observed at a highly aerated condition (2 vvm). The EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor. The morphological study revealed that the fungus grows in mainly three different yeast-like forms: ovoid, elongated, and double yeast forms. The high population of the elongated yeast has a very close relationship to high EPS production. The EPS were protein-bound polysaccharides consisted of mainly mannose, xylose, and fucose. The molecular weights of EPS were determined to be (1.3-1.5)x10(6).     

Microbial biomass production from rice straw hydrolysate in airlift bioreactors

Rice straw is a by-product of rice production, and a great bioresource as raw biomass material for manufacturing value-adding protein for animal feedstock, which has been paid more and more attention. In the present work, utilizing rice straw hydrolysate as a substrate for microbial biomass production in 11.5L external-loop airlift bioreactors was investigated. Rice straw hydrolysate obtained through acid-hydrolyzing rice straw was used for the culture of yeast Candida arborea AS1.257. The influences of gas flow rate, initial liquid volume, hole diameter of gas sparger and numbers of sieve plates on microbial biomass production were examined. The best results in the external-loop airlift bioreactor were obtained under 9.0 L initial liquid volume, 1.1 (v/v)/min gas flow rate during culture time of 0-24 h and 1.4 (v/v)/min gas flow rate of 24-48 h at 29+/-1 degrees C. The addition of the sieve plates in the riser of the external-loop airlift bioreactor increased productivity. After 48 h, under optimized operation conditions, crude protein productivity with one sieve and two sieves were 13.6 mg/mL and 13.7 mg/mL, respectively, comparing 12.7 mg/mL without sieves in the airlift bioreactor and 11.7 mg/mL in the in the 10-L mechanically stirred tank bioreactor. It is feasible to operate the external-loop airlift bioreactors and possible to reduce the production cost for microbial biomass production from the rice straw hydrolysate.     

Pilot-scale culture of somatic embryos of Eleutherococcus senticosus in airlift bioreactors for the production of eleutherosides

Purpose of work

To establish pilot scale bioreactor cultures of somatic embryos of Siberian ginseng for the production of biomass and eleutherosides. Somatic embryos of Eleutherococcus senticosus were cultured in airlift bioreactors using Murashige and Skoog medium with 30 g sucrose l^?1 for the production of biomass and eleutherosides. Various parameters including the type of bioreactor, aeration volume, and inoculum density were optimized for 3 l capacity bioreactors. Balloon-type airlift bioreactors, utilizing a variable aeration volume of 0.1–0.3 vvm and an inoculum of 5 g l^?1, were suitable for biomass and eleutheroside production. In 500 l balloon-type airlift bioreactors, 11.3 g dry biomass l^?1, 220 µg eleutheroside B l^?1, 413 µg eleutheroside E l^?1, and 262 µg eleutheroside E1 l^?1 were produced.     

High-density spore production of Piriformospora indica, a plant growth-promoting endophyte, by optimization of nutritional and cultural parameters

Piriformospora indica is an axenically cultivable root endophytic fungus which exerts plant growth promoting effects on its host plants. To enable commercial production of its spores, the medium composition and culture conditions have been optimized in a 14 L bioreactor such that they result in maximum biomass during growth phase and in maximum spore yield during subsequent sporulation phase. Maximum spore yields were obtained with modified Kaefer medium using a glucose deprivation strategy. An enhancement of 100% in overall biomass productivity (0.18 g L(-1) h(-1)) and reduction of about 70% in the time (60 h) required to achieve the maximum spore yield (9.25×10(7) spores/mL) was achieved in comparison to the original Kaefer medium. The high spore yield obtained in the present study seems to be economical for commercial production of P. indica.     

