New sensor allowing continuous water activity measurement of submerged or solid-substrate fermentations

Water activity of the substrate is an important and acute factor for growth as well as for metabolic production of microorganisms or for biocatalyst systems. A sensor has been designed in order to control this parameter on-line during submerged and solid-substrate fermentations. This sterilizable sensor allows the measurement of the relative humidity of the atmosphere in a small chamber by means of a capacitive element separated from the medium by a thin ethylenepolytetrafluoride membrane. For high a(w) values (>0.90) a sequential circulation of a dried gas prevents the sensor saturation. Measurements are rapid and accurate, and control of the water activity of a fermentation medium has been carried out for 5 days using this sensor.     

Effects of controlled gas environments in solid-substrate fermentations of rice

The gas environment is solid-substrate fermentations of rice significantly affected levels of biomass and enzyme formation by a fungal species screened for high amylase production. Constant oxygen and carbon dioxide partial pressures were maintained at various levels in fermentations by Aspergillus oryzae. Control of the gas phase was maintained by a “static” aeration system admitting oxygen on demand and stripping excess carbon dioxide during fermentation. Constant water vapor pressures were also maintained by means of saturated salt solutions. High Oxygen pressures stimulated amylase productivity significantly. On the other hand, amylase production was severely inhibited at high carbon dioxide pressures. While relatively insensitive to oxygen pressure, maximum biomass productivities were obtained at an intermediate carbon dioxide pressure. High oxygen transfer rates were obtained at elevated oxygen pressures, suggesting, in view of the stimulatory effect of oxygen on amylase production, a stringent oxygen requirement for enzyme synthesis. Solid-substrate fermentations were highly advantageous as compared with submerged cultures in similar gas environments. Not only were amylase productivities significantly higher, but the enzyme was highly concentration in the aqueous phase of the semisolid substrate particles and could be extracted in a small volume of liquid. Results of this work suggest that biomass and product formation in microbial processes may be amenable to control by the gas environment. This is believed to offer an interesting potential for optimizing selected industrial fermentation processes with respect to productivity and energy consumption.     

Mycelium differentiation and antibiotic production in submerged cultures of Streptomyces coelicolor

Despite the fact that most industrial processes for secondary metabolite production are performed with submerged cultures, a reliable developmental model for Streptomyces under these culture conditions is lacking. With the exception of a few species which sporulate under these conditions, it is assumed that no morphological differentiation processes take place. In this work, we describe new developmental features of Streptomyces coelicolor A3(2) grown in liquid cultures and integrate them into a developmental model analogous to the one previously described for surface cultures. Spores germinate as a compartmentalized mycelium (first mycelium). These young compartmentalized hyphae start to form pellets which grow in a radial pattern. Death processes take place in the center of the pellets, followed by growth arrest. A new multinucleated mycelium with sporadic septa (second mycelium) develops inside the pellets and along the periphery, giving rise to a second growth phase. Undecylprodigiosin and actinorhodin antibiotics are produced by this second mycelium but not by the first one. Cell density dictates how the culture will behave in terms of differentiation processes and antibiotic production. When diluted inocula are used, the growth arrest phase, emergence of a second mycelium, and antibiotic production are delayed. Moreover, pellets are less abundant and have larger diameters than in dense cultures. This work is the first to report on the relationship between differentiation processes and secondary metabolite production in submerged Streptomyces cultures.     

Growth estimation of solid-state koji by covering a cellophane membrane on the mash

In a solid-substrate fermentation system, fungal growth within a solid mash is an important index for the efficiency of the saccharification and production of metabolites. Estimation of fungal mass in such a heterogeneous solid-substrate systems is difficult and tedious. In this work, the comparison of Aspergillus oryzae which is a common strain for the wine-brewing process cultured on a cellophane membrane placed on a koji juice agar medium and a small scale of steamed rice koji culture was conduted. Experimental results showed that the cellophane membrane technique resembled the steamed rice koji culture and is considered as a convenient and effective way for investigating the growth characteristics and cytology for solid-substrate koji system.     

Microbial growth on peptones from fish industrial wastes

Industrial fish peptone was an excellent substrate for biomass production in solid and submerged fermentations. The maximal growth rates of several microorganisms were two to three times higher than those grown on beef (bacto)peptones and the final biomass concentrations were almost twice as great as those grown on beef peptones. Fish peptones did not increase the production of secondary metabolites relative to those produced on beef peptones in non-optimized media. Fish peptone has promising potential as a substrate for biomass production.     

