Effects of glucose and phenol on soluble microbial products (SMP) in sequencing batch reactor systems

Soluble microbial products (SMP) are ubiquitously present in the effluents of biological wastewater treatment systems. In sequencing batch reactor (SBR) systems, effects of influent concentration and temperature on the amount and the molecular weight (MW) distribution of SMP were investigated for the two substrates, glucose and phenol. The values of effluent SMP/S₀ of phenol were higher than those of glucose at different influent concentrations and temperatures. It was found that the effluent SMP (S_e) was linearly correlated to the influent total organic carbon (TOC) (S₀) for both substrates. The slope and intercept of the equation were affected by the temperature. According to the analysis of the MW distribution, it was shown that there exists a bimodal pattern with the majority of SMP having a MW<1 kDa or >10 kDa. The low MW fraction (<1 kDa) amounts to 47.3–70.4% of the effluent SMP. The high MW fraction (>10 kDa) slightly fluctuates in the range of 21.2–32.8% of the effluent SMP.     

Ecotoxicological Evaluation of a Bench-Scale Bioslurry Treating Explosives-Spiked Soil

The ecotoxicological effects of four bioslurry reactors treating 2,4,6-trinitotoluene (TNT)- and 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX)-spiked soil were evaluated. A control bioslurry reactor was used to assess the endogenous toxicity of the bioslurry operation conditions. A battery of ecotoxicity tests was used: Microtox, green algae growth inhibition, bacterial genotoxicity and mutagenicity, and earthworm mortality and growth inhibition. Bioslurry soluble and solid phases were separated by centrifugation in order to identify toxicity and possible toxicants associated with each phase. Microtox toxicity values were initially very high in both bioslurry reactors spiked with TNT, in relation with TNT concentration. Initial toxicity was also detected by algal growth inhibition, earthworm lethality, genotoxicity and mutagenicity tests. An endogenous toxicity was detected in the control bioreactor using the Microtox and the SOS Chromotest. The soluble phase of the control bioslurry was genotoxic, suggesting that some potentially genotoxic agents were induced in the bioslurry samples. At the end of the bioremediation treatment, data showed that toxicity was reduced using all of the bioassays, except for earthworm lethality and growth inhibition tests in both RDX-spiked bioslurries. This study demonstrates the usefulness of a battery of toxicity tests to monitor bioremediation processes.     

A novel continuous toxicity test system using a luminously modified freshwater bacterium

An automated continuous toxicity test system was developed using a recombinant bioluminescent freshwater bacterium. The groundwater-borne bacterium, Janthinobacterium lividum YH9-RC, was modified with luxAB and optimized for toxicity tests using different kinds of organic carbon compounds and heavy metals. luxAB-marked YH9-RC cells were much more sensitive (average 7.3-8.6 times) to chemicals used for toxicity detection than marine Vibrio fischeri cells used in the Microtox assay. Toxicity tests for wastewater samples using the YH9-RC-based toxicity assay showed that EC50-5 min values in an untreated raw wastewater sample (23.9 +/- 12.8%) were the lowest, while those in an effluent sample (76.7 +/- 14.9%) were the highest. Lyophilization conditions were optimized in 384-multiwell plates containing bioluminescent bacteria that were pre-incubated for 15 min in 0.16 M of trehalose prior to freeze-drying, increasing the recovery of bioluminescence and viability by 50%. Luminously modified cells exposed to continuous phenol or wastewater stream showed a rapid decrease in bioluminescence, which fell below detectable range within 1 min. An advanced toxicity test system, featuring automated real-time toxicity monitoring and alerting functions, was designed and finely tuned. This novel continuous toxicity test system can be used for real-time biomonitoring of water toxicity, and can potentially be used as a biological early warning system.     

