Response surface methodological approach for Rhizomucor miehei lipase-mediated esterification of α-terpineol with propionic acid and acetic anhydride

Esterification of α-terpineol with acetic anhydride or propionic acid mediated by Rhizomucor miehei lipase was subjected to a response surface study in order to optimize conditions for maximum esterification. The variables were enzyme/substrate (acid) ratio, α-terpineol concentration and incubation period using lipase from R. miehei. Between acetic anhydride and propionic acid, the former showed better yields at lower enzyme/substrate ratios than the latter. Yields predicted by the models for α-terpinyl propionate and α-terpinyl acetate formation were found to be in agreement with the experimentally determined ones. This revised version was published online in July 2006 with corrections to the Cover Date.     

Steady-state and transient behavior in microbial methanification: I. Experimental results

The process of methanification of volatile fatty acids (VFA) was studied to elucidate its kinetics. An upflow anaerobic sludge bed (UASB) system was used to perform the experiments. At residence times of less than 2.5 h the UASB system was found to exhibit hysteresis with respect to acetic and propionic acid consumption but not with respect to butyric acid consumption. These hysteretic effects could be attributed to the manner in which the various VFA-consuming cultures were structured inside the flocculated biomass in light of the cross-inhibitory effects of the acetic- and propionic-acid-consuming fractions of the total culture. (Butyric acid proved to be non-interactive.) Production of methane was found to respond almost instantaneously to changes in the inlet conditions of the UASB system. This indicated that methane is not primarily growth associated, as has often been assumed, but is related to changes in the culture's maintenance energy requirements. Reactor operation was found to be stable even when the concentration of each VFA in the feed was simultaneously changed by 50%. Even at very high organic throughput rates (35 kg COD/day m(3)-reactor) conversions of 82% were observed.     

Controlled shear affinity filtration (CSAF): a new technology for integration of cell separation and protein isolation from mammalian cell cultures

Up-flow anaerobic sludge blanket (UASB) reactors are being used with increasing regularity all over the world, especially in India, for a variety of wastewater treatment operations. Consequently, there is a need to develop methodologies enabling one to determine UASB reactor performance, not only for designing more efficient UASB reactors but also for predicting the performance of existing reactors under various conditions of influent wastewater flows and characteristics. This work explores the feasibility of application of an artificial neural network-based model for simulating the performance of an existing UASB reactor. Accordingly, a neural network model was designed and trained to predict the steady-state performance of a UASB reactor treating high-strength (unrefined sugar based) wastewater. The model inputs were organic loading rate, hydraulic retention time, and influent bicarbonate alkalinity. The output variables were one or more of the following, effluent substrate concentration (Se), reactor bicarbonate alkalinity, reactor pH, reactor volatile fatty acid concentration, average gas production rate, and percent methane content of the gas. Training of the neural network model was achieved using a large amount of experimentally obtained reactor performance data from the reactor mentioned above as the training set. Training was followed by validation using independent sets of performance data obtained from the same UASB reactor. Subsequently, simulations were performed using the validated neural network model to determine the impact of changes in parameters like influent chemical oxygen demand (COD) concentration and hydraulic retention time on the reactor performance. Simulation results thus obtained were carefully analyzed based on qualitative understanding of UASB process and were found to provide important insights into key variables that were responsible for influencing the working of the UASB reactor under varying input conditions.     

