Influence of physiological conditions on EDTA degradation

Aerobic biodegradation of the chelating agent EDTA by a mixed bacterial culture was investigated. Bacterial growth and degradation of the substrate required the presence of sufficient metal ions in the culture fluid. Uncomplexed EDTA interacted negatively with the cell walls of the bacteria and completely inhibited bacterial growth, whereas Mg(II)/Ca(II)-EDTA was degraded up to an initial concentration of 4.7 g/l. Therefore, concentrations of metal ions must be stoichiometric to that of EDTA or higher. Specific degradation rates ranged between 120 mg EDTA g^–1 (cell dry weight) h^–1 and 285 mg EDTA g^–1 h^–1. In contrast, complexes with high thermodynamic stability constants such as Fe(III)-EDTA remained as inert compounds in the solution. Specific growth rates of the mixed culture were found to vary between 0.03 h^–1 and 0.07 h^–1, which could be explained by population dynamics within the synergistic mixed community. Growth was significantly accelerated by the addition of vitamins.     

Influence of temperature rise on kinetic parameters during batch propagation of Kluyveromyces fragilis in cheese whey under ambient conditions

Three 5 l working volume fermenters were used to investigate the growth of the yeast Kluyveromyces fragilis in acid cheese whey under ambient temperature in order to assess the specific growth rate and yield, the lactose and oxygen uptake rates during the various phases of batch culture, the effect of increasing temperature on the various kinetic parameters, and the need for a cooling unit for single cell production batch systems. The initial dissolved oxygen in the medium was 5.5 mg l^–1 and the pH was maintained at 4.5. The observed lag phase, specific growth rate and maximum cell number were 4 h, 0.2 h^–1 and 8.4 × 10⁸ cells ml^–1, respectively. About 99% of the lactose in cheese whey was utilized within 20 h, 85% during the exponential growth phase. The specific lactose utilization rates by K. fragilis were 0.20 × 10^–12, 1.457 × 10^–12, 0.286 × 10^–12 and 0.00 g lactose cell^–1 h^–1, for the lag, exponential, stationary and death phases, respectively. The dissolved oxygen concentration in the medium decreased as the cell number increased. The lowest oxygen concentration of 1.2 mg l^–1 was observed during the stationary phase. The volumetric oxygen transfer coefficient was 0.41 h^–1 and the specific oxygen uptake rates were 0.32 × 10^–12, 2.14 × 10^–12, 0.51 × 10^–12 and 0.003 × 10^–12 mg O₂ cell^–1 h^–1, for the lag, exponential, stationary and death phases, respectively. The maximum temperature recorded for the medium was 33 °C, indicating that a cooling unit for batch production of single cell protein at ambient temperature is not needed for this type of bioreactor. The increase in medium temperature affected the cell growth and the lactose and oxygen uptake rates.     

The endogenous metabolism ofFibrobacter succinogenes and its relationship to cellobiose transport,viability and cellulose digestion

Fibrobacter succinogenes S85 digested ballmilled cellulose at a rapid rate (0.10 h^–1), but there was a long lag time if the culture was not transferred daily. WhenF. succinogenes was starved for 100h, a large fraction of the cells (>30%) still bound to cellulose, but the lag time was 150h. The lag time was similar for either cellulose- or cellobiose-grown inocula, and lag times were highly correlated (r ² = 0.91) with a decrease in viable cell number. The number of viable cells declined from 10⁸ to 10⁶ in the first 30h of starvation, and by 72h the viable cell number was less than 10³/ml. Cells growing exponentially on cellobiose had a large pool of polysaccharide, and continuous culture experiments indicated that polysaccharide accumulation was not significantly influenced by the growth rate of the culture (approximately 0.7 mg polysaccharide mg^–1 protein). When the cellobiose was depleted, cellular polysaccharide decreased at first order rate of 0.09 h^–1. The rate of endogenous metabolism was initially 0.08mg polysaccharide mg^–1 protein h^–1, and there was little decline in viability until the rate of endogenous metabolism was less than 0.01 mg polysaccharide mg^–1 protein h^–1. When the rate was less than 0.01 mg polysaccharide mg^–1 protein h^–1, the cells could not maintain a sodium gradient, transport cellobiose or grow. The endogenous metabolic rate needed for cell survival was 20 fold less than the maintenance energy of cells growing in continuous culture (0.01 versus 0.232mg carbohydrate mg^–1 protein h^–1).     