Production of <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis> strain PS-2001 biomass in submerged culture

Mushrooms or fruiting bodies of many basidiomycetes are commonly produced in solid-state fermentation, generally after 20-60 days of growth. However, it is also possible to produce biomass from these fungi, in submerged fermentation in shorter time. This work was aimed at evaluating biomass production with the basidiomycete Pleurotus sajor-caju, in a submerged process and to determine the proportion of chemical components of this biomass. Initially, an optimization of the culture medium was done to produce a faster growth of microbial mass by changing the concentrations of ammonium sulfate, soy protein and yeast extract. Using the optimized culture medium, values of approximately 5.5 g L(-1) of biomass in a medium with 10 g L(-1) of glucose were attained. When the optimized culture medium was tested in a 5-L stirred tank bioreactor, using 10 g L(-1) of glucose or sucrose as carbon source, values of 8.18 and 5.94 g L(-1) of biomass concentration were obtained, respectively. In the medium with glucose, high yields (0.82 g g(-1)) and productivity of 0.085 g L(-1) h(-1) were obtained. The exopolysaccharide content (1.58 g dry matter L(-1)) in the culture was higher in the fermentation with sucrose. The nutritional composition of the biomass obtained in the submerged fermentation was similar to that of the fruiting body in terms of quantities of total carbohydrates, ash and calories, but total fat and protein were higher.     

Growth and exopolysaccharide production during free and immobilized cell chemostat culture of Lactobacillus rhamnosus RW-9595M

AIMS: Biomass and exopolysaccharide (EPS) production were studied during chemostat cultures in whey permeate medium with Lactobacillus rhamnosus RW-9595M-free cells and cells immobilized on solid porous supports (ImmobaSil). METHODS AND RESULTS: A continuous culture with free cells was conducted for 9 days at dilution rates (D) between 0.3 and 0.8 h(-1) in yeast extract (YE)/mineral supplemented whey permeate. Maximum EPS production (1808 mg l(-1)) and volumetric productivity (542.6 mg l(-1) h(-1)) were obtained for a low D of 0.3 h(-1). A continuous fermentation in a two-stage bioreactor system, composed of a first stage with immobilized cells and a second stage inoculated with free cells produced in the first reactor, was carried out for 32 days. The influence of YE concentration, temperature and dilution rate, and their interactions on biomass, EPS and lactic acid production was investigated. A statistically significant model was found only for lactic acid production. Marked cell morphological and physiological changes led to the formation of very large cell-containing aggregates and a low mean soluble EPS production (138 mg l(-1)). Aggregate volumetric productivity of the two-stage system varied between 5.7 and 49.5 g l(-1) h(-1) for different fermentation conditions and times. Aggregates contained a very high biomass concentration, estimated at 74% of aggregate dry weight by nitrogen analysis and 4.3 x 10(12) CFU g(-1) by a DNA extraction method and a high nonsoluble polysaccharide content (14.2%). At age 24 days, insoluble EPS concentration and volumetric productivity were 1250 mg l(-1) and 2240 mg l(-1) h(-1) respectively. The physiological changes were shown to be reversible when cells were incubated during three successive batch cultures. CONCLUSIONS: EPS production and volumetric productivity during continuous free-cell chemostat cultures with L. rhamnosus RW-9595M are among the highest values reported for lactobacilli in literature. Immobilization and continuous culture resulted in low soluble EPS production and large morphological and physiological changes of L. rhamnosus RW-9595M, with formation of macroscopical aggregates mainly composed of biomass and nonsoluble EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on continuous EPS production by immobilized LAB. Immobilization and culture time-induced cell aggregation and could be used to produce new synbiotic products with very high viable cell and EPS concentrations.     

