Modeling of olive oil degradation and oleic acid inhibition during chemostat and batch cultivation of Bacillus thermoleovorans IHI-91

Olive oil degradation by the thermophilic lipolytic strain Bacillus thermoleovorans IHI-91 in chemostat and batch culture was modeled to obtain a general understanding of the underlying principles and limitations of the process and to quantify its stoichiometry. Chemostat experiments with olive oil as the sole carbon source were successfully described using the Monod chemostat model extended by terms for maintenance requirements and wall growth. Maintenance requirements and biomass yield coefficients were in the range reported for mesophiles. For a chemostat experiment at D = 0.3 h(-1) the model was validated up to an olive oil feed concentration of about 3.0 g L(-1) above which an inhibitory effect occurred. Further analysis showed that the liberated oleic acid is the main cause for this inhibition. Using steady-state oleic acid concentrations measured in chemostat experiments with olive oil as substrate it was possible to derive a kinetic expression for oleic acid utilization, showing that a concentration of 430 mg L(-1) leads to a complete growth inhibition. Oleic acid accumulation observed during batch fermentations can be predicted using a model involving growth-associated lipase production and olive oil hydrolysis. Simulations confirmed that this accumulation is the cause for the sudden growth cessation occurring in batch fermentations with higher olive oil start concentrations. Further, an oscillatory behavior, as observed in some chemostat experiments, can also be predicted using the latter model. This work clearly demonstrates that thermophilic lipid degradation by Bacillus thermoleovorans IHI-91 is limited by long-chain fatty acid beta-oxidation rather than oil hydrolysis.     

Esterase and lipase activity in Jatropha curcas L. seeds

Two new esterases (JEA and JEB) and a lipase (JL) were extracted from the seeds of Jatropha curas L. Lipase activity was only found during germination of the seeds and increased to a maximum after 4 days of germination. All enzymes were found to be most active in the alkaline range at around pH 8 and the purified (fractionated precipitation with ethanol and gel filtration) esterases were very stable at high temperatures. The molecular weight (SDS-PAGE) of both esterases was determined to be 21.6-23.5 kDa (JEA) and 30.2 kDa (JEB) and the isoelectric point was 5.7-6.1 for esterase JEA and 9.0 for esterase JEB. Most ions caused a negative influence on the activity of both esterases. Using p-nitrophenyl butyrate as a substrate JEA showed a K(m) of 0.02 mM and a v(max) of 0.26 micromol mg(-1) min(-1). Under the same conditions JEB showed a K(m) of 0.07 mM and a v(max) of 0.24 micromol mg(-1) min(-1). Both esterases hydrolyzed tributyrin, nitrophenyl esters up to a chain length of =C4 and naphtylesters up to a chain length =C6. In transesterification reactions, JL was found to be most active at very low water activities (0.2) and in high water activities, the lipase hydrolysed triglycerides into conversions above 80%. The lipase hydrolysed both short chain and long chain triglycerides at about the same rate but was inactive on alpha-methylbenzyl acetate. JL is a potentially useful biocatalyst in the hydrolysis of triglycerides in organic solvents.     

Influence of the thermochemical sludge pretreatment on the nitrification of A/O reactor with the removal of phosphorus by simultaneous precipitation

In the present study, a laboratory scale anoxic/oxic (A/O) reactor is used for the removal of nutrient and sludge reduction. Phosphorus removal was achieved through simultaneous precipitation, and sludge production was reduced through thermochemical pretreatment. The main objective of the study was to investigate the influence of sludge pretreatment on the nitrification rate. Total phosphorus in the effluent was maintained around 0.5 ～ 1.0 mg/L by simultaneous precipitation, using coagulant alum at 2.2 mole ratio. Before simultaneous precipitation, the nitrification rate of the A/O reactor was found to be 0.050 g N-NH₄ ⁺/g MLVSS.d. The thermochemical sludge pretreatment began on the 120^th day at pH 11 and 80°C. The initiation of sludge pretreatment brought about a significant reduction of the A/O reactor nitrification rate, which fell to 0.038 g N-NH₄ ⁺/g MLVSS/day. The effect of sludge pretreatment was reflected in the reduction of the nitrogen removal efficiency from 85 to 74%. Recycling of the thermochemically pretreated sludge accounted for 57% sludge reduction, which had an adverse influence on the nitrification rate of the system.     

