Cellulose degradation and carboxymethylcellulase production by Sporocytophaga myxococcoides grown in an air-lift fermenter

Summary Sporocytophaga myxcoccoides was grown in a 31 air-lift fermenter using a medium containing 2% w/v insoluble cellulose. The cellulose content of the medium reduced the k_La of the fermenter but during growth the dissolved oxygen concentration did not fall below 75% saturation. Rates of cellulose degradation and extracellular enzyme production were similar to those reported for a stirred-tank fermenter.     

Effects of dissolved oxygen concentration on biodegradation of 2,4-dichlorophenoxyacetic acid

Batch experiments were conducted to examine the effects of dissolved oxygen concentration on the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by an enrichment culture of 2,4-D-utilizing bacteria. A modified Monod equation was found to describe the relationship between the specific growth rate and the concentrations of both the organic substrate and dissolved oxygen. Values for the maximum specific growth rate, yield, and Monod coefficient for growth on 2,4-D were 0.09 h-1, 0.14 g/g, and 0.6 mg/liter, respectively. The half-saturation constant for dissolved oxygen was estimated to be 1.2 mg/liter. These results suggest that dissolved oxygen concentrations below 1 mg/liter may be rate limiting for the biodegradation of chlorinated aromatic compounds such as 2,4-D, which have a requirement for molecular oxygen as a cosubstrate for metabolism.     

Effects of dissolved oxygen concentration on biodegradation of 2,4-dichlorophenoxyacetic acid.

Batch experiments were conducted to examine the effects of dissolved oxygen concentration on the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by an enrichment culture of 2,4-D-utilizing bacteria. A modified Monod equation was found to describe the relationship between the specific growth rate and the concentrations of both the organic substrate and dissolved oxygen. Values for the maximum specific growth rate, yield, and Monod coefficient for growth on 2,4-D were 0.09 h-1, 0.14 g/g, and 0.6 mg/liter, respectively. The half-saturation constant for dissolved oxygen was estimated to be 1.2 mg/liter. These results suggest that dissolved oxygen concentrations below 1 mg/liter may be rate limiting for the biodegradation of chlorinated aromatic compounds such as 2,4-D, which have a requirement for molecular oxygen as a cosubstrate for metabolism.     

Microcalorimetric study of aerobic growth of Cellulomonas sp. 21399 on various carbohydrates

Summary Microcalorimetry was used to study the energetic aerobic growth of Cellulomonas sp. 21399 on glucose, cellobiose and amorphous and crystalline cellulose. The thermochemical aspect of growth on glucose was established with regard to the anabolic contribution. The results obtained allowed the use of glucose as a reference substrate for cellulose degradation. The experimental enthalpy change and the maximum catabolic activity, calculated from the maximum power evolved by the culture, were, respectively,-1079 kJ/mol and 0.85 mmol glucose per hour per dry weight of cells. The growth response on amorphous cellulose was equivalent to that demonstrated on glucose. However, on crystalline cellulose media, Cellulomonas sp. 21399 exhibited eight times less power and the quantity of heat evolved during growth showed that 50% of the cellulose was degraded. Quantitative results and the shape of power-time curves achieved indicate that the structural features of cellulose strongly influence its microbial degradability.     

Feasibility study of exponential feeding strategy in fed-batch cultures for phenol degradation using Cupriavidus taiwanensis

An indigenous phenol-degrading bacterial isolate Cupriavidus taiwanensis R186 was used to degrade phenol from an aqueous solution under fed-batch operation. An exponential feeding strategy combined with dissolved oxygen control was applied based on kinetic characteristics of cell growth and phenol degradation to meet sufficient metabolic needs for cellular growth and achieve the best phenol removal efficiency. Without the stress of phenol inhibition, the optimal set point of specific growth rate of exponential feeding for fed-batch phenol degradation was found to be 0.50–0.55μ_max (μ_max denotes the maximum specific growth rate from Monod model). Meanwhile, the sufficient set point of dissolved oxygen for maximal phenol degradation efficiency was approximately at 10–55% air saturation. With the optimal operation conditions, the best phenol degradation rate was 0.213 g phenol h⁻¹, while a shortest treatment time of 15 h was achieved for complete degradation of 11.35 mM (ca. 3.20 g) of phenol.     