Modeling of two-phase anaerobic process treating traditional Chinese medicine wastewater with the IWA Anaerobic Digestion Model No. 1

The aim of the study was to implement a mathematical model to simulate two-phase anaerobic digestion (TPAD) process which consisted of an anaerobic continuous stirred tank reactor (CSTR) and an upflow anaerobic sludge blanket (UASB) reactor in series treating traditional Chinese medicine (TCM) wastewater. A model was built on the basis of Anaerobic Digestion Model No. 1 (ADM1) while considering complete mixing model for the CSTR, and axial direction discrete model and mixed series connection model for the UASB. The mathematical model was implemented with the simulation software package MATLABTM/Simulinks. System performance, in terms of COD removal, volatile fatty acids (VFA) accumulation and pH fluctuation, was simulated and compared with the measured values. The simulation results indicated that the model built was able to well predict the COD removal rate (−4.8–5.0%) and pH variation (−2.9–1.4%) of the UASB reactor, while failed to simulate the CSTR performance. Comparing to the measured results, the simulated acetic acid concentration of the CSTR effluent was underpredicted with a deviation ratios of 13.8–23.2%, resulting in an underprediction of total VFA and COD concentrations despite good estimation of propionic acid, butyric acid and valeric acid. It is presumed that ethanol present in the raw wastewater was converted into acetic acid during the acidification process, which was not considered by the model. Additionally, due to the underprediction of acetic acid the pH of CSTR effluent was overestimated.     

Growth characteristics of Geotrichum ingens on propionic acid and acetic acid in batch and continuous culture

Growth of Geotrichum ingens in batch cultures was completely inhibited by 47 g acetic acid/l or 33 g propionic acid/I. With mixtures of acetic and propionic acids, however, growth only ceased at 55 g/l. Acetic acid inhibited growth linearly, whereas propionic acid inhibited growth non-linearly. In continuous culture, two steady states at each dilution rate were observed at high dilution rates for acetic acid and propionic acid. The highest yield coefficient (0.69 g cells/g substrate) was achieved with propionic acid as substrate. On both substrates and their mixtures, the protein content of the biomass increased when the dilution rate was increased.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa     

A model to describe the performance of the UASB reactor

A dynamic model to describe the performance of the Upflow Anaerobic Sludge Blanket (UASB) reactor was developed. It includes dispersion, advection, and reaction terms, as well as the resistances through which the substrate passes before its biotransformation. The UASB reactor is viewed as several continuous stirred tank reactors connected in series. The good agreement between experimental and simulated results shows that the model is able to predict the performance of the UASB reactor (i.e. substrate concentration, biomass concentration, granule size, and height of the sludge bed).     

Anaerobic acidogenesis of a complex wastewater: I. The influence of operational parameters on reactor performance

The influence of operational, parameters, such as hydraulic retention time, organic loading rate, influent substrate concentration, pH, and temperature, on the performance of the first phase of anaerobic digestion has been investigated. A complex substrate based on beef extract was used, and six series of experimental runs were conducted, each one showing the effect of one operational variable. The predominant fermentation products were always acetic and propionic acid, independent of the values of the operational parameters. For initial COD concentrations and hydraulic retention times above the critical values identified as 3 g/L and 6 h, respectively, the degree of acidification achieved was between 30 and 60%. The degree of acidification was found to increase with the hydraulic retention time and decrease with the influent substrate concentration and organic loading rate, while the opposite held true for the rate of product formation. Furthermore, it has been demonstrated that acidification is primarily determined by the hydraulic retention time and the rate of product formation by the influent substrate concentration. The concentration of the acetic acid produced was found to depend on the operational parameters. However, the concentration of propionic acid produced depended only on the substrate availability with a consistent proportion of 8% initial COD converted to it. The optimum pH and temperature were 7 and 40 degrees C, respectively. The percentage of acetic acid as a proportion of the total volatile fatty acids produced was found to increase with increasing pH and temperature, while the percentage of propionic acid seemed to decrease accordingly. Finally the effect of the temperature on the rate of acidification followed an Arrhenius type equation with an activation energy equal to 4739 cal/mol.     

Inhibitory effect of medium-chain-length fatty acids on synthesis of polyhydroxyalkanoates from volatile fatty acids by Ralstonia eutropha

Medium-chain-length fatty acids, such as nonanoic (9:0) and octanoic (8:0) acids, are more toxic to Ralstonia eutropha than volatile fatty acids such as acetic, propionic and butyric acids. Nonanoic acid was degraded to acetic and propionic acids via -oxidation by Ralstonia eutropha for cell growth and synthesis of polyhydroxyalkanoates (PHAs). In a mixture of the fatty acids, utilization of nonanoic acid was depressed by acetic and propionic acids, and vice versa. The PHA accumulation from the volatile fatty acids was decreased from 53% (w/w) of dry cell mass to 23% due to the nonanoic acid. Similar phenomena were also observed with octanoic acid and its metabolic intermediates, acetic and butyric acids.     

Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate

In this study, local sewage sludge was acclimated to establish H2-producing enrichment cultures, which were used to convert sucrose to H2 with continuously stirred anaerobic bioreactors. The steady-state behaviors of cell growth, substrate utilization, and product formation were closely monitored. Kinetic models were developed to describe and predict the experimental results from the H2-producing cultures. Operation at dilution rates (D) of 0.075-0.167 h(-1) was preferable for H2 production, resulting in a H2 concentration of nearly 0.02 mol/l. The optimal hydrogen production rate was 0.105 mol/h occurring at D=0.125 h(-1). The major volatile fatty acid produced was butyric acid (HBu), while acetic acid and propionic acid were also produced in lesser quantities. The major solvent product was ethanol, whose concentration was only 15% of that of HBu, indicating that the metabolic flow favors H2 production. The proposed model was able to interpret the trends of the experimental data. The maximum specific growth rate (mu(max)), Monod constant (Ks), and yield coefficient for cell growth (Y(x/s)) were estimated as 0.172 h(-1), 68 mg COD/l, and 0.1 g/g, respectively. The model study also suggests that product formation in the continuous hydrogen-producing cultures was essentially a linear function of biomass concentration.     

Influence of chain ordering on the selectivity of dipalmitoylphosphatidylcholine bilayer membranes for permeant size and shape.

The effects of lipid chain packing and permeant size and shape on permeability across lipid bilayers have been investigated in gel and liquid crystalline dipalmitoylphosphatidylcholine (DPPC) bilayers by a combined NMR line-broadening/dynamic light scattering method using seven short-chain monocarboxylic acids (formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and trimethylacetic acid) as permeants. The experimental permeability coefficients are compared with the predictions of a bulk solubility diffusion model in which the bilayer membrane is represented as a slab of bulk hexadecane. Deviations of the observed permeability coefficients (Pm) from the values predicted from solubility diffusion theory (Po) lead to the determination of a correction factor, the permeability decrement f (= Pm/Po), to account for the effects of chain ordering. The natural logarithm of f has been found to correlate linearly with the inverse of the bilayer free surface area with slopes of 25 +/- 2, 36 +/- 3, 45 +/- 8, 32 +/- 12, 33 +/- 4, 49 +/- 12, and 75 +/- 6 A2 for formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and trimethylacetic acid, respectively. The slope, which measures the sensitivity of the permeability coefficient of a given permeant to bilayer chain packing, exhibits an excellent linear correlation (r = 0.94) with the minimum cross-sectional area of the permeant and a poor correlation (r = 0.59) with molecular volume, suggesting that in the bilayer interior the permeants prefer to move with their long principal axis along the bilayer normal. Based on these studies, a permeability model combining the effects of bilayer chain packing and permeant size and shape on permeability across lipid membranes is developed.     

Fed-batch fermentation with and without on-line extraction for propionic and acetic acid production byPropionibacterium acidipropionici