Influence of solids retention time on membrane fouling: characterization of extracellular polymeric substances and soluble microbial products

The objective of this study was to investigate the influence of solids retention time (SRT) on membrane fouling and the characteristics of biomacromolecules. Four identical laboratory-scale membrane bioreactors (MBRs) were operated with SRTs for 10, 20, 40 and 80 days. The results indicated that membrane fouling occurred faster and more readily under short SRTs. Fouling resistance was the primary source of filtration resistance. The modified fouling index (MFI) results suggested that the more ready fouling at short SRTs could be attributed to higher concentrations of soluble microbial products (SMP). Fourier transform infrared (FTIR) spectra indicated that the SRT had a weak influence on the functional groups of the total extracellular polymeric substances (TEPS) and SMP. However, the MBR under a short SRT had more low-molecular-weight (MW) compounds (<1 kDa) and fewer high-MW compounds (>100 kDa). Aromatic protein and tryptophan protein-like substances were the dominant groups in the TEPS and SMP, respectively.     

Studies on biodegradation of a mixture of toxic and nontoxic pollutant using Arthrobacter species

The effect of a nontoxic easily degradable substrate, glucose, on the biodegradation of toxic pollutant, phenol, was studied in batch reactors using a phenol degrading culture (Arthrobacter species). The effect of glucose on phenol degradation was determined at different glucose concentrations. The effect of different inoculum on substrate removal in a phenol and glucose mixture was also studied. Results indicated that when a mixed substrate (phenol and glucose) was used, phenol acclimated population showed an initial preference for phenol and utilised glucose after phenol removal. However phenol degradation rate was reduced in the presence of glucose. It was also observed that phenol degradation was completely inhibited when the glucose concentration exceeds 2 g/l. The substrate removal pattern changed completely when inoculum was drawn from mixed substrate acclimatised culture. The glucose utilisation started immediately and the rate of glucose utilisation was not affected by the presence of phenol. The phenol degradation also started simultaneously. In presence of phenol only, the rate of phenol degradation for the culture acclimatised to mixed substrates was lower than that of phenol acclimatised culture. These results indicate that nontoxic substrate can affect the biodegradation of toxic pollutants is suitable and acclimatisation may be necessary for biodegradation of mixed substrate.     

Color and toxicity removal following tyrosinase-catalyzed oxidation of phenols

The products of phenol oxidation catalyzed by mushroom tyrosinase (polyphenol oxidase, EC 1.14.18.1) were assessed in terms of their residual color and toxicity. The addition of aluminum sulfate had little effect on the removal of colored products from phenol solutions treated with tyrosinase. Although chitosan was used successfully to remove the color when added before the reaction initiation or after the reaction completion, the required dose of chitosan was lower when it was added after the reaction. In this case, the minimum doses of chitosan required to achieve 90% color removal were proportional to the logarithm of the initial concentration of phenol. The color removal induced by chitosan addition appeared to be the result of chemical interaction followed by a coagulation mechanism. All treated solutions of phenol and chlorophenols, except 2,4-dichlorophenol, had substantially lower toxicities than their corresponding initial toxicities, as measured using the Microtox assay. Chitosan addition significantly enhanced the reduction in toxicity. The toxicities of the phenol solutions treated with tyrosinase were markedly lower than previously reported toxicities of solutions treated with peroxidase enzymes.     

Effects of glucose on phenol biodegradation by heterogeneous populations

The effect of the presence of more easily degradable alternative carbon sources on the biodegradation of toxic waste components is of great practical importance. In this work, a mixed phenol/glucose waste was fed to two heterogeneous populations acclimated to different conditions: one was acclimated to phenol as a sole source of carbon and one to a mixed phenol/glucose substrate. Batch substrate utilization experiments were performed under both growth and nonproliferating (no medium nitrogen source) conditions in order to assess substrate removal patterns at the levels of enzyme production and enzyme function. The results indicated that the substrate removal pattern exhibited by the cells was significantly influenced by the acclimation characteristics of the culture. The phenol acclimated cells showed an initial preference for phenol, but the presence of glucose hindered phenol removal rate under both growth and nonproliferating conditions. The cells acclimated to the mixed phenol/glucose waste demonstrated rapid initial glucose removal with a slower concomitant utilization of phenol; acclimation to the mixed waste evidently had a significant impact on the substrate removal pattern for this mixed substrate system.     