Production of antifungal metabolites by the ectomycorrhizal fungusPisolithus tinctorius strain SMF

Summary An ectomycorrhizal fungus,Pisolithus tinctorius strain SMF, isolated from a basidiocarp removed from the roots of a recently fallen old growth fir in the Smoky Mountains of Tennessee, was characterized for its in vitro production of antifungal metabolites. On solid mediumP. tinctorius SMF strongly inhibited growth of strains ofFusarium solani, Geotrichum candidum, Phanerochaete chrysosporium, andVerticillium dahliae, all species known to be plant pathogens. Evidence from paired colony growth inhibition studies on agar plates indicated that production of antifungal agents byP. tinctorius SMF may be enhanced by close physical contact with other fungi. The antifungal activity ofP. tinctorius SMF was much greater than that of several culture collection strains ofP. tinctorius. The culture collection strains either showed no or very limited activity. The antifungal activity was associated with an apparently inducible metabolism ofP. tinctorius SMF and with the production of darkly colored water soluble phenolic metabolites. Small scale fermentation studies showed that the phenolics are readily producible by submerged culture fermentation. This is the first report of submerged culture production of antifungal metabolites by an ectomycorrhizal fungus.     

Nutrition and bioprocess development for efficient biosynthesis of an antitumor compound from marine-derived fungus

An integrated nutrition and bioprocess strategy was developed for improving the biosynthesis of an antitumor compound, 1403C, by a marine-derived fungus, Halorosellinia sp. (no. 1403). First, statistical design strategies were synthetically applied to optimize the nutritional composition. The resulting 1403C production reached 2.07 g/l, which was 143.5 % higher than the original production. However, it only produced 0.44 g/l of 1403C in 5-l bioreactor fermentation. Thus, the operating parameters including culture pH, dissolved oxygen, agitation speed, impeller type and inoculum level were considered to improve the fermentation process, and an effective control strategy for 1403C production by Halorosellinia sp. submerged in a 5-l bioreactor was established. When inoculating 0.22 g/l dry biomass, controlling dissolved oxygen not lower than 30 % during the growth phase but ranging between 30 and 40 % during the stationary phase, using a double-layer six-flat-blade Rushton disc turbine agitated at 400 rpm, keeping short-term low pH and rapid-rising pH with glucose starvation, the highest 1403C production was finally obtained at 1.32 g/l, which was promoted by 200 % compared to before optimization. Fermentation scale-up was finally performed in a 500-l bioreactor, and 1403C production of 1.09 g/l was obtained.     

Kinetic and growth parameters of Arthrospira (Spirulina) platensis cultivated in tubular photobioreactor under different cell circulation systems

Arthrospira platensis was cultivated in tubular photobioreactor in order to evaluate growth and biomass production at variable photosynthetic photon flux density (PPFD = 60, 120, and 240 μmol photons m(-2)s(-1)) and employing three different systems for cell circulation, specifically an airlift, a motor-driven pumping and a pressurized system. The influence of these two independents variables on the maximum cell concentration (X(m)), cell productivity (P(x)), nitrogen-to-cell conversion factor (Y(X/N) ), photosynthetic efficiency (PE), and biomass composition (total lipids and proteins), taken as responses, was evaluated by analysis of variance. The statistical analysis revealed that the best combination of responses' mean values (X(m) = 4,055 mg L(-1), P(x) = 406 mg L(-1)day(-1), Y(X/N) = 5.07 mg mg(-1), total lipids = 8.94%, total proteins = 30.3%, PE = 2.04%) was obtained at PPFD = 120 μmol photons m(-2)s(-1); therefore, this light intensity should be considered as the most well-suited for A. platensis cultivation in this photobioreactor configuration. The airlift system did not exert any significant positive statistical influence on the responses, which suggests that this traditional cell circulation system could successfully be substituted by the others tested in this work.     