Nitrogen removal from wastewater with a low COD/N ratio at a low oxygen concentration

The goal of the study was to determine the effectiveness of nitrification and denitrification and the kinetics of ammonia removal from a mixture of wastewater and anaerobic sludge digester supernatant in an SBR at limited oxygen concentration. In addition, the COD removal efficiency and sludge production were assessed.In the SBR cycle alternating aerobic and anaerobic phases occurred; in the aeration phase the dissolved oxygen (DO) concentration was below 0.7 mg O₂/L. The low DO concentration did not inhibit ammonia oxidation-nitrification and the efficiency was ca. 96-98%. However, a relatively high COD concentration in the effluent was detected. The values of K_m and V_max, calculated from the Michaelis-Menten equation, were 43 mg N-NH₄/L and 15.64 mg N-NH₄/L h, respectively. Activated sludge production was almost stable (0.62-0.66 g MLVSS/g COD). A high net biomass production resulted from a low specific biomass decay rate of 0.0015 d⁻¹.     

Feasibility of using ultrasonic irradiation to recover active biomass from waste activated sludge

Under typical operating conditions, the microbial fraction of activated sludge flocs is approximately 40% by weight. The objective of this research is to evaluate the feasibility of using ultrasonic irradiation to disrupt activated sludge flocs allowing for the subsequent separation of active and inactive fractions. If separation of floc components is possible, then methods may be incorporated into wastewater treatment plant operations whereby only the inactive fraction of floc is wasted (i.e., of waste activated sludge, WAS), which in turn could increase the overall effective biological solids retention time, leading to increased process robustness with no net increase in reactor size. The results indicate that ultrasonic irradiation of WAS at 800 Wl(-1) followed by 30 min of settling can produce a supernatant with heterotrophic specific oxygen uptake rates (SOURs) of over two times the SOUR measured in the bulk mixed liquor. Under these conditions 26% of the initial heterotrophic activity was recovered within only 11% of the initial volatile mass. Similarly, autotrophic analysis revealed that nitrifying organisms, while sensitive to the effects of ultrasonic irradiation, can be separated from the activated sludge floc and recovered. An irradiation density of 200 Wl(-1) with an exposure time between 1 and 2 min produced a supernatant with a specific ammonia removal rate of over two times the initial mixed liquor rate.     

Kinetic modeling of aerobic biodegradation of high oil and grease rendering wastewater

Batch scale activated sludge kinetic studies were undertaken for the treatment of pet food wastewater characterized by oil and grease concentrations of up to 21,500 mg/L, COD and BOD concentrations of 75,000 and 60,000 mg/L, respectively as well as effluent from the batch dissolved air flotation (DAF) system. The conducted kinetics studies showed that Haldane Model fit the substrates and biomass data better than Monod model in DAF-pretreated wastewater, while the modified hydrolysis Monod model better fit the raw wastewater kinetic data. For the DAF pretreated batches, Haldane Model kinetic coefficients k, K(S), Y and Ki values of 1.28-5.35 g COD/g VSS-d, 17,833-23,477 mg/L, 0.13-0.41 mg VSS/mg COD and 48,168 mg/L, respectively were obtained reflecting the slow biodegradation rate. Modified hydrolysis Monod model kinetic constants for the raw wastewater i.e., k, K(S), Y, and K(H) varied from 1-1.3 g COD/g VSS-d, 5580-5600 mg COD/l, 0.08-0.85 mg VSS/mg COD, and 0.21-0.66 d(-1), respectively.     

Simultaneous nitrification, denitrification, and phosphorus removal from nutrient-rich industrial wastewater using granular sludge

The biological removal of nitrogen and phosphorus from nutrient-rich abattoir wastewater using granular sludge has been investigated. A lab-scale sequencing batch reactor, seeded with granular sludge developed using synthetic wastewater, was operated for 13 months under alternating anaerobic and aerobic conditions. It is demonstrated that the granules could be sustained and indeed further developed with the use of abattoir wastewater. The organic, nitrogen, and phosphorus loading rates applied were 2.7 gCOD L(-1) day(-1), 0.43 gN L(-1) day(-1), and 0.06 gP L(-1) day(-1), respectively. The removal efficiency of soluble COD, soluble nitrogen and soluble phosphorus were 85%, 93%, and 89%, respectively. However, the high suspended solids in the effluent limited the overall removal efficiency to 68%, 86%, and 74% for total COD, TN, and TP, respectively. This good nutrient removal was achieved through the process known as simultaneous nitrification, denitrification, and phosphorus removal, likely facilitated by the presence of large anoxic zones in the center of the granules. The removal of nitrogen was likely via nitrite optimizing the use of the limited COD available in the wastewater. Accumulibacter spp. were found to be responsible for most of the denitrification, further reducing the COD requirement for nitrogen and phosphorus removal. Mineral precipitation was evaluated and was not found to significantly contribute to the overall nutrient removal. It is also shown that the minimum HRT in a granular sludge system is not governed by the sludge settleability, as is the case with floccular sludge systems, but likely by the limitations associated with the transfer of substrates in granules.     

Incorporation of ultrasonic cell disintegration into a membrane bioreactor for zero sludge production

For the prevention of excess sludge production from a membrane bioreactor (MBR), an ultrasonic cell disintegration process was incorporated. The results of this study showed that excess sludge production could be prevented using an ultrasound hybrid (MBR-US) system at an organic loading of around 0.91 kg BOD₅/m³ per day. Under the same organic loading rate, the mixed liquor suspended solid (MLSS) of MBR-US system was maintained at 7000–8000 mg/l while the MLSS of a conventional MBR increased from 7000 to 13,700 mg/l during the experimental period. While sludge production was completely prevented, the effluent quality of the MBR-US system slightly deteriorated. The additional organic loading caused by disintegrated sludge return was considered to be a reason. With sonication the volume of the average particle size of the sludge in the aeration tank decreased from 132 to 95 μm. In the MBR-US system, around 25–30% of total phosphorus removal was achieved without sludge removal from the aeration tank.     

Characteristics of immobilized lipase-catalyzed hydrolysis of olive oil of high concentration in reverse phase system

Candida rugosa lipase immobilized by adsorption on swollen Sephadex LH-20 could almost completely hydrolyze 60% (v/v) olive oil in isooctane. Kinetic analysis of the lipase-catalyzed hydrolysis reaction was found to be possible in this system. Amount of fatty acids produced was linearly proportional to the enzyme concentration of 720 mug/g wet gel. The specific enzyme activity was 217 units/mg protein at 60% (v/v) olive oil concentration. When the initial rate is plotted versus concentration of olive oil, this system did not follow Michaelis-Menten kinetics. Maximum activity was obtained at pH 7, but optimum temperature shifted towards higher one with the increase of olive oil concentration. Among the various chemical compounds tested, Hg(2+) and Fe(2+) inhibited the lipase seriously. As the concentration of olive oil increased, the rate of the hydrolysis also increased, but degree of the hydrolysis was observed to decrease. The supply of water from the inside of the gel to the surface of the gel was the main factor for the control of the rate of hydrolysis in batch hydrolysis. The immobilized lipase was used to hydrolyze olive oil two times. Achievement of chemical equilibrium took a longer time with the addition of water and the degree of hydrolysis decreased in the second consecutive trial. After the second hydrolysis trial, the gels were regenerated in a packed column first by eluting out both residual fatty acids around the gel particles and the accumulated glycerol with ethanol and then with 0.05M phosphate buffer, pH 7. The immobilized lipase on the regenerated gel showed the same hydrolysis activity as the original one.     

Accelerating enzymatic hydrolysis of chitin by microwave pretreatment

Response surface analysis was used to determine optimum conditions [2% (w/v) chitin, 57.5 degrees C, 38 min] for microwave irradiation of chitin to improve its enzymatic hydrolysis. V(max)/K(m) of cabbage chitinase toward untreated and microwave-irradiated chitin was found to be 21.1 and 31.7 nmol h(-1) mg(-2) mL, respectively. Similar improvement was observed in the case of pectinase in its unusual catalytic activity of chitin degradation. It was found that a greater extent of chitin hydrolysis by chitinase was possible after the substrate chitin was irradiated with microwaves.     