Use of direct-infusion electrospray mass spectrometry to guide empirical development of improved conditions for expression of secondary metabolites from actinomycetes

A major barrier in the discovery of new secondary metabolites from microorganisms is the difficulty of distinguishing the minor fraction of productive cultures from the majority of unproductive cultures and growth conditions. In this study, a rapid, direct-infusion electrospray mass spectrometry (ES-MS) technique was used to identify chemical differences that occurred in the expression of secondary metabolites by 44 actinomycetes cultivated under six different fermentation conditions. Samples from actinomycete fermentations were prepared by solid-phase extraction, analyzed by ES-MS, and ranked according to a chemical productivity index based on the total number and relative intensity of ions present in each sample. The actinomycete cultures were tested for chemical productivity following treatments that included nutritional manipulations, autoregulator additions, and different agitation speeds and incubation temperatures. Evaluation of the ES-MS data from submerged and solid-state fermentations by paired t test analyses showed that solid-state growth significantly altered the chemical profiles of extracts from 75% of the actinomycetes evaluated. Parallel analysis of the same extracts by high-performance liquid chromatography-ES-MS-evaporative light scattering showed that the chemical differences detected by the ES-MS method were associated with growth condition-dependent changes in the yield of secondary metabolites. Our results indicate that the high-throughput ES-MS method is useful for identification of fermentation conditions that enhance expression of secondary metabolites from actinomycetes.     

Beneficial effects of enhanced aeration using perfluorodecalin in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cultures for lipase production

The inadequate supply of oxygen to biomass is a critical factor to the productivity of most aerobic submerged fermentations. This happens because oxygen is sparingly soluble in the aqueous media. The use of a second liquid phase of perfluorocarbon (PFC), an oxygen-carrying compound, in the culture medium can increase the availability of oxygen to the microorganisms. The effect of perfluorodecalin on Yarrowia lipolytica cultures was investigated in shake-flask cultures. It was found that the specific growth rate of Y. lipolytica, a strictly aerobic yeast, increases with increasing PFC concentration. Extracellular lipase production was increased with 20% (v/v) of PFC and agitation of 250 rev/min. It was shown that the PFC presence benefitted lipase production and not just its secretion to the extracellular medium.     

Improved secondary metabolite production in the genus Streptosporangium by optimization of the fermentation conditions

The cultivation of strains of the genus Streptosporangium in batch fermentations demonstrated that the optimal conditions for secondary metabolite production are completely different to those of the closely related genus Streptomyces. The dissolved oxygen tension (pO(2)) was identified as an important parameter for optimal production of secondary metabolites in submerged cultures. Extreme variations of this parameter by changes in aeration (gas flow), agitation system and stirrer speed showed a tremendous impact in production yields of all investigated strains. Finally, a 20-fold increase in productivity was observed by conditions of controlled oxygen excess compared to optimal fermentation conditions for Streptomyces strains.     

Classification and measurement of fungal pellets by automated image analysis

An automated image analysis method for classifying and measuring pellets of filamentous fungi growing in submerged fermentations has been developed. The method discriminates between pelleted mycelial growth and loose aggregates of dispersed hyphae. Pellets are classified into smooth and hairy types. In both cases, the core of the pellet is identified and its shape and size characterized. For hairy pellets the annular region is also characterized. The method was tested on pellets of Aspergillus niger ATCC 11414 grown in a defined medium in shake flasks. This rapid method makes practical extensive studies on the morphology of pellets in submerged fermentations and the influence of fermentation conditions on that morphology.     

Differentiation and anaerobiosis in standing liquid cultures of Streptomyces coelicolor

Streptomyces coelicolor differentiates on solid agar media by forming aerial hyphae that septate into spores. We here show that differentiation also occurs in standing liquid minimal media. After a period of submerged growth, hyphae migrate to the air interface, where they become fixed by a rigid reflecting film. Colonies that result from these hyphae form sporulating aerial hyphae. In addition, submerged hyphae in the liquid minimal medium may attach to the surface. Liquid standing cultures easily become anoxic only 1 to 2 mm below the surface. Yet, biomass increases, implying the existence of metabolic pathways supporting anaerobic growth.     