Relative importance of the endomycorrhizal and (or) ectomycorrhizal associations in Allocasuarina and Casuarina genera

This work was carried out to determine the relative importance of the endomycorrhizal and (or) ectomycorrhizal association in species of Casuarina and Allocasuarina. Under axenic conditions, Pisolithus and Scleroderma isolates formed ectomycorrhizas with a mantle and a Hartig net on Allocasuarina verticillata but failed to form a Hartig net on Casuarina glauca. In a controlled soil system, C. glauca was inoculated with the endomycorrhizal fungus Glomus intraradices Schenck & Smith, and A. verticillata was inoculated with Pisolithus albus IR100 Bougher & Smith and (or) G. intraradices. Both symbionts significantly stimulated growth in both plant species. For A. verticillata, its growth response to ectomycorrhizal inoculation was higher than to endomycorrhizal inoculation. When both symbionts were inoculated, antagonism among the fungal isolates was observed with a higher ectomycorrhizal colonization. These results showed that A. verticillata was ectomycorrhizal dependent, whereas C. glauca was endomycorrhizal dependent. From a practical point of view, this study shows the importance of selecting compatible mycorrhizal fungi for developing successful inoculation programmes. In addition, it would help to further research and determine the effect of ecto- and endo-mycorrhizal symbiosis on the formation and function of N2-fixing actinorhizal nodules.     

Beta-glucan production by Botryosphaeria rhodina in different bench-top bioreactors

AIMS: Evaluation of the technical feasibility of transferring beta-glucan production by Botryosphaeria rhodina DABAC-P82 from shaken flasks to bench-top bioreactors. METHODS AND RESULTS: Three different bioreactors were used: 3 l stirred tank reactor (STR-1) equipped with two different six-blade turbines; STR as above but equipped with a three-blade marine propeller plus draft-tube (STR-2); 2 l air-lift column reactor (ALR) equipped with an external loop. STR-1, tested at three different stirrer speeds (300, 500 and 700 rev min(-1)) appeared to be less suitable for beta-glucan production by the fungus, being maximum production (19.4 g l(-1)), productivity (0.42 g l(-1) h(-1)) and yield (0.48 g g(-1) of glucose consumed) markedly lower than those obtained in shaken culture (29.7 g l(-1), 1.23 g l(-1) h(-1) and 0.61 g g(-1), respectively). Better performances were obtained with both STR-2 and ALR. With the latter, in particular, the increase of production was accompanied by reduced fermentation time (25.7 g l(-1) after only 22 h); productivity and yield were highest (1.17 g l(-1) h(-1) and 0.62 g g(-1) of glucose consumed, respectively). CONCLUSION: Using an air-lift reactor with external loop, the scaling up from shaken flasks to bench-top bioreactor of the beta-glucan production by B. rhodina DABAC-P82 is technically feasible. SIGNIFICANCE AND IMPACT OF THE STUDY: Although culture conditions are still to be optimized, the results obtained using the ARL are highly promising.     

Propionic acid production in an in situ cell retention bioreactor

Continuous fermentations were conducted with in situ cell retention using spin filters (pore size 5 microm and 10 microm) for propionic acid production by Propionibacterium acidipropionici. Continuous fermentation with a 5-microm pore size spin filter resulted in 50% cell retention. Propionic acid productivity was enhanced (0.9 g l(-1) h(-1)) by approximately four-fold compared to conventional batch fermentation (0.25 g l(-1) h(-1)). The in situ cell retention (5-microm pore size spin filter) bioreactor was operated continuously and smoothly for 8 days at a dilution rate of D=0.05 h(-1).     

Investigation of the bacitracin biosynthesis in an airlift bioreactor

Results of pilot plant studies using an external-loop airlift bioreactor (170 l fermentation volume, liquid height-to-riser diameter: 27, loop-to-tower cross-section-area: 0.1225) have proven the relative merits of such a system in the bacitracin biosynthesis produced by the Bacillus licheniformis submerged aerobic cultivation. The results were compared to those obtained in a pilot-scale stirred-tank bioreactor with the same values of k_La. Excepting the aeration rate of 0.2 vvm, the fermentation process performed at 0.5 vvm and 1/0 vvm, respectively, unfolded similarly in the two fermentation devices with respect to the cell mass production, substrate utilization and bacitracin production during the fermentation process. In the riser section of the airlift bioreactor, the dissolved oxygen levels were higher, while in the downcomer section they were lower than those realized in the stirred tank bioreactor. Power requirements of the airlift fermenter were by 17–64% lower than those for a mechanically agitated system, depending on the aeration rates, which led to an important energy saving. Moreover, the lack of mechanical devices in the airlift system provides safety and a more gentle environment for the cultivation of microorganisms.     