Pilot trials of the microbial degradation of Christos-Bitas water in oil emulsion (chocolate mousse) and BP llandarcy gas oil using venturi aeration

Oil residues arising from the Christos-Bitas spillage were found to contain 28% of oil extractable by carbon tetrachloride; the remainder consisted of water and undefined solids. Christos-Bitas mousse was added to 1.18 m(3) liquor inoculated with oil-contaminated marine mud, and aerated with a 1.5-hp vortex pump and venturi nozzle (12.5 mm) in a cylindrical tank. After 70 days, oil degradation reached 7 mg oil/L/h. About 98% of the solvent extractable oil added was degraded over 83 days. Analysis of oil residues harvested at the end of this experiment showed that there was a decreasing trend in percent degradation in the following order: aromatics > saturates > heterocyclics > asphalts. No less than 94% of any fraction analysed was degraded.In the second pilot trial, oil degradation was carried out in a cylindrical jacket tank containing 6.82 m(3) liquor inoculated with oil-contaminated marine mud from Penarth, South Wales, UK, together with pure cultures derived from the same source, and aerated with a 7.5-hp vortex pump and venturi nozzle (18 mm diameter). Mixing of the oil was inhomogeneous for the first 100-110 days. The overall degree of substrate dispersion and total oil balance was determined by sampling at different depths. Degradation by the mixed culture was achieved at the rate of 164 mg oil/L/h. After 224 days, this was equivalent to 9.6 x 10(3)/kg(-1)/yr;(214 kg/wk) for 6.82 m(3) of liquor. The degradation rate continued to rise as the feed rate was increased by means of an automatic, timed pump. A lag phase of five to six months was necessary to allow the mixed population to build up to an exploitable level.     

Immobilization of activated sludge by PVA-boric acid method

A new method (polyvinyl alcohol-boric acid method) for an inexpensive and effective immobilization of activated sludge was developed. Using activated sludge immobilized by this PVA-boric acid method, synthetic waste-water was treated at a high loading rate of 0.5-2.35 kg TOC/m(3) day. Total organic carbon and total nitrogen were removed at efficiencies of 93 and 30-40%, respectively. The kinetic constants Y and b for this immobilized activated-sludge process were determined to be 0.594g mixed liquor suspended solids (MLSS)/g TOC and 0.0219 day(-1), respectively. The cost calculation of chemicals required for the immobilization of activated sludge by this PVA-boric acid method was proved to be extremely inexpensive for the immobilization of activated sludge.     

Lipid accumulation and growth of Chlorella zofingiensis in flat plate photobioreactors outdoors

Culturing microalgae using natural sunlight is an effective way to reduce the cost of microalgae-based biodiesel production. In order to evaluate the feasibility of culturing Chlorella zofingiensis outdoors for biodiesel production, effects of nitrogen limitation and initial cell concentration on growth and lipid accumulation of this alga were investigated in 60 L flat plate photobioreactors outdoors. The highest μmax and biomass productivity obtained was 0.994 day(-1) and 58.4 mg L(-1)day(-1), respectively. The lipid content was much higher (54.5% of dry weight) under nitrogen limiting condition than under nitrogen sufficient condition (27.3%). With the increasing initial cell concentrations, the lipid contents declined, while lipid concentrations and productivities increased. The highest lipid content, lipid concentration, and lipid productivity obtained was 54.5%, 536 mg L(-1) and 22.3 mg L(-1)day(-1), respectively. This study demonstrated that it was possible to culture C. zofingiensis under outdoor conditions for producing biodiesel feedstock.     

Lipolytic activity of suspended and membrane immobilized lipase originating from indigenous Burkholderia sp. C20

In this work, a simple, inexpensive, and efficient method of preparing immobilized lipase is presented. The lipase originating from a newly isolated indigenous strain Burkholderia sp. C20 was immobilized onto cellulose nitrate (CN) membrane via filtration. The CN-immobilized lipase was able to retain 60% of its original activity after repeated uses for nine times. The thermal stability of the lipase was also slightly improved after immobilization. The optimal reaction conditions of CN-lipase were pH 9.0 and 55 degrees C, which are similar to those for the suspended lipase. Both suspended and immobilized lipase could hydrolyze the six oil substrates examined, while immobilized lipase displayed less specificity over the oil substrates. Kinetic analysis shows that the dependence of lipolytic activity of both suspended and immobilized lipase on oil substrate concentration can be described by Michaelis-Menten model with good agreement. The estimated kinetic constants for suspended lipase (v(max)=243.9 U/mg, K(m)=0.024 mM) and immobilized lipase (v(max)=32.8 U/mg, K(m)=5.61 mM) were quite different. Employment of immobilization seemed to result in a decrease in v(max) and an increase in K(m), most likely due to the mass transfer resistance arising from formation of micelles during the lipase immobilization process.     

Simultaneous sludge reduction and nutrient removal (SSRNR) with interaction between Tubificidae and microorganisms: a full-scale study

A symbiotic ecosystem between Tubificidae and microorganisms was established at a full-scale wastewater treatment plant (WWTP). In this ecosystem Tubificidae were inoculated, and then adhered to the outer layers of carrier materials in an oxidation tank. During the long-term treatment of sewage volumes of 20,000 m(3)d(-1), the excess sludge production rate was reduced from 0.21 to 0.051 kg m(-3) and sludge settleability was significantly improved. When the influent concentrations of COD, NH(4)(+)-N, PO(4)(-)-P, and SS were in the ranges of 130.0-459.0 mg L(-1), 14.2-27.5 mg L(-1), 1.6-7.0 mg L(-1), and 60.0-466.0 mg L(-1), respectively, the COD and SS removal efficiency was increased by 8.7% and 13.6% within the symbiotic system compared to the control without Tubificidae. In addition, NH(4)(+)-N and phosphorus removal efficiency can also be improved. The results showed that both sludge reduction and nutrient removal were enhanced simultaneously significantly within the system utilizing the symbiotic interactions of Tubificidae and microorganisms.     

Direct effect of temperature on the catalytic activity of nitric oxide synthases types I, II, and III.

The effect of temperature (between 5.0 and 45.0 degrees C) on the catalytic activity of nitric oxide synthases types I, II, and III (NOS-I, NOS-II, and NOS-III, respectively) has been investigated, at pH 7.5. The value of V(max) for NOS-I activity increases from 1.8 x 10(1) pmol min(-1) mg(-1), at 5.0 degrees C, to 1.8 x 10(2) pmol min(-1) mg(-1), at 45.0 degrees C; on the other hand, the value of K(m) (=4.0 x 10(-6) M) is temperature independent. Again, the value of V(max) for NOS-II activity increases from 8.0 pmol min(-1) mg(-1), at 7.0 degrees C, to 5.4 x 10(1) pmol min(-1) mg(-1), at 40.0 degrees C, the value of K(m) (=1.8 x 10(-5) M) being unaffected by temperature. Temperature exerts the same effect on NOS-I and NOS-II activity, as shown by the same values of DeltaH(V(max)) (=4.2 x 10(1) kJ mol(-1)), DeltaH(K(m)) (=0 kJ mol(-1)), and DeltaH((V(max))(/K(m))()) (=4.2 x 10(1) kJ mol(-1)). On the contrary, the value of K(m) for NOS-III activity decreases from 3.8 x 10(-5) M, at 10.0 degrees C, to 1.6 x 10(-5) M, at 40.0 degrees C, the value of V(max) (=6.8 x 10(1) pmol min(-1) mg(-1)) being temperature independent. Present results indicate that temperature influences directly NOS-I and NOS-II activity independently of the substrate concentration, the values of K(m) being temperature independent. However, when l-arginine level is higher than 2 x 10(-4) M, as observed under in vivo conditions, NOS-III activity is essentially unaffected by temperature, the substrate concentration exceeding the value of K(m). As a whole, although further studies in vivo are needed, these observations seem to have potential physiopathologic implications.     