Enhanced submerged Aspergillus ficuum phytase production by implementation of fed-batch fermentation

Phytase is an important feed and food additive, which is both used in animal and human diets. Phytase has been used to increase the absorption of several divalent ions, amino acids, and proteins in the bodies and to decrease the excessive phosphorus release in the manure to prevent negative effects on the environment. To date, microbial phytase has been mostly produced in solid-state fermentations with insignificant production volumes. There are only a few studies in the literature that phytase productions were performed in submerged bench-top reactor scale. In our previous studies, growth parameters (temperature, pH, and aeration) and important fermentation medium ingredients (glucose, Na-phytate, and CaSO₄) were optimized. This study was undertaken for further enhancement of phytase production with Aspergillus ficuum in bench-top bioreactors by conducting fed-batch fermentations. The results showed that addition of 60 g of glucose and 10 g of Na-phytate at 96 h of fermentation increased phytase activity to 3.84 and 4.82 U/ml, respectively. Therefore, the maximum phytase activity was further enhanced with addition of glucose and Na-phytate by 11 and 40 %, respectively, as compared to batch phytase fermentations. It was also reported that phytase activity increased higher in early log stage additions than late log stage additions because of higher microbial activity. In addition, the phytase activity in fed-batch fermentation did not drop significantly as compared to the batch fermentation. Overall, this study shows that fungal phytase can be successfully produced in submerged fed-batch fermentations.     

Use of Direct-Infusion Electrospray Mass Spectrometry To Guide Empirical Development of Improved Conditions for Expression of Secondary Metabolites from Actinomycetes

A major barrier in the discovery of new secondary metabolites from microorganisms is the difficulty of distinguishing the minor fraction of productive cultures from the majority of unproductive cultures and growth conditions. In this study, a rapid, direct-infusion electrospray mass spectrometry (ES-MS) technique was used to identify chemical differences that occurred in the expression of secondary metabolites by 44 actinomycetes cultivated under six different fermentation conditions. Samples from actinomycete fermentations were prepared by solid-phase extraction, analyzed by ES-MS, and ranked according to a chemical productivity index based on the total number and relative intensity of ions present in each sample. The actinomycete cultures were tested for chemical productivity following treatments that included nutritional manipulations, autoregulator additions, and different agitation speeds and incubation temperatures. Evaluation of the ES-MS data from submerged and solid-state fermentations by paired t test analyses showed that solid-state growth significantly altered the chemical profiles of extracts from 75% of the actinomycetes evaluated. Parallel analysis of the same extracts by high-performance liquid chromatography–ES-MS–evaporative light scattering showed that the chemical differences detected by the ES-MS method were associated with growth condition-dependent changes in the yield of secondary metabolites. Our results indicate that the high-throughput ES-MS method is useful for identification of fermentation conditions that enhance expression of secondary metabolites from actinomycetes.     

Hydrolysis of pectin: an enzymatic approach and its application in banana fiber processing

Sediment samples were collected from different estuarine and marine areas along the West coast of India. Eighteen actinomycete cultures were isolated using starch casein agar and were screened for polygalacturonase activity by growing them on pectin-agar plates. Clear zones were visualized using 1% cetrimide. Out of the 18 strains screened ten cultures could effect hydrolysis of pectin. The above cultures were subjected to secondary screening under submerged fermentation. The actinomycete strain of Streptomyces lydicus was found to be a potent producer of polygalacturonase. Different growth media were screened for enzyme production and the best medium was selected for further studies. The crude enzyme was used for the treatment of raw banana fibers.     

Fermentative production of self-toxic fungal secondary metabolites

Fungi are well known for their vast diversity of secondary metabolites that include many life-saving drugs and highly toxic mycotoxins. In general, fungal cultures producing such metabolites are immune to their toxic effects. However, some are known to produce self-toxic compounds that can pose production optimization challenges if the metabolites are needed in large amounts for chemical modification. One such culture, LV-2841, was identified as the lead for one of our exploratory projects. This culture was found to be a slow grower that produced trace amounts of a known metabolite, cercosporamide, under the standard flask fermentation conditions, and extensive medium optimization studies failed to yield higher titers. Poor growth of the culture in liquid media was attributed to the self-toxicity of cercosporamide to the producing organism, and the minimum inhibitory concentration (MIC) of cercosporamide was estimated to be in the range of 8–16 μg/ml. Fermentations carried out in media containing Diaion^® HP20 resin afforded significantly higher titers of the desired compound. While several examples of resin-based fermentations of soil streptomyces have been published, this approach has rarely been used for fungal fermentations. Over a 100-fold increase in the production titer of cercosporamide, a self-toxic secondary metabolite, was achieved by supplementing the production medium with a commercially available neutral adsorbent resin.     

Statistical optimization of growth medium for the production of the entomopathogenic and phytotoxic cyclic depsipeptide beauvericin from Fusarium oxysporum KFCC 11363P

The production of the entomopathogenic and phytotoxic cyclic depsipeptide beauvericin (BEA) was studied in submerged cultures of Fusarium oxysporum KFCC 11363P isolated in Korea. The influences of various factors on mycelia growth and BEA production were examined in both complete and chemically defined culture media. The mycelia growth and BEA production were highest in Fusarium defined medium. The optimal carbon and nitrogen sources for maximizing BEA production were glucose and NaNO3, respectively. The carbon/ nitrogen ratio for maximal production of BEA was investigated using response surface methodology (RSM). Equations derived by differentiation of the RSM model revealed that the production of BEA was maximal when using 108 mM glucose and 25 mM NaNO3.     

Iron deficiency-induced tetracycline production in submerged cultures by Streptomyces aureofaciens

Streptomyces aureofaciens ATCC 10762 grown in rotary-shaken submerged cultures produced substantial amounts of tetracycline only when the defined medium was deprived of iron. The biosynthesis of tetracycline was inhibited either by free iron at concentrations above 1–2 μmol l⁻¹, or by chelated iron provided by the siderophores of this bacterial strain. Late static iron-containing cultures allowed cell differentiation and sporulation and led to tetracyclines synthesis. A nitrosoguanidine-induced mutant able to synthesize tetracycline in the presence of iron in shaken submerged cultures was isolated and compared to the wild-type strain. However, no constitutive siderophore-mediated iron transport occurred in the mutant. These results suggest the involvement of a putative iron-controlled repressor in the biosynthesis of these secondary metabolites during vegetative growth and primary metabolism of the bacterium.     

The growth conditions of cephalosporium eichhorniae for the conversion of starch substrates to protein

The protein synthesis by Cephalosporium eichhorniae on substrates containing starch was evaluated at different pH, moisture content, ammonium sulphate and potassium dihydrogen phosphate supplementation. The optimum pH level was about 4.2 and the moisture content 65%. The optimum level of supplementation of medium containing sweet potato substrate with (NH₄)₂SO₄ was smaller than that for cassava substrate (5.2 g/l < 6.7 g/l in submerged culture, and 4.0% < 5.2% in solid state fermentation). The total crude protein yields were about 7.6 g/l for submerged cultures and 12% DM for solid state fermentations.     

Acylceramides and lanosterol-lipid markers of terminal differentiation in cultured human keratinocytes: Modulating effect of retinoic acid

Summary Epidermal differentiation is accompanied by profound changes in the synthesis of a variety of intracellular proteins and intercellular lipids. In conventional, submerged culture keratinocytes have been shown to lose the ability to synthesize the protein markers of differentiation. They re-express them, however, when they are cultured in medium supplemented with delipidized [retinoic acid (RA)-depleted] serum or in air-exposed cultures using de-epidermized dermis (DED) as a substrate. Recent studies have revealed that acylceramides (AC) and lanosterol (LAN), which are present only in trace amounts in cultures of keratinocytes grown under submerged conditions on DED in medium supplemented with normal serum, become expressed in significant amounts when the culture is lifted to the air-liquid interface. Inasmuch as culture conditions may markedly affect the extent of keratinocyte differentiation, the present study aimed to investigate the effect of normal (RA-containing) or delipidized (RA-depleted) serum and of RA administration on lipid composition (especially of the AC and LAN contents) in cells cultured under submerged and air-exposed conditions. To test a possible effect of dermal substrate (used in the air-exposed model), the lipid composition of keratinocytes grown under submerged conditions on a plastic and on a dermal substrate (de-epidermized dermis, DED) has also been compared. The results revealed that under all culture conditions, RA deprivation of fetal bovine serum resulted in a marked increase of total ceramide content. Even under submerged conditions, the presence of both AC and LAN could be detected. In air-exposed culture, the content of these lipids was markedly increased. Addition of RA at 1 μM concentration to cultures grown in RA-depleted medium induced marked changes in lipid composition under all culture conditions tested. In cells grown under submerged conditions (both on plastic and on DED) AC and LAN were no longer present in detectable amounts. Also in air-exposed culture, a marked decrease in the content of these lipids was observed. These results suggest that liposoluble serum components, like RA, control the synthesis of lipids that are present in later stages of epidermal differentiation.