Production of extracellular protease from crude substrates with dregs in an external-loop airlift bioreactor with lower ratio of height to diameter

Bacillus subtilis AS1.398 was cultivated in a 11.5-L total volume external-loop airlift bioreactor with a low height-to-diameter ratio of 2.9 and a riser-to-downcomer diameter ratio of 6.6 for the production of protease from crude substrates with dregs. The influence of aeration rate, liquid volume, and sparger hole diameter on protease production was investigated. An average of 8197 u/mL protease activity was obtained after a total fermentation time of 32 h in the external-loop airlift bioreactor with a liquid volume of 8.5 L, air flow rate of 1.5 vvm, and sparger hole diameter of 1.5 mm. The addition of one stainless steel sieve plate in the riser of the airlift bioreactor increased productivity of protease. After 32 h of fermentation, an average of 8718 u/mL protease activity was achieved in the external-loop airlift bioreactor with one sieve plate and an air flow rate of 1.2 vvm, liquid volume of 8.5 L, and gas sparger hole diameter of 1.5 mm. This was 9.0% higher than the typical averages of about 8000 u/mL protease activity in the mechanically stirred tank bioreactors of the enzyme factory using the same microorganism. It is possible to make a scale-up of the external-loop airlift bioreactor and feasible to operate it for production of protease from crude substrate with dregs.     

Improvement of Panax notoginseng cell culture for production of ginseng saponin and polysaccharide by high density cultivation in pneumatically agitated bioreactors

A Panax notoginseng cell culture was successfully scaled up from shake flask to 1.0-L bubble column reactor and concentric-tube airlift reactor. High-density bioreactor batch cultivation was carried out using a modified MS medium. The maximum cell density in batch cultures reached 20.1, 21.0 and 24.1 g/L in the shake flask, bubble column and airlift reactors, respectively, and their corresponding biomass productivity was 950, 1140 and 1350 mg/(L x d) for each. The productivity of ginseng saponin was 70, 96 and 99 mg/(L x d) in the flask, bubble column and airlift reactors, respectively; and the polysaccharide productivity reached 104, 119 and 151 mg/(L x d) for each. Furthermore, a fed-batch cultivation strategy was developed on the basis of specific oxygen uptake rate (SOUR), i.e., sucrose feeding before a sharp decrease of SOUR, and the highest cell density of 29.7 g/L was successfully achieved in the airlift bioreactor on day 17 with a very high biomass productivity of 1520 mg/(L x d). The concentrations of ginseng saponin and polysaccharide reached about 2.1 and 3.0 g/L, respectively, and their productivity was 106 (saponin) and 158 mg/(L x d) (polysaccharide). This work successfully demonstrated the high-density bioreactor cultivation of P. notoginseng cells in pneumatically agitated bioreactors and the reproduction of the shake flask culture results in bioreactors. The cell density, biomass productivity, production titer and productivity of both ginseng saponin and polysaccharide obtained here were the highest that have been reported on a reactor scale for all the ginseng species.     

Indole 3-acetic acid production by ectomycorrhizal fungi.

Ability of 8 ectomycorrhizal fungi to synthesise indole 3-acetic acid from L-tryptophan and their growth rate were studied. Differences in the levels of IAA synthesis and biomass production among the 8 mycorrhizal fungi were observed. A positive correlation was recorded between IAA level and mycelial growth. The synthesis of IAA and mycelial biomass were maximum on 30th day after incubation. Pisolithus tinctorius and Laccaria laccata exhibited higher amounts of IAA production than other fungi, whereas Amanita muscaria and Rhizopogon luteolus showed least quantity of IAA.