Microbial transformation of biomacromolecules in a membrane bioreactor: implications for membrane fouling investigation

Background

The complex characteristics and unclear biological fate of biomacromolecules (BMM), including colloidal and soluble microbial products (SMP), extracellular polymeric substances (EPS) and membrane surface foulants (MSF), are crucial factors that limit our understanding of membrane fouling in membrane bioreactors (MBRs).

Findings

In this study, the microbial transformation of BMM was investigated in a lab-scale MBR by well-controlled bioassay tests. The results of experimental measurements and mathematical modeling show that SMP, EPS, and MSF had different biodegradation behaviors and kinetic models. Based on the multi-exponential G models, SMP were mainly composed of slowly biodegradable polysaccharides (PS), proteins (PN), and non-biodegradable humic substances (HS). In contrast, EPS contained a large number of readily biodegradable PN, slowly biodegradable PS and HS. MSF were dominated by slowly biodegradable PS, which had a degradation rate constant similar to that of SMP-PS, while degradation behaviors of MSF-PN and MSF-HS were much more similar to those of EPS-PN and EPS-HS, respectively. In addition, the large-molecular weight (MW) compounds (>100 kDa) in BMM were found to have a faster microbial transformation rate compared to the small-MW compounds (<5 kDa). The parallel factor (PARAFAC) modeling of three-dimensional fluorescence excitation-emission matrix (EEM) spectra showed that the tryptophan-like PN were one of the major fractions in the BMM and they were more readily biodegradable than the HS. Besides microbial mineralization, humification and hydrolysis could be viewed as two important biotransformation mechanisms of large-MW compounds during the biodegradation process.

Significance

The results of this work can aid in tracking the origin of membrane foulants from the perspective of the biotransformation behaviors of SMP, EPS, and MSF.     

Behavior of microbial communities developed in the presence/reduced level of soluble microbial products

Soluble microbial products (SMP) are organics produced by microorganisms as they degrade substrates. The available literature does not reveal how SMP affect and regulate microbial activities. In this study, we monitored variations in pH, dissolved oxygen concentration, soluble biological and chemical oxygen demands (sBOD₅ and sCOD) as a measure of microbial activity in synthetic wastewater. Aerobic degradation tests were carried out under the following conditions: aeration, 1,500 cm³ /min; initial sBOD₅, 515±5 mg/l; initial sCOD, 859±6 mg/l; initial biomass concentration (defined as mixed liquor suspended solids), 1,200±25 mg/l; sludge retention time, 24 h; and temperature, 20±1°C. The study involved non-acclimated biomass (R0 flora), biomass developed in the presence of SMP (R1 flora), and biomass developed in reduced level of SMP (R2 flora). We also determined which of these flora produced more refractory SMP. The results showed that R2 flora utilized the synthetic feed more quickly, and produced less refractory organic matter than R0 and R1 flora. The production of more refractory organics by R0 and R1 flora shows that not all the biomass was active. R1 flora degraded the substrates irregularly, suggesting that some microbes were dependent on the metabolic products of those that could utilize the feed components. These results show that production of SMP also depends on the prior substrates and on the ability of the flora to respond to changes in substrate composition.     