Fed-batch propionic and acetic acid fermentations were performed in semi-defined laboratory medium and in corn steep liquor withPropionibacterium acidipropionici strain P9. On average, over four experiments, 34.5 g/l propionic acid and 12.8 g/l acetic acid were obtained in about 146 h in laboratory medium with 79 g/l glucose added over five feeding periods. The highest concentration of propionic acid, 45 g/l, was obtained when the glucose concentration was not allowed to drop to zero. In corn steep liquor 35 g/l propionic acid and 11 g/l acetic acid were produced in 108 h from 59.4 g/l total lactic acid provided as seven feedings of corn steep liquor. Extractive fed-batch fermentations were conducted in semi-defined medium using either flat-sheet-supported liquid membranes or hollow-fiber membrane extraction to remove organic acids from the culture medium. As operated during the course of the fermentation, these systems extracted 25% and 22% of the acetic acid and 36.5% and 44.5% of the propionic acid, respectively, produced in the fermentation. Total amounts of acids produced were about the same as in comparable nonextractive fermentations: 30–37 g/l propionic acid and 13 g/l acetic acid were produced in 150 h. Limitations on acid production can be attributed to limited substrate feed, not to failure of the extraction system.Journal paper J-16303 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 3122.     

The influence of LBG addition to carrageenan on the mechanical stability of the gel and the fermentative activity of immobilized propionic acid bacteria

In the first part of the experiments, the mechanical properties of 1%, 2% and 3% carrageenan and 1%, 2% and 3% carrageenan/locust bean gum (LBG) gels stored in various concentrations of propionic and acetic acids and their mixtures were examined. The stability of these materials was measured by uniaxial compression between two parallel plates using the Instron Universal Testing Machine. A mathematical model explaining the dependence of the destroying force on the storage time was chosen for data analysis. Using this model, the average rate of gel deterioration was calculated. The structural properties of the examined gels were most influenced by the highest concentration of propionic and acetic acids and their mixtures (1% acetic acid and 2% propionic acid). The addition of LBG to carrageenan decreased the gel destroying force and increased its resistance to acids. In the second part of the experiments, the Propionibacterium freudenreichii subsp. shermanii NCFB 1081 and NCFB 566 were immobilized in a living state in 1%, 2% and 3% carrageenan and 1%, 2% and 3% carrageenan/LBG gels. The ammonia consumption, glucose utilization, production of propionic and acetic acids and the biosynthesis of vitamin B₁₂ were examined. An increase in the productivity of propionic acid and a significant decrease in the vitamin B₁₂ produced in the biosynthesis were observed when immobilized cells were used. The immobilization of cells enhanced the productivity of propionic acid by up to 40% compared to free cells. The best results were obtained for the second and third applications of immobilized cells in all concentrations of carrageenan gels and 2% and 3% carrageenan/LBG gels The results showed that carrageenan/LBG is a better support material for the immobilization of propionic acid bacteria than the pure carrageenan.     

The effect of propionic to acetic acid ratio on anaerobic-aerobic (low dissolved oxygen) biological phosphorus and nitrogen removal

In this paper, three lab-scale sequencing batch reactors (SBR-A, B, and C) operated with anaerobic/aerobic (low dissolved oxygen, 0.15-0.45 mg L(-1)) configuration were long-term cultured, respectively with single acetic acid and propionic/acetic acid of 1/1 and 2/1 (carbon molar ratio), and the comparisons of anaerobic and aerobic transformations of phosphorus and nitrogen among them were made. With the increase of propionic/acetic acid, lower anaerobic phosphorus release and higher phosphorus release to short-chain fatty acids uptake ratio were observed, and less anaerobic and aerobic transformations of glycogen and poly-3-hydroxybutyrate as well as total polyhydroxyalkanoates occurred, but the transformations of poly-3-hydroxyvalerate and poly-3-hydroxy-2-methyvalerate increased. The phosphorus removal efficiency was respectively 81, 94 and 97% in SBR-A, B and C. Almost all ammonium was removed and no significant nitrite was accumulated at different propionic/acetic acid ratios. However, the nitrate accumulation and total nitrogen removal were observed to be affected by propionic/acetic acid ratio. The total nitrogen removal efficiency was 61, 68 and 82%, and the aerobic end nitrate concentration was 8.05, 6.40 and 3.54 mg L(-1) in three SBRs, respectively. All the above studies indicated that the sole acetic acid caused more nitrate accumulation than propionic and acetic acids mixture, and a pertinent increase of wastewater propionic/acetic acid ratio was of benefit to both nitrogen and phosphorus removal in an anaerobic/aerobic (low dissolved oxygen) biological wastewater treatment process.     