Influence of glucose and oxygen on the production of ethyl acetate and isoamyl acetate by a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain during alcoholic fermentation

The effect of glucose and dissolved oxygen in a synthetic medium simulating the standard composition of grape juice on the production of ethyl acetate and isoamyl acetate by a Saccharomyces cerevisiae strain during alcoholic fermentation was studied. The specific in vitro activity of alcohol acetyltransferase (AATase, EC 2.3.1.84) and esterases (ESase, EC 3.1.1.1; hydrolysis and synthesis of esters) in cell-free extracts was also examined. The specific activity of AATase for ethyl acetate was found to peak at the beginning of the exponential growth phase and that for isoamyl acetate at its end. While the glucose concentration only affected the maximum specific activity of AATase, and only slightly, oxygen inhibited such activity, to a greater extent for isoamyl acetate than for ethyl acetate. On the other hand, esterases were found to catalyse the synthesis of ethyl acetate only at a low or medium glucose concentration (50 or 100 g l^-1, respectively), and to reach their maximum hydrolytic activity on isoamyl acetate during the stationary growth phase. The highest ethyl acetate and isoamyl acetate concentrations in the medium were obtained with a glucose concentration of 250 g l^-1 and semianaerobic conditions.     

Influence of hyperbaric oxygen tension on growth parameters of Candida tropicalis and Rhodococcus erythropolis

Summary Oxygen-limited growth was avoided by means of oxygen-enriched aeration in aerobic fermentation processes. Studies were carried out with Candida tropicalis (Cast.) Berkhout and Rhodococcus erythropolis (DSM 43215). The effect of hyperbaric dissolved oxygen tension on growth parameters was examined by varying the dissolved oxygen concentration and the carbon source (glucose, ethanol, and n-alkanes). Up to an oxygen concentration of 40 mg/l in the culture suspension no impairment of the economic coefficients and no promotion of cell lysis was found. It was observed that raised oxygen concentrations in the aeration gas led to enhanced specific growth rates. At cell concentrations above 20 g/l dry weight an uncoupling of carbon source dissimilation and biomass production was observed even at non-limiting oxygen concentrations.     

Studies on quantitative physiology of Trichoderma reesei with two-stage continuous culture for cellulose production

By employing a two-stage continuous-culture system, some of the more important physiological parameters involved in cellulose biosynthesis have been evaluated with an ultimate objective of designing an optimally controlled cellulose process. The two-stage continuous-culture system was run for a period of 1350 hr with Trichoderma reesei strain MCG-77. The temperature and pH were controlled at 32°C and pH 4.5 for the first stage (growth) and 28°C and pH 3.5 for the second stage (enzyme production). Lactose was the only carbon source for the both stages. The ratio of specific uptake rate of carbon to that of nitrogen, Q(C)/Q(N), that supported good cell growth ranged from 11 to 15, and the ratio for maximum specific enzyme productivity ranged from 5 to 13. The maintenance coefficients determined for oxygen, M_O, and for carbon source, M_C, are 0.85 mmol O₂/g biomass/hr and 0.14 mmol hexose/g biomass/hr, respectively. The yield constants determined are: Y_X/O = 32.3 g biomass/mol O₂, Y_X/C = 1.1 g biomass/g C or Y_X/C = 0.44 g biomass/g hexose, Y_X/N = 12.5 g biomass/g nitrogen for the cell growth stage, and Y_X/N = 16.6 g biomass/g nitrogen for the enzyme production stage. Enzyme was produced only in the second stage. Volumetric and specific enzyme productivities obtained were 90 IU/liter/hr and 8 IU/g biomass/hr, respectively. The maximum specific enzyme productivity observed was 14.8 IU/g biomass/hr. The optimal dilution rate in the second stage that corresponded to the maximum enzyme productivity was 0.026 ～ 0.028 hr^?1, and the specific growth rate in the second stage that supported maximum specific enzyme productivity was equal to or slightly less than zero.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line off-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the specific growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the specific growth rate, μ, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum specific growth rate, μ _max, measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q _O2,max, of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly differ from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-specific rates are up to 1.3 times higher in continuous cultures. Received: 8 February 1999 / Accepted: 17 February 1999     

The induction and repression of benzene and catechol oxidizing capacity of Pseudomonas putida ML2 studied in perturbed chemostat culture