Effect of Co‐Substrates on Aerobic Phenol Degradation by Acclimatized and Non‐acclimatized Enrichment Cultures

Aerobic degradation of 7 mmol/L phenol in the presence of alternative carbon sources (7 mmol/L glucose or acetate or 1–2 mmol/L 2‐chlorophenol) was investigated using non‐acclimatized and acclimatized sewage sludges and enrichment cultures. The substrates represented an intermediate of phenol degradation (acetate), an independent substrate (glucose) or a “precursor‐substrate” of phenol degradation (2‐chlorophenol). Bacteria from sewage sludge, not pre‐adapted to phenol (2 mmol/L), rapidly respired acetate and glucose in the presence of phenol, whereas phenol was only bioconverted to any unknown aromatic metabolite after 24 h. In the presence of phenol and 2‐chlorophenol, no removal of both substances was observed when using the unacclimatized sludge. Sludge that was acclimatized to the degradation of phenol showed an initial preference for easily degradable co‐substrates such as glucose or acetate with only a slow concomitant respiration of phenol. Respiration of phenol increased rapidly after the co‐substrates were depleted. The highest phenol degradation rates were 51.6 mmol/L d, when phenol was the sole carbon substrate. Vice versa, phenol was preferentially respired in the presence of a less easily degradable co‐substrate such as 2‐chlorophenol at a rate of around 7 mmol/L d. Further studies with an enrichment culture that was obtained after 7 successive transfers of phenol‐adapted sludge into mineral medium with phenol as the only carbon source indicated that the acetate and glucose‐degrading capabilities were diminished or almost completely lost. In these enrichment cultures, phenol degradation was not affected by the presence of glucose, but glucose was not degraded. In contrary, the presence of acetate slightly slowed down the phenol degradation rate of the enrichment culture. Growth of the microorganisms apparently occurred at the expense of phenol and acetate respiration. The result of this work may be of practical importance in determining the feeding strategy, which is the key factor for most biological wastewater treatment systems. When acetate was present together with phenol in a wastewater, the phenol degradation rates were influenced by acetate, since acetate was an intermediate of phenol degradation. Glucose as an “independent substrate” was apparently degraded by other bacteria via acetate, and in this way it also influenced the phenol degradation rates. Glucose‐degrading bacteria could be “washed out” from the acclimatized sludge during several transfers into mineral medium with phenol as the sole carbon source. If later on, glucose was added again, it remained undegraded and did not influence phenol degradation. 2‐Chlorophenol degradation also requires other bacteria than phenol degraders.     

Removal of phenolic compounds from a petrochemical effluent with a methanogenic consortium.

A methanogenic consortium was used to degrade phenol and ortho- (o-) cresol from a specific effluent of a petrochemical refinery. This effluent did not meet the local environmental regulations for phenolic compounds (178 mg/L), oils and greases (61 mg/L), ammoniacal nitrogen (75 mg/L) or sulfides (3.2 mg/L). The consortium, which degrades phenol via its carboxylation to benzoic acid, was progressively adapted to the effluent. Despite the very high effluent toxicity (EC50 of 2% with Microtox), the adapted consortium degraded 97% of 156 mg/L phenol in the supplemented effluent after 13 days in batch cultures (serum bottle). The addition of proteose peptone to the effluent is essential for phenol degradation. o-cresol was also transformed but not meta- or para-cresols. A continuous flow fixed-film anaerobic bioreactor was developed with the consortium. Treating the effluent with the bioreactor reduced phenol and phenolic compounds concentrations by 97 and 83%, respectively, for a hydraulic residence time of 6 h. This treatment also reduced by about half the effluent toxicity. Oils and greases and ammoniacal nitrogen were not affected. Similar microbiological forms were observed in serum bottles and in the bioreactors with or without the petrochemical effluent. These results indicate that this methanogenic consortium can treat efficiently the phenolic compounds in this specific petrochemical effluent.     

Cryoprotectants for the vitrification of corneal endothelial cells

The objective of this work was to select and test systematically possible cryoprotective agents (CPAs) and to obtain a suitable formula for vitrification of corneal endothelial cells (CECs). Fresh bovine CECs were isolated and tested with an optimized vitrification protocol with multi-step CPA loading and removal. Three types of CPAs components, i.e. the penetrating CPAs, sugars and macromolecular compounds, were experimentally evaluated using the viability assayed by trypan blue. Dimethyