The application of air-lift fermenters to the cultivation of filamentous fungi

Summary An investigation of the performance of air-lift fermenters showed that the value of the oxygen mass transfer coefficient (K_La) increased with both the aeration rate and vessel size. Although some change in the liquid circulation pattern occurred with increasing superficial gas velocity, there was no transition from bubbly to slug flow over the range of superficial gas velocities studied. Increases in broth viscosity caused an increase in gas hold up and a reduction in the values for K_La, although this reduction was not as great as that observed in mechanically agitated fermenters. Under conditions of aeration and agitation which gave comparable K_La values similar biomass yields of Aspergillus oryzae were obtained in 7.25 l and 100 l air-lift fermenters, and in a 3.5 l mechanically agitated fermenter.     

A comparison of laboratory and pilot-scale fermentations in winemaking conditions

We investigated the influence of the fermenter size on alcoholic fermentation. Experiments were carried out at pilot scale, in 100-L fermenters, and at laboratory scale, in stirred and static 1-L fermenters. Two musts, Grenache blanc and Sauvignon, were fermented with and without the addition of solid particles from grape musts. Highly clarified must fermentation kinetics was strongly affected by the scale of the experiment, with slower fermentation occurring in the 100-L fermenter. Alcohol, ester, and thiol synthesis in clarified sauvignon must fermentation was also strongly correlated with the fermentation scale. Addition of solid particles from grape tended to reduce the effects on kinetics associated with increasing the scale of the fermentation, by increasing the maximum rate of CO₂ production, and by shortening the duration of fermentation. The addition of such particles also decreased the effects of scaling up the fermentation on the concentration of some volatile compounds, i.e., isoamyl acetate, ethyl octanoate, but did not decrease this effect for other compounds, such as isobutyl acetate, isobutanol, and 3-mercaptohexanol.     

Nitrogen fixation as evidence for the reducing nature of the early biosphere

Probably the first nitrogen fixers were anaerobic, non-photosynthetic, bacteria, i.e. fermenters. During the evolution of N₂ fixation they still needed nitrogen on the oxidation level of ammonia. Because of the complexities in structure and function of nitrogenase this evolution must have required a long time. The photosynthetic and later the respiring bacteria inherited the capacity for N₂ fixation from the fermenters, but the process did not change a great deal when it was taken over.Because of the long need for NH₃, which is unstable in a redoxneutral atmosphere, a long-persisting reducing atmosphere was needed. The transition to a redoxneutral atmosphere, dominated by CO₂, H₂O and N₂, cannot have been rapid, and the NH₃ in it was recycled. Probably the atmosphere contained for a long time, as was suggested by Urey but is often denied now, a great deal of methane as a reductant. The recycling occurred with participation of intermediates like cyanide, through energy input as UV radiation or as electric discharges. A stationary state was set up.The hypothesis is recalled that coloured, photosynthetic, NH₃ bacteria, analogous to coloured sulphur bacteria, may have existed, or may still exist, in reducing conditions. A few remarks are made about the origin of nitrification in the later, oxidizing atmosphere.     

Effect of a combination of inulin and polyphenol-containing adzuki bean extract on intestinal fermentation in vitro and in vivo

The effect of a combination of inulin (INU) and polyphenol-containing adzuki bean extract (AE) on intestinal fermentation was examined in vitro using fermenters for 48 h and in vivo using rats for 28 d. The total short-chain fatty acid concentrations in the fermenters were decreased by a combination of INU and AE, but the concentration in the INU + AE group was higher than the cellulose (CEL) and CEL + AE groups. The cecal propionate concentration was increased by a combination of INU and AE compared with their single supplement. The ammonia-nitrogen concentration in the fermenters and rat cecum was decreased by INU and AE. Cecal mucin levels were increased by INU and AE respectively. Therefore, our observations suggested that the combination of INU and AE might be a material of functional food that includes several healthy effects through intestinal fermentation.     

Production of bacteriocin jenseniin G by Propionibacterium is pH sensitive

Bacteriocin jenseniin G is detected typically in 50-fold concentrated 10 d cultures of the producer, Propionibacterium thoenii ( jensenii ) P126 at low concentrations (32–64 AU ml⁻¹). The effect of pH on production was examined by growing the producer in sodium lactate broth, both statically and in stirred fermenters, with or without pH control. Activity was detected in unconcentrated static producer cultures at day 7 through to day 14. Maximal jenseniin G activity (21 AU ml⁻¹) was observed in concentrated supernates at day 9, remaining relatively constant through to day 14. Producer cultures grown in stirred fermenters without pH control yielded detectable activity in 50-fold concentrated supernates at day 3 and maximal activity at day 11. Producer growth in stirred fermenters at controlled pH yielded maximum production at pH 6·40 and maximum activity (160 AU ml⁻¹) at day 13. Jenseniin G activity at pH 6·40 represented a fivefold increase over previous reports. Medium pH was critical to jenseniin G production.     

Fibre digestibility in large herbivores as related to digestion type and body mass — An in vitro approach

The coexistence of different ungulate species in a given ecosystem has been the focus of many studies. Differences between ruminant foregut fermenters and hindgut fermenters were remarkable for example in the way they ingest and digest high fibre diets. Digestion trials based on total collections are difficult to conduct or are sometimes even not possible for wild animals in the field or in zoos. To gain information on the fibre digestion achieved by these animals and the influence of body mass (BM) thereon, a method using spot sampling is desirable. In this study, in vitro fermentation of faecal neutral detergent fibre (NDF) was used as a measure of fibre digestion in large ungulates. Food and faecal samples of 10 ruminant foregut fermenting and 7 hindgut fermenting species/breeds were collected. All animals received 100% grass hay with ad libitum access. The NDF of food and faeces was fermented in vitro in a Hohenheim gas test (HGT) for 96 h. The digestion type generally had an effect on the gas production (GP) of faecal NDF in the HGT with hindgut fermenters showing higher values than ruminant foregut fermenters. At any time interval of incubation, BM had no influence on GP. The results are in accordance with both findings that ruminant foregut fermenters have longer mean retention times and more comprehensive particle reduction and findings of a lack of influence of BM on digesta mean retention time. It can be stated that the HGT (96 h) is a useful and quick method to show also small differences within groups in fibre digestion.     

A kLa identification technique for a dynamic model of the coupled system: Air-lift fermenter — oxygen probes

The time delay of oxygen probe response to the signal from a fermenter makes identification of the volumetric oxygen transfer coefficient k_La by the dynamic method more complicated. A coupled model involving the transient-state oxygen balance of the fermenter together with the dynamic model of the oxygen probe must be then formulated, solved and identified. In this paper two simple models of air-lift loop fermenters have been proposed and a coupled mathematical model of the fermenter – oxygen probe system has been developed. The identification procedure was used to estimate k_La values in the fermenter with internal circulation flow on the basis of experimental measurements. A comparison of evaluated and experimental indications of the probes placed at various heights of the column proves that the model presented gives a possibility of the first-step approximation of k_La in loop fermenters.     

The negligible role of carbon dioxide and ethylene in ajmalicine production by Catharanthus roseus cell suspensions

Summary Removal of gaseous metabolites in an aerated fermenter affects ajmalicine production by Catharanthus roseus negatively. Therefore, the role of CO₂ and ethylene in ajmalicine production by C. roseus was investigated in 3 l fermenters (working volume 1.8 l) with recirculation of a large part of the exhaust air. Removal of CO₂, ethylene or both from the recirculation stream did not have an effect on ajmalicine production. Inhibition of ethylene biosynthesis in shake flasks with Co²⁺, Ni²⁺ or aminooxyacetic acid did not affect ajmalicine production. However, the removal of CO₂ did enhance the amount of extracellular ajmalicine.     

Shifts in microbial populations in Rusitec fermenters as affected by the type of diet and impact of the method for estimating microbial growth (15N v. microbial DNA)

Rusitec fermenters are in vitro systems widely used to study ruminal fermentation, but little is known about the microbial populations establishing in them. This study was designed to assess the time evolution of microbial populations in fermenters fed medium- (MC; 50% alfalfa hay : concentrate) and high-concentrate diets (HC; 15 : 85 barley straw : concentrate). Samples from solid (SOL) and liquid (LIQ) content of fermenters were taken immediately before feeding on days 3, 8 and 14 of incubation for quantitative polymerase chain reaction and automated ribosomal intergenic spacer analysis analyses. In SOL, total bacterial DNA concentration and relative abundance of Ruminococcus flavefaciens remained unchanged over the incubation period, but protozoal DNA concentration and abundance of Fibrobacter succinogenes, Ruminococcus albus and fungi decreased and abundance of methanogenic archaea increased. In LIQ, total bacterial DNA concentration increased with time, whereas concentration of protozoal DNA and abundance of methanogens and fungi decreased. Diet×time interactions were observed for bacterial and protozoal DNA and relative abundance of F. succinogenes and R. albus in SOL, as well as for protozoal DNA in LIQ. Bacterial diversity in SOL increased with time, but no changes were observed in LIQ. The incubated diet influenced all microbial populations, with the exception of total bacteria and fungi abundance in LIQ. Bacterial diversity was higher in MC-fed than in HC-fed fermenters in SOL, but no differences were detected in LIQ. Values of pH, daily production of volatile fatty acids and CH₄ and isobutyrate proportions remained stable over the incubation period, but other fermentation parameters varied with time. The relationships among microbial populations and fermentation parameters were in well agreement with those previously reported in in vivo studies. Using ¹⁵N as a microbial marker or quantifying total microbial DNA for estimating microbial protein synthesis offered similar results for diets comparison, but both methods presented contrasting results for microbial growth in SOL and LIQ phases. The study showed that fermentation parameters remained fairly stable over the commonly used sampling period (days 8 to 14), but shifts in microbial populations were detected. Moreover, microbial populations differed markedly from those in the inocula, which indicates the difficulty of directly transposing results on microbial populations developed in Rusitec fermenters to in vivo conditions.     

Nickel as an accelerator of biogas production in water hyacinth (Eichornia crassipes solms.)

The effect of nickel ions on biogas production has been investigated in 3-litre fermenters. It was observed that nickel is stimulatory up to 5 ppm, with an optimum at 2·5 ppm, in a water hyacinth-bovine excreta substrate. A similar effect with nickel was also observed at 5 ppm in bovine excreta.     

Importance of carbon dioxide for the cultivation of Thermoactinomyces vulgaris from spores

Thermoactinomyces vulgaris has been used for the production of the serine protease Thermitase [EC 3.4.21.14] by fermentation. Spore germination of this strain is influenced by the gas composition of the medium. Since the outgrowth of the germ tubes occurs only in the presence of a raised CO₂ concentration, the cultivation in aerated stirred fermenters is not reproducible. However, if cultivation takes place in a closed fermentation system, initially unaerated, in which the CO₂ formed by spore respiration accumulates, reproducible germination can be obtained. From an O₂-balance, indirect conclusions can be drawn with regard to the effect of CO₂, and the process of germination can be described. The cultivation method described permits almost complete germination of all inoculated spores and reproducible rates of biomass and enzyme production in the subsequent fermentation. Thus, stabilization of the procedure with increased enzyme yields is ensured.     

Some laboratory investigations of anaerobic waste-water treatment process control in two different types of filter fermenters

Anaerobic degradation of a model waste water was investigated in two different types of fermenters (filter and hybride filter) under batch and continuous conditions. Gas production, COD removal, formation of fatty acids and the CoF₄₂₀–content of the biomass were monitored. At both modes of action the filter showed more process stability.     

Iron reduction and gley formation by nitrogen-fixing clostridia

Summary Studies on iron reduction and the mechanism of gley formation by nitrogen-fixing clostridia are reported. Up to 10⁶ cells/g soil of anaerobic, nitrogen-fixing clostridia, capable of reducing iron (III)-oxide, were counted in samples taken from various top soils. In a gleyed subsoil as many as 10⁵ bacteria per g soil, capable of reducing and fixing nitrogen, were enumerated using the most probable number technique. In general, the ratio of the auxotrophic iron reducing clostridia (glucose+yeast extract fermenters) to the prototrophic iron reducing flora (glucose fermenters) was found much larger in the top soil samples than in those derived from various gleyed subsoils.An enrichment method for the isolation of nitrogen-fixing, iron reducing clostridia of the butyric-butyl type is described. The iron reducing capacity of this type of clostridia as well as of Clostridium pasteurianum was determined quantitatively. Generally, the presence of soil or soil extract enhanced the amount of dissolved ferrous iron, both with butyric acid fermenters and with Cl. pasteurianum.When enriched iron reducing clostridia were incubated anaerobically under N₂-atmosphere in a sterile, red-colored, lateritic type of soil with glucose, intense gleying occurred within a few days. Microscopic observations indicated the presence of sporeforming bacteria of the Clostridium butyricum type or related species.The biological and chemical mechanism of gley formation is discussed.This research was started at the Institut für Landwirtschaftliche Mikrobiologie, Justus Liebig-Universität, Giessen, Germany.     

Shedding light on biogas: Phototrophic biofilms in anaerobic digesters hold potential for improved biogas production

Conventional anaerobic digesters intended for the production of biogas usually operate in complete darkness. Therefore, little is known about the effect of light on their microbial communities. In the present work, 16S rRNA gene amplicon Nanopore sequencing and shotgun metagenomic sequencing were used to study the taxonomic and functional structure of the microbial community forming a biofilm on the inner wall of a laboratory-scale transparent anaerobic biodigester illuminated with natural sunlight. The biofilm was composed of microorganisms involved in the four metabolic processes needed for biogas production, and it was surprisingly rich in Rhodopseudomonas faecalis, a versatile bacterium able to carry out photoautotrophic metabolism when grown under anaerobic conditions. The results suggested that this bacterium, which is able to fix carbon dioxide, could be considered for use in transparent biogas fermenters in order to contribute to the production of optimized biogas with a higher CH₄:CO₂ ratio than the biogas produced in regular, opaque digesters. To the best of our knowledge, this is the first study characterising the phototrophic biofilm associated with illuminated bioreactors.     

The optimization of L-lysine fermentation and a mass transfer model

The efficiency of L-lysine biosynthesis is essentially determined by the power input and aeration ratio in the stirred fermenter. A mass transfer model was developed by means of the results of lysine fermentations in four geometrically similar fermenters with working volumes of 10 1, 50 1, 100 1 and 2500 1 which allows the optimization of lysine fermentation from the energetical point of view. The usefullness of this k_La-model is demonstrated with an example where the power input for an unknown fermenter is calculated.     

Dietary polysaccharides: fermentation potentials of a primitive gut ecosystem

The alimentary canal of the earthworm is representative of primitive gut ecosystems, and gut fermenters capable of degrading ingested biomass-derived polysaccharides might contribute to the environmental impact and survival of this terrestrial invertebrate. Thus, this study evaluated the postulation that gut microbiota of the model earthworm Lumbricus terrestris ferment diverse biomass-derived polysaccharides. Structural polysaccharides (e.g. cellulose, chitin) had marginal impact on fermentation in anoxic gut content treatments. In contrast, nonstructural polysaccharides (e.g. starch, glycogen) greatly stimulated (a) the formation of diverse fermentation products (e.g. H₂, ethanol, fatty acids) and (b) the facultatively fermentative families Aeromonadaceae and Enterobacteriaceae. Despite these contrasting results with different polysaccharides, most saccharides derived from these biopolymers (e.g. glucose, N-acetylglucosamine) greatly stimulated fermentation, yielding 16S rRNA gene-based signatures of Aeromonadaceae-, Enterobacteriaceae- and Fusobacteriaceae-affiliated phylotypes. Roots and litter are dietary substrates of the earthworm, and as proof-of-principle, gut-associated fermenters responded rapidly to root- and litter-derived nutrients including saccharides. These findings suggest that (a) hydrolysis of certain ingested structural polysaccharides may be a limiting factor in the ability of gut fermenters to utilize them and (b) nonstructural polysaccharides of disrupted biomass are subject to rapid fermentation by gut microbes and yield fatty acids that can be utilized by the earthworm.     

Membrane inlet mass spectrometry — measurement of dissolved gases in fermentation liquids

The measurement of gas concentrations in fermenters is vital for monitoring and controlling a great variety of biotechnological processes. At present, most systems analyse the content of inflowing and outflowing gas streams. The information obtained in this way has proved to be very useful, but gas phase analyses suffer from a considerable disadvantage; the gas composition of the outflow does not precisely reflect the gas composition of the microbes' environment at the time the measurements are made. To overcome this problem, new systems have been developed which measure the concentration of gases (particularly O₂, CO₂ and H₂) dissolved in fermentation liquids.     

Characterisation of liquid and gas flow in jet loop reactors

The fluid dynamics in jet loop fermenters has been subject to experimental and theoretical investigation. It is demonstrated that by determination of Euler number, Bodenstein number, and residence time distribution for the gas phase it is possible to perform a reliable characterization of the fermenters. It can be shown that the investigated jet loop fermenters with internal loop closely resemble ideally mixed tanks.     

Model for the growth of aerobic microorganisms under oxygen limiting conditions

A simple dynamic model is proposed which will allow fermenters to be run at throughputs which fully utilize the mass transfer capabilities of the fermenters while not decreasing the yield from the substrate. The model is compared with one previously proposed, which was originally formulated for double substrate limitation when both substrates were supplied in the feed. Computer solutions of the model are given which show the effects of the parameters used. Experimental results from growing Candida utilis on a high concentration of glucose were found to be similar to those predicted by the model.     

Comparison of alternative kinetic models for estimating the specific growth rate of Gibberella fujikuroi by image analysis techniques

Summary A new equation is proposed in order to obtain a consistent and accurate correlation between morphometric data of growing mycelia of Gibberella fujikuroi in solid media and its observed specific growth rates ( _obs) in stirred fermenters. A critical analysis is made of previous data and models.