A study of acetic acid production by immobilized Acetobacter cells: Product inhibition effects

Acetobacter living cells were immobilized by adsorption onto a ceramic support. The effects of acetic acid inhibition have been studied in a fixed-bed pulsed-flow reactor. Provided that sufficient oxygen was available, the concentration of acetic acid was found to be independent of the flow rate. Damped oscillations of acetic acid concentration were observed after step changes in dilution rate. The theoretical model developed from steady-state data was successful in predicting the experimental dynamic behavior of the reactor.     

In vitro gas production kinetics and short-chain fatty acid production from rumen incubation of diets supplemented with hop cones (Humulus lupulus L.)

The aim of this study was to assess the effects of hop cones (Humulus lupulus L.) from two varieties Aurora and Dana, differing in their α- and β-acid contents, on rumen microbial activity measured with in vitro gas production kinetics and short-chain fatty acids (SCFA) production. Hop cones were added to the total mixed dairy cow ration (CONT) in concentrations simulating a cow’s daily intake of 50, 100 and 200 g of hop cones – the concentrations of hop cones expressed on a substrate basis were 43, 82 and 153 mg/g of substrate. Substrates were anaerobically incubated in glass syringes, and gas production kinetic parameters were determined by fitting data with the Gompertz model. Gas produced after 24 h (Gas24), maximum fermentation rate (MFR) and time of maximum fermentation rate (TMFR) were calculated from the estimated gas production kinetic parameters. After 24 h of incubation, the fermentation liquids of each substrate were taken for the determination of SCFA. Increasing the hop cone concentration decreased the total potential gas production, Gas24, MFR and shortened TMFR. The highest hop cone concentration significantly decreased acetic and butyric acid productions and total SCFA production after 24 h of incubation, but not propionic acid production, resulting in a decreased ratio between acetic acid and propionic acid.     

Assessment of toxicity of volatile fatty acids to Photobacterium phosphoreum

The toxicity of four volatile fatty acids (VFAs) as anaerobic digestion (AD) intermediates was investigated at pH 7. Photobacterium phosphoreum T3 was used as an indicator organism. Binary, ternary and mixtures of AD intermediates were designated by letters A (acetic acid + propionic acid), B (acetic acid + butyric acid), C (acetic acid + ethanol), D (propionic acid + butyric acid), E (propionic acid + ethanol), F (butyric acid + ethanol), G (acetic acid + propionic acid + butyric acid), H (acetic acid + propionic acid + ethanol), I (acetic acid + butyric acid+ ethanol), J (propionic acid + butyric acid + ethanol) and K (acetic acid + propionic acid + butyric acid + ethanol) to assess the toxicity through equitoxic mixing ratio method. The IC₅₀ values of acetic acid, propionic acid, butyric acid and ethanol were 9.812, 7.76, 6.717 and 17.33 g/L respectively, displaying toxicity order of: butyric acid > propionic acid > acetic acid > ethanol being additive in nature. The toxic effects of four VFAs could be designated as synergistic and one additive in nature.     

Whey fermentation by immobilized cells of Propionibacterium shermanii

Growth of Propionibacterium shermanii B-123 was faster on media containing lactate than on that containing lactose. Cheese whey was therefore fermented with Lactobacillus helveticus and neutralized with NaOH or Ca(OH)₂, before inoculation with B-123. Fermentation rate by immobilized propionibacteria was best in Ca(OH)₂-neutralized whey, and at lactate concentrations between 1 and 2%. Calcium propionate concentrations of 1 and 3% reduced fermentation rates by 40% and 55% respectively. Optimum temperature for propionate fermentation by immobilized cells was 37°C. Ratios of propionic acid to acetic acid increased as incubation temperature was increased. Agitation increased propionic acid fermentation rates but lowered the ratio of propionic acid to acetic acid. Beads containing immobilized propionibacteria were re-utilized for ten consecutive fermentations. Fermentation rates increased upon re-utilization. Escherichia coli and Staphylococcus aureus were inhibited by the propionic fermentation but did not die; they kept growing at a reduced rate.     

Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate

Summary Batch propionic acid fermentations by Propionibacterium acidipropionici with lactose, glucose, and lactate as the carbon source were studied. In addition to propionic acid, acetic acid, succinic acid and CO₂ were also formed from lactose or glucose. However, succinic acid was not produced in a significant amount when lactate was the growth substrate. Compared to fermentations with lactose or glucose at the same pH, lactate gave a higher propionic acid yield, lower cell yield, and lower specific growth rate. The specific fermentation or propionic acid production rate from lactate was, however, higher than that from lactose. Since about equimolar acid products would be formed from lactate, the reactor pH remained relatively unchanged throughout the fermentation and would be easier to control when lactate was the growth substrate. Therefore, lactate would be a preferred substrate over lactose and glucose for propionic acid production using continuous, immobilized cell bioreactors. Correspondence to: S. T. Yang     

The oxidation of hemins by microsomal heme oxygenase. Structural requirements for the retention of substrate activity

The substrate specificity of microsomal heme oxygenase from rat liver was studied by introducing systematic structural changes in the array of substituents of the protohemin IX rings. Replacement of the vinyls by methyl groups resulted in hemins which were excellent substrates of the heme oxygenase. Replacement of the 4-vinyl group by a propionic acid chain (harderohemin), decreased substrate activity to 40%. The replacement of the vinyls by formyl residues strongly decreased substrate activity but the hemins were still substrates of heme oxygenase. The oxidation rates of Spirographis hemin and of 2,4-diformyldeuterohemin IX showed a time lag which was absent when isoSpirographis hemin was used as a substrate. This lag could be attributed to the formation of a transient hemiacetal between the 2-formyl group and the alpha-mesohydroxy residue. The isomeric protohemins I, XI, and XIV (Fischer's notation) were examined as possible substrates of microsomal heme oxygenase. In these protohemins the array of substituents of rings A and B was the same as in protohemin IX, but the methyl and propionic acid residues of rings C and D were at different positions from those of protohemin IX. None of them had substrate activity, indicating that the presence of two vicinal propionic acid side-chains at C6 and C7 was necessary for substrate activity. A hemin with only one propionic acid residue at C5 was not a substrate of the enzyme, either. When the propionic acid residues of protohemin IX were replaced by butyric acid residues, substrate activity decreased to 50% (as compared to protohemin IX), while when they were replaced by acetic acid residues, the substrate activity was entirely suppressed. The addition of dimethyl sulfoxide (25 mM) to the incubation mixture enhanced the oxidation of hemins with non-polar substituents in rings A and B by about 35%, while it was without effect on hemins with polar substituents in the same rings.     

Improvement of vitamin B(12) fermentation by reducing the inhibitory metabolites by cell recycle system and a mixed culture

The major problem in vitamin B(12) production using Propionibacterium is the growth inhibition of the cell due to the accumulation of inhibitory metabolites such as propionic acid and acetic acid. In the present paper, we considered several approaches of controlling the propionic acid concentration at low level. Namely: (1) the periodic cultivation of Propionibacterium where dissolved oxygen (DO) concentration was alternatively changed between 0 and 1ppm; (2) cell recycle system using hollow fiber module; and (3) mixed culture using Propionibacterium and Ralstonia eutropha where the latter microorganism assimilates the propionic acid produced by the former. It was found that the productivity of vitamin B(12) was the highest for the cell recycle system, while if the performance was evaluated based on the amount of vitamin B(12) produced per medium used, the mixed culture system gave the far highest value.