Microbial production of propionic acid and vitamin B12 using molasses or sugar

With a cell concentration of 125 g dry biomass 1^–1 and a dilution rate of 0.1 h^–1,Propionibacterium acidipropionici produces 30 g propionic acid 1^–1 from sugar with a productivity of 3 g 1^–1 h^–1. The yield of propionic acid is approx. 0.36–0.45 g propionic acid g^–1 sucrose and is independent of the dilution rate and cell concentration. Acetic acid is an unwanted by-product in the production of propionic acid. The concentration of acetic acid only increases slightly when the cell concentration is increased. A two-stage fermentation process was developed for the conversion of sugar or molasses of various types to propionic acid and vitamin B₁₂. By fermentation of blackstrap molasses (from sugar beet and sugar cane) in the first fermentation stage 17.7 g propionic acid 1^–1 with a yield of 0.5 g propionic acid g^–1 carbohydrate was produced with a dilution rate of 0.25 h^–1. In the second stage 49 mg vitamin B₁₂ 1^–1 was produced at a dilution rate of 0.03 h^–1.     

Adsorption of Ni2+ on the surface of molecularly imprinted adsorbent from Penicillium chysogenum mycelium

The adsorption capacity for Ni²⁺ on to the surface molecular imprinting adsorbent on Penicillium chysogenum mycelium (the surface-imprinted adsorbent) was 40–45 mg g^–1 (using 200 mg Ni²⁺ l^–1), two times of the mycelium adsorbent. The surface-imprinted adsorbent had good stability at pH 28. The optimal concentration of EDTA for desorption was 0.1 to 0.5 g l^–1. The surface imprinted adsorbent could be reused 15 times without losing its uptake.     

Extraction of extracellular polymeric substances from the photosynthetic bacterium Rhodopseudomonas acidophila

Among the four methods for extracting extracellular polymeric substances (EPS) from Rhodopseudomonas acidophila (EDTA, NaOH, H₂SO₄, heating/centrifugation), EDTA extraction was found to be the most effective. The contents of the major components of EPS from R. acidophila, i.e., carbohydrate, protein and nucleic acid, were 6.5, 58.4 and 5.4 mg g^–1 dry cells, respectively. The optimum extraction time was 1–3 h and the EDTA dosage was approximately 2.8 g g^–1 dry cells. Under these conditions, no cell lysis was observed. The EPS content and the percentage of the three main components were greatly dependent on the extraction method. The intensity of absorption peaks for photosynthetic pigments in the UV–visible spectrum of bacteria remained unchanged prior to and after EDTA extraction; and no pigment peaks appeared in the EPS spectrum. This suggests that few cells were destroyed and lysis did not occur. UV–visible spectrum analysis, an easy and rapid technique, could be used to monitor cell lysis during EPS extraction from R. acidophila.     

Batch xylitol production from wheat straw hemicellulosic hydrolysate using Candida guilliermondii in a stirred tank reactor

Batch production of xylitol from the hydrolysate of wheat straw hemicellulose using Candida guilliermondii was carried out in a stirred tank reactor (agitation speed of 300 rpm, aeration rate of 0.6 vvm and initial cell concentration of 0.5 g l^–1). After 54 h, xylitol production from 30.5 g xylose l^–1 reached 27.5 g l^–1, resulting in a xylose-to-xylitol bioconversion yield of 0.9 g g^–1 and a productivity of 0.5 g l^–1 h^–1.     

Effects of stream order and season on mineralization of [14C]-phenol in streams

Mineralization of trace levels of [¹⁴C]-phenol by heterotrophic microorganisms was quantified at 4 sites along a river continuum in southwestern Virginia. Significant phenol mineralization rates were detected in surface sediment and seston samples at all sites from August 1985 through May 1986. Phenol degradation was strongly affected by season (ANOVA; P < 0.0001). From a baseline rate in August (range: 1.19 × 10^-5 to 897 × 10^-4 mg phenol mineralized mg AFDW^-1 h^-1) phenol mineralization rose to a yearly maximum in October (range: 1.21 × 10^-4 to 1.16 × 10^-3 mg phenol mineralized mg AFDW^-1 h^-1) despite decreasing stream temperatures. This autumnal peak in phenol degradation was attributed to the pulsed input of allochthonous detritus, especially leaf litter, which contains substantial quantities of phenols and related compounds. Although phenol mineralization was significant in these streams, phenols were metabolized at much slower rates than more labile compounds present in the dissolved organic matter (DOM) pool. Estimates of turnover rates for three major components of DOM revealed that glucose and glutamate turnover rates (0.064–0.140 h^-1 mg sediment AFDW^-1 and 0.140–0.610 h^-1 mg sediment AFDW^-1, respectively) were, respectively, 2.2–4.7 × and 9.6–16.9 × greater than phenol turnover rates (0.015–0.064 h^-1 mg sediment AFDW^-1). Although the relatively low rates of utilization of refractory phenolic materials suggest that these compounds may accumulate and become more prevalent components of the DOM pool, phenol concentrations at the 4 study sites remained below detectable levels (i.e., < 1 g 1^-1) throughout the study. Consequently, it seems that although phenolic materials are metabolized more slowly than labile DOM, phenols are degraded at rates which preclude accumulation in the water column.     

Factors affecting the growth form of Aspergillus terreus NRRL 1960 in relation to itaconic acid fermentation

The effect of medium composition on the growth form of Aspergillus terreus NRRL 1960 in relation to itaconic acid fermentation has been studied. Four types of mycelial pellets were obtained under the conditions used and may be classified as (a) frayed and loose with 0.1–0.5 mm diameter (b) compact with 0.1–0.5 mm diameter (c) loose with 0.5–2.0 mm diameter and (d) compact with 0.5–2.0 mm diameter. Their respective maximum specific rates of formation and yields of itaconic acid, based on 100 g sucrose supplied, were (a) 1.25 mol mg^–1h^–1 and 55–59 g, (b) 0.27–0.43 mol mg^–1 h^–1 and 26–38 g, (c) 0.75–0.90 mol mg^–1 h^–1 and 45–51 g and (d) 0.12 mol mg^–1 h^–1 and 10 g. The presence of Ca²⁺, Zn²⁺ and Fe²⁺ in the basal medium at concentrations of 23.3 mg/100 ml, 0.01 mg/100 ml and 0.006 mg/100 ml respectively were found to be adequate and crucial in obtaining the desired outgrowth for both high production rates and consistent yields of itaconic acid. The further addition of either commercial plaster of Paris or analytical-reagent-grade CaSO₄, especially when activated by heating to 530°C and present in excess of solubility, results in small and frayed pellets, which lead to itaconic acid yields of 55–59 g acid/100 g sugar supplied.     

Biodegradation and detoxification of nicotine in tobacco solid waste by a Pseudomonas sp.

A Pseudomonas sp. grew with nicotine optimally 3 g l^–1 and at 30 °C and pH 7. Nicotine was fully degraded within 10 h. The resting cells degraded nicotine in tobacco solid waste completely within 6 h in 0.02 m sodium phosphate buffer (pH 7) at maximally 56 mg nicotine h^–1 g dry cell^–1.     

Pentachlorophenol removal from slightly acidic mineral salts,commercial sand,and clay soil by recovered Arthrobacter strain ATCC 33790

Arthrobacter strain ATCC 33790, a pentachlorophenol (PCP)-metabolizer isolated by the author, has been recovered after 10 years of storage. The freeze-dried preparation grown on half-strength Trypticase Soy Broth adapted to utilize PCP within 1 week. Cultures grown on PCP-nutrient agar were found to utilize PCP in mineral salts medium within 2–3 days. The culture was prepared for continuous growth at pH 6.5 by successive feeding of 100–110 mg solid aliquots of PCP to a 1-l culture initially grown at pH 7.4. Continuous culture growth at pH 6.5 was possible on a mineral salts feed containing 1800 ppm PCP. Continuous cultures grown at pH 6.7 on mineral salts feeds containing 500 and 340 mg PCP/l were especially efficient in removing PCP. Less than 4 mg PCP/l were detected in the effluent at dilution rates near washout. In batch culture studies at pH 6.5 the PCP utilization kinetics were found to be similar at low PCP concentration to those at pH 7.4 for the approximately same inoculum size. Utilization of 35 mg PCP/l was very slow at pH 6.0. Growth rates at pH 6.5 at controlled PCP concentration ranges of 5–35 and 75–115 mg/l were 0.09 h^–1 and 0.05 h^–1, respectively. The ability of strain ATCC 33790 to utilize PCP in mineral salts media containing naphthalene, methylnaphthalenes, and cresols was examined. Naphthalene, 1-, and 2-methylnaphthalenes at their solubility limit, and o- and m-cresols at 900–1000 mg/l prevented utilization of 80–90 mg PCP/l. PCP was rapidly removed from both commercial sand at 30°C and from clay soil at room temperature. Estimated inoculum sizes of 6.6 × 10⁶, 6.6 × 10⁴, and 656 cells/g were found to be effective in removing approximately half the starting amount of PCP from sand in 3, 19, and 42 h, respectively. Nearly complete disappearance of extractable PCP was observed after 1 day in clay soil inoculated with 6 × 10⁶ cells/g.     

Production of RNA by a marine photosynthetic bacterium, Rhodovulum sp.

The marine photosynthetic bacterium, Rhodovulum sp. PS88, produces RNA not only in cells but also as an extracellular polymeric substance during aerobic continuous cultivation in the dark. At a dilution rate of 0.32–0.5 h^–1, the maximum RNA production was 460 mg RNA l^–1 broth (200 mg RNA g^–1 suspended solids) which is a value about 2–3 times more than that of yeast cells.     

Continuous solvent production by Clostridium beijerinckii BA101 immobilized by adsorption onto brick

The performance of a continuous bioreactor containing Clostridium beijerinckii BA101 adsorbed onto clay brick was examined for the fermentation of acetone, butanol, and ethanol (ABE). Dilution rates from 0.3 to 2.5 h^–1 were investigated with the highest solvent productivity of 15.8 g l^–1 h^–1 being obtained at 2.0 h^–1. The solvent yield at this dilution rate was found to be 0.38 g g^–1 and total solvent concentration was 7.9 g l^–1. The solvent yield was maximum at 0.45 at a dilution rate of 0.3 h^–1. The maximum solvent productivity obtained was found to be 2.5 times greater than most other immobilized continuous and cell recycle systems previously reported for ABE fermentation. A higher dilution rate (above 2.0 h^–1) resulted in acid production rather than solvent production. This reactor was found to be stable for over 550 h. Scanning electron micrographs (SEM) demonstrated that a large amount of C. beijerinckii cells were adsorbed onto the brick support.     

Potential of Thiobacillus ferrooxidans for waste gas purification. Part 1. Kinetics of continuous ferrous iron oxidation

Kinetic data of ferrous iron oxidation by Thionacillus ferrooxidans were determined. The aim was to remove H₂S (<0.5 ppm) from waste gas by a process proposed earlier. Kinetic data necessary for industrial scale-up were investigated in a chemostat airlift reactor (dilution rate 0.02–0.12 h^–1; pH 1.3). Due to the low pH, ferric iron precipitation and wall growth could be avoided. The maximum ferrous iron oxidation rate of submersed bacteria was 0.77 g 1^–1 h^–1, the maximum specific growth rate about 0.12 h^–1 and the yield coefficient was found to be 0.007 g g^–1 Fe²⁺. The specific O₂ demand of an exponentially growing, ironoxidizing batch culture was 1.33 mg O₂ mg^–1 biomass h^–1. The results indicate that a pH of 1.3 has no negative influence on the kinetics of iron oxidation and growth. Correspondence to: W. Schäfer-Treffenfeldt     

Food consumption of Sarotherodon mossambicus (Trewaves) exposed to sublethal concentration of diammonium phosphate

S. mossambicus was exposed to toxic and sublethal concentrations of the fertilizer diammonium phosphate (0.2 to 1.0 g l^–1). Mortality, food utilization and growth were studied. At a concentration of 0.6 g l^–1 DAP, 100% mortality was observed within 96 h; no mortality occurred at 0.5 g l^–1; LC50 was 0.55 g l^–1. Rearing the fish in increasing sublethal concentrations of DAP, it was found that the feeding rate decreased from 25.4 mg g^–1 fish^–1 d^–1 (fish reared in DAP-free water) to 10.1 mg g^–1 d^–1 at the highest sublethal concentration (0.5 g l^–1). Growth rate was drastically reduced. At high sublethal concentrations of DAP, the fish lost reserve energy, in addition to the energy obtained from food intake for survival, as a result of increased swimming activity and opercular beats.     

Effect of the dilution rate on the biomass yield ofBacillus thuringiensis and determination of its rate coefficients under steady-state conditions

Depending on the biomass yield on glucose and the cell morphology ofBacillus thuringiensis, three different metabolic states were observed in continuous culture. At dilution rates between 0.18 h^–1 and 0.31 h^–1 vegetative cells, sporulating bacteria and spores coexisted, while glucose and amino acids were consumed. Only vegetative cells were observed at dilution rates between 0.42 h^–1 and 0.47 h^–1 and glucose was used as the main carbon and energy source. AtD = 0.50 h^–1 the biomass yield on glucose decreases sharply. To define better the specific growth rate range in which the microorganism uses mainly glucose, a dilution rate of 0.25–0.45 h^–1 was studied. The experimental data could be adjusted to a Monod model and the following rate coefficients and growth yields were determined: maximum specific growth rate 0.54 h^–1, saturation constant 0.56 mg glucose ml^–1, biomass growth yields 0.43 g cells (g glucose)^–1, and 0.76 g cells (g oxygen)^–1, and maintenance coefficients 0.065 g glucose (g cells)^–1 h^–1 and 0.039 g oxygen (g cells)^–1 h^–1.     