Cloning, expression, purification, and properties of an endoglucanase gene (glycosyl hydrolase family 12) from Aspergillus niger VTCC-F021 in Pichia pastoris

A gene coding for an endoglucanase (EglA), of the glycosyl hydrolase family 12 and derived from Aspergillus niger VTCC-F021, was cloned and sequenced. The cDNA sequence, 717 bp, and its putative endoglucanase, a 238 aa protein with a predicted molecular mass of 26 kDa and a pI of 4.35, exhibited 98.3-98.7% and 98.3-98.6% identities, respectively, with cDNA sequences and their corresponding endoglucanases from Aspergillus niger strains from the GenBank. The cDNA was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 1.59 U/ml culture supernatant, after 72 h of growth in a YP medium induced with 1% (v/v) of methanol. The molecular mass of the purified EglA, determined by SDS-PAGE, was 33 kDa, with a specific activity of 100.16 and 19.91 U/mg toward 1% (w/v) of beta-glucan and CMC, respectively. Optimal enzymatic activity was noted at a temperature of 55°C and a pH of 5. The recombinant EglA (rEglA) was stable over a temperature range of 30- 37°C and at pH range of 3.5-4.5. Metal ions, detergents, and solvents tested indicated a slightly inhibitory effect on rEglA activity. Kinetic constants (K(m), V(max), k(cat), and k(cat)/ K(m)) determined for rEglA with beta-glucan as a substrate were 4.04 mg/ml, 102.04 U/mg, 2,040.82 min-1, and 505.05, whereas they were 10.17 mg/ml, 28.99 U/mg, 571.71 min-1, and 57.01 with CMC as a substrate, respectively. The results thus indicate that the rEglA obtained in this study is highly specific toward beta-glucan. The biochemical properties of rEglA make it highly valuable for downstream biotechnological applications, including potential use as a feed enzyme.     

Fate in sewage of a recombinantEscherichia coli K-12 strain used in the commercial production of bovine somatotropin

Summary The fate of a derivative ofEscherichia coli strain W3110G [pBGH1], a strain used for production of bovine somatotropin, was examined in semi-continuous activated sludge (SCAS) units. A nalidixic acid-resistant derivative of W3110G [pBGH1], strain LBB270 [pBGH1], was used to facilitate tracking. SCAS units (300 ml) containing municipal mixed liquor were operated on a daily cycle of 23 h aeration and 1 h settling followed by decanting of clear supernatant (175 ml) and refilling with fresh primary effluent. SCAS units were inoculated with two concentrations ofE. coli LBB270 [pBGH1] and operated for 200 h. Viable levels ofE. coli LBB270 [pBGH1] were measured daily in aerated mixed liquor and decanted supernatant. Viable counts in the mixed liquor decreased from 10000- to 100000-fold in less than 200 h. Losses ofE. coli LBB270 [pBGH1] in decanted supernatants accounted for less than 2-fold of the total losses observed in the SCAS units. TheE. coli LBB270 [pBGH1] was not evenly distributed in the mixed liquor, but became preferentially associated with the settleable floc. These results show thatE. coli LBB270 [pBGH1] was unable to survive in municipal sludge even when inoculated at concentrations greater than, or comparable to, levels of indigenous microorganisms.     

Expression, purification, and characterization of recombinant Metarhizium anisopliae acid trehalase in Pichia pastoris

The mature peptide of Metarhizium anisopliae acid trehalase (ATM1) (EC3.2.1.28) was successfully expressed in Pichia pastoris at high levels under the control of AOX1 promoter. The recombinant ATM1 (reATM1) was secreted into culture medium. After 48-h 0.5% methanol induction, the activity of reATM1 in the culture supernatant reached the peak, 5.35 U/mg. Enzyme with a histidine sequence appended to the C terminus was still active and was purified using metal-chelate affinity chromatography. The yield of purified reATM1 was 2.5 mg from 1L supernatant. The purified reATM1 exhibited a molecular mass of approximately 170 kDa on SDS-PAGE. The optimum temperature and pH of reATM1 were 30 degrees C and 6.0, respectively, and the K(m) and V(max) values for reATM1 were 2.6 mM and 0.305 mmol/min/mg, respectively. Studies showed that the enzymatic properties of reATM1 were similar to those of the native ATM1.     

Purification, and properties of a bovine uricase

Uricase from bovine kidney, purified to homogeneity level, had a molecular weight of 70 kDa. The apparent K(m) and V(max) values for uric acid hydrolysis were 0.125 mM and 102 IU mg(-1) protein respectively. The activation energy requirement for uric acid hydrolysis by uricase and inactivation of enzyme were 11.6 and 14.5 kJ/M respectively. Both enthalpy (Delta H*) and entropy of activation (Delta S*) for uricase activity were lower than those reported for some thermostable enzymes.