The oxidation of catechol, an intermediate in benzene catabolism, was studied using transient variations in dissolved oxygen tension (DOT) when a succinate limited steady state culture of Pseudomonas putida ML2 was perturbed with a pulse of another substrate. A model was developed and tested for the effect of fluctuations in oxidizing enzyme activity on DOT. It was found that the rate of induction of catechol oxidizing enzymes was independent of dilution rate up to a relative growth rate /_max of 0.75. Only at higher dilution rates was catabolite repression observed.Abbreviations DOT dissolved oxygen tension - K _L a gas transfer coefficient - specific growth rate - _max maximum specific growth rate - K_s substrate saturation constant     

Protease production by Bacillus subtilis in oxygen-controlled,glucose fed-batch fermentations

Summary An open-loop, on-off control system using the dissolved oxygen level to control a glucose feed was used in a study of growth and production of protease by Bacillus subtilis CNIB 8054. With this system, both glucose and oxygen were controlled at low concentrations. In batch fermentations, protease activity in the fermentation broth was maximum when growth had stopped. During oxygen-controlled, glucose fed-batch fermentations, growth and the production of protease activity continued during glucose feeding. Oxygen-controlled, glucose fed-batch fermentations produced more protease activity than batch fermentations, depending upon the set point for dissolved oxygen. These results indicate that control of glucose and oxygen concentrations can result in improvements in protease production.     

Dependency of growth yield,maintenance and K s-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown diazotrophically in sucrose-limited chemostat cultures at either 12, 48, 108, 144 or 192 M dissolved oxygen. Steady state protein levels and growth yield coefficients (Y) on sucrose increased with increasing dilution rate (D). Specific rate of sucrose consumption (q) increased in direct proportion to D. Maintenance coefficients (m) extrapolated from plots of q versus D, as well as from plots of 1/Y versus 1/D exhibited a nonlinear relationship to the dissolved oxygen concentration. Constant maximal theoretical growth yield coefficients (Y ^G) of 77.7 g cells per mol of sucrose consumed were extrapolated irrespective of differences in ambient oxygen concentration. For comparison, glucose-, as well as acetate-limited cultures were grown at 108 M oxygen. Fairly identical m- and Y ^G-values, when based on mol of substrate-carbon with glucose and sucrose grown cells, indicated that both substrates were used with the same efficiency. However, acetate-limited cultures showed significantly lower m- and, at comparable, D, higher Y-values than cultures limited by either sucrose or glucose. Substrate concentrations (K _s) required for half-maximal growth rates on sucrose were not constant, they increased when the ambient oxygen concentration was raised and, at a given oxygen concentration, when D was decreased. Since biomass levels varied in linear proportion to K _s these results are interpreted in terms of variable substrate uptake activity of the culture.Abbreviations D dilution rate - K _s substrate concentration required for half maximal growth rate - m maintenance coefficient - q specific rate of substrate consumption - Y growth yield coefficient - Y ^G maximum theoretical growth yield coefficient     

Evaluation of kinetic parameters and mass transfer of glucose-fed granules under hypoxic conditions

This study demonstrated that the availability of oxygen influenc the kinetic parameters of sludge granules for the utilization and mass transfer of substrates. Batch experiments revealed that substrate utilization of the coupled sludge granules followed Monod’s kinetic model under hypoxic conditions and at initial substrate concentrations ranging from 1,350 to 4,456 mg/L. The corresponding kinetic coefficients of ks (maximum specific substrate glucose utilization rate), Ks (half saturation coefficient), and Y (growth yield) were 5.6 ∼ 7.8/day, 58 ∼ 64 mg/L, and 0.11 ∼ 0.17 mg of MLSS/mg of COD, respectively. Low dissolved oxygen content suppressed the activity of aerobic enzymes, which resulted in a ks value between those of aerobic granules and anaerobic granules. The maximum oxygen consumption rate (ko = 0.89/day) was relatively higher while the half-saturation constant (Ko = 1.71 mg/L) was significantly lower than those of aerobic granules. These results imply that dissolved oxygen was used more efficiently under hypoxic conditions. Thiele modulus (ϕ) and effectiveness factor (η) analysis revealed that the activity of microorganisms inside the granules was limited by the availability of oxygen. These properties differed from those found in aerobic granules, anaerobic granules, and activated sludge.     

Production of antibacterial violet pigment by psychrotropic bacterium RT102 strain

The antibacterial action of violet pigment, a mixture of violacein and deoxyviolacein, isolated from phychrotrophic bacterium RT102 strain was examined, and the operational conditions for the effective production of violet pigment were studied. The antibacterial activity of the violet pigment was confirmed for several bacteria such asBacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, andPseudomonas aeruginosa, and the high concentration of violet pigment, above about 15 mg/L, caused not only growth inhibition but also death of cells. The growth properties of RT102 strain were clarified under various incubation conditions such as pH, temperature, and dissolved oxygen concentration. The maximum violet pigment concentration,i.e. 3.7 g/L, and the maximum productivity of violet pigment,i.e. 0.12 g L⁻¹h⁻¹, were obtained in a batch culture of pH 6, 20°C, and 1 mg/L of dissolved oxygen concentration.     

Scale-up of naringinase production process based on the constant oxygen transfer rate for a novel strain of Bacillus methylotrophicus

Naringinase bioprocess based on Bacillus methylotrophicus was successfully scaled up based on constant oxygen transfer rate (OTR) as the scale-up criterion from 5-L bioreactor to 20-L bioreactor. OTR was measured in 5 and 20-L bioreactor under various operating conditions using dynamic method. The operating conditions, where complete dispersion was observed were identified. The highest OTR of 0.035 and 0.04?mMol/L/s was observed in 5 and 20-L bioreactor, respectively. Critical dissolved oxygen concentration of novel isolated strain B. methylotrophicus was found to be 20% of oxygen saturation in optimized medium. The B. methylotrophicus cells grown on sucrose had maximum oxygen uptake rate of 0.14?mMol/L/s in optimized growth medium. The cells produced the maximum naringinase activity of 751 and 778?U/L at 34?hr in 5 and 20-L bioreactors, respectively. The maximum specific growth rate of about 0.178/hr was observed at both the scales of operations. The maximum naringinase yield of 160 and 164?U/g biomass was observed in 5 and 20-L bioreactors, respectively. The growth and production profiles at both scales were similar indicating successful scale-up strategy for B. methylotrophicus culture.     

Effect of substrate concentration on ethanol production by Zymomonas mobilis on cellulose hydrolysate

Summary A cellulose hydrolysate from Aspen wood, containing mainly glucose, was fermented into ethanol by a thermotolerant strain MSN77 of Zymomonas mobilis. The effect of the hydrolysate concentration on fermentation parameters was investigated. Growth parameters (specific growth rate and biomass yield) were inhibited at high hydrolysate concentrations. Catabolic parameters (specific glucose uptake rate, specific ethanol productivity and ethanol yield) were not affected. These effects could be explained by the increase in medium osmolality. The results are similar to those described for molasses based media. Strain MSN77 could efficiently ferment glucose from Aspen wood up to a concentration of 60 g/l. At higher concentration, growth was inhibited.Nomenclature S glucose concentration (g/l) - X biomass concentration (g/l) - P ethanol concentration (g/l) - C conversion of glucose (%) - t fermentation time (h) - q_S specific glucose uptake rate (g/g.h) - q_p specific ethanol productivity (g/g.h) - YINX/S biomass yield (g/g) - Y_p/S ethanol yield (g/g) - specific growth rate (h^-1)     

Enhanced production of cellulase usingAcidothermus cellulyticus in fed-batch culture

Summary Fed-batch fermentations of Acidothermus cellulolyticus utilizing mixtures of cellulose and sugars were investigated for potential improvements in cellulase enzyme production. In these fermentations, we combined cellulose from several sources with various simple sugars at selected concentrations. The best source of cellulose for cellulase production was found to be ball-milled Solka Floc at 15 g/l. Fed-batch fermentations with cellobiose and Solka Floc increased cell mass only slightly, but succeeded in significantly enhancing cellulase synthesis compared to batch conditions. Maximum cellulase activities obtained from fermentations initiated with 2.5 g cellobiose/l and 15 g Solka Floc/l were 0.187 units (U)/ml, achieved by continuous feeding to maintain <0.1 g cellobiose/l, and 0.215 U/ml using the same initial medium when 2.5 g cellobiose/l was step-fed after the sugar was nearly consumed. In batch, dual-substrate systems consisting of simple sugars with Solka Floc, substrate inhibition was evident in terms of specific growth rates, specific productivity values, and maximum enzyme yields. Limiting concentrations of glucose or sucrose at 5 g/l, and cellobiose at 2.5 g/l, in the presence of Solka Floc, yielded cellulase activities of 0.134, 0.159, and 0.164 U/ml, respectively. Offprint requests to: M. E. Himmel     

Rheological,Mass Transfer,and Mixing Characterization of Cellulase-Producing Trichoderma reesei Suspensions

Steady and dynamic shear measurements are utilized to characterize the rheological behavior of Trichoderma reesei RUT-C30 fungal suspensions during batch growth on xylose (soluble substrate) or cellulose (particulate solid substrate) at three different fermentor impeller speeds (250, 400, and 550 rpm). Biomass concentrations versus time were unimodal on xylose and bimodal on cellulose. This behavior is consistent with relatively rapid, early growth on easily metabolized growth medium components (yeast extract), followed by a second, slower growth phase due to hydrolysis of recalcitrant cellulose by increasing cellulase concentrations. Critical dissolved oxygen (DO) concentration for T. reesei growth on cellulose was found to be 0.073 mmol/L. The DO was kept above this level by supplementing the air feed with pure oxygen, implying that mass transfer limitations were not the cause of bimodal cell growth. Steady shear rheological data showed shear thinning behavior and a yield stress for all broth samples regardless of substrate. Casson and Herschel−Bulkley constitutive equations fit steady shear data well. Dynamic shear measurements on broth suspensions indicated “gel-like” behavior at low strains, with microstructural breakdown at larger displacements. Time variations of the Casson model parameters (yield stress and Casson viscosity) and dynamic moduli (elastic and viscous modulus) followed both cell mass and morphology: a single maximum in all rheological variables resulted when cells were grown on xylose or on cellulose at impeller speeds of 400 or 550 rpm, and dual maxima were observed for cellulose-grown cells at 250 rpm.     

Physiological properties of Cellulomonas fermentans,a mesophilic cellulolytic bacterium

Summary The fermentation of cellobiose, glucose and cellulose MN 300 by Cellulomonas fermentans was studied. The molar growth yields (i.e. grams of cells per mole of hexose equivalent) were similar on cellobiose and cellulose at low sugar consumption levels (47.8 and 46.5 respectively), but was lower on glucose (38.0). The occurrence of cellobiose phosphorylase activity, detected in cellobiose- and cellulose-grown cells, might explain this result. The specific growth rates measured in cultures on cellobiose, glucose and cellulose were 0.055 h^-1, 0.040 h^-1 and 0.013 h^-1 respectively. Growth inhibition was observed, and a drop in Y_H occurred after relatively low but different quantities of hexose were consumed (2.2 mM, 5 mM and 8 mM hexose equivalent with cellulose, glucose and cellobiose respectively), which coincided with a change in the fermentative metabolism from a typical mixed acid metabolism (1 ethanol, 1 acetate and 2 formate synthesized by consumed hexose) to a more ethanolic fermentation. When growth ceased in cellulose cultures, consumption of cellulose continued, as did production of ethanol.Molar growth yields of C. fermentans were similar in anaerobic and aerobic cellobiose cultures (47.8 g/mol and 42.2 g/mol respectively). Specific growth rates were also quite similar under both culture conditions (0.055±0.013 h^-1 and 0.070±0.007 h^-1 respectively). Aerobic metabolism was studied using ¹⁴C glucose. During the exponential growth phase, acetate, succinate and nonidentified compound(s) accumulated in the supernatant, but no ¹⁴CO₂ was produced. During the stationary phase, acetate was oxidized and ¹⁴CO₂ produced, but without any further biomass synthesis. It seems that a blocking of metabolite oxidation may have occurred in C. fermentans except in the case of acetate, but acetate oxidation was apparently not coupled with production of energy utilizable in biosynthesis.     

DO-stat fed-batch production of 2-keto-d-gluconic acid from cassava using immobilized Pseudomonas aeruginosa

Bioconversion of cassava-derived glucose to 2-keto-d-gluconic acid (2-KDG) using resting cells of immobilized Pseudomonas aeruginosa IFO 3448 was investigated. The tuberous roots of cassava were selected as the feedstock as they are inexpensive and widely available, and possess high amounts of starch (approximately 70% (w/w) of dry mass). Immobilized bacteria was used in a fed-batch fermenter and recycled over a period of 2 weeks. Given that the formation of 2-KDG from glucose requires oxygen as a reagent, and that high glucose concentrations are detrimental to the production yield of 2-KDG by resting cells, a DO-stat control strategy was used, whereby the feed rate of cassava hydrolysate was regulated by coupling it with the control variable, dissolved oxygen. For 319 h of operation including three cycles of repeated fed batch, 72 g of 2-KDG was produced from hydrolysate derived from 110 g of dried cassava at a maximum production rate of 0.55 g/L/h and an average concentration of 35 g/L.