Anaerobic degradation of phenol in batch and continuous cultures by a denitrifying bacterial consortium

Summary The anaerobic degradation of phenol under denitrifying conditions by a bacterial consortium was studied both in batch and continuous cultures. Anaerobic degradation was dependent on NO_f3 ^p– and concentrations up to 4 mm phenol were degraded within 2–5 days. During continuous growth in a fermenter, steady states could be maintained at eight dilution rates (D) corresponding to residence times between 12.5 and 50 h. Culture wash-out occurred at D=0.084 h^–1. The kinetic parameters obtained for anaerobic degradation of phenol under denitrifying conditions by the consortium were: maximam specific growth rate = 0.091 h^–1; saturation constant = 4.91 mg phenol/l; true growth yield = 0.57 mg dry wt/mg phenol; maintenance coefficient = 0.013 mg phenol/mg dry wt per hour. The Haldane model inhibition constant was estimated from batch culture data giving a value of 101 mg/l. The requirement of CO₂ for the anaerobic degradation of phenol with NO_f3 ^p– indicates that phenol carboxylation to 4-hydroxybenzoate was the first step of phenol degradation by this culture. 4-Hydroxybenzoate, proposed as an intermediate of phenol carboxylation under these conditions, was detected only in continuous cultures at very low growth rates (D=0.02 h^–1), but was never detected as a free intermediary metabolite either in batch or in continuous cultures. Correspondence to: N. Khoury     

Propionic acid production by immobilized cells of a propionate-tolerant strain of Propionibacterium acidipropionici

Cells of the propionate-tolerant strain Propionibacterium acidipropionici P200910, immobilized in calcium alginate beads, were tested for propionic and acetic acid production both in a semidefined laboratory medium and in corn steep liquor in batch, fed-batch, and continuous fermentation. Cell density was about 9.8 × 10⁹ cells/g (wet weight) of beads, and beads were added to the medium at 0.1 g (wet weight) beads/ml. Beads could be reused for several consecutive batch fermentations; propionic acid production in the tenth cycle was about 50%–70% of that in the first cycle. In batch culture complete substrate consumption (glucose in semidefined medium, lactate in corn steep liquor) and maximum acid production were seen within 36 h, and acid yields from the substrate were higher than in free-cell fermentations. Fed-batch fermentations were incubated up to 250 h. Maximum propionic acid concentrations obtained were 45.6 g/l in corn steep liquor and 57 g/l in semidefined medium; this is the highest concentration achieved to date in our laboratory. Maximum acetic acid concentrations were 17 g/l and 12 g/l, respectively. In continuous fermentation of semidefined medium, dilution rates up to 0.31 h^–1 could be used, which gave higher volumetric productivities (0.96 g l^–1 h^–1 for propionic acid and 0.26 g l^–1 h^–1 for acetic acid) than we have obtained with free cells. Corn steep liquor shows promise as an inexpensive medium for production of both acids by immobilized cells of propionibacteria.Journal paper no. J- 15614 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project no. 3122     

Enhancement of single cell oil production by Yarrowia lipolytica growing in the presence of Teucrium polium L. aqueous extract

Yarrowia lipolytica, grown in a nitrogen-limited continuous culture (D = 0.032 h^–1), produced 9.3 g dry biomass l^–1, which contained 0.25 g oil g^–1. When an aqueous extract from Teucrium polium L. was added, the biomass concentration remained constant while the oil content increased to 0.33 g oil g^–1 dry weight. The specific rate of oil formation increased from 7.9 to 10.6 mg oil g^–1 biomass h.     

Propionic acid and biomass production using continuous ultrafiltration fermentation of whey

Summary This paper presents a study of propionic acid and propionibacteria production from whey by usingPropionibacterium acidi-propionici in continuous fermentation with cell recycle. The highest propionic acid volumetric productivity achieved was 5 g.l^–1.h^–1 with no biomass bleeding. A maximal biomass concentration of 130 g.l^–1 was achieved before initiating biomass bleeding to give a biomass volumetric productivity of 3.2 g.l^–1.h^–1 with a biomass of 75 g.l^–1 and a propionic acid productivity of 3.6 g.l^–1.h^–1 (for about 100 hours i.e. more than 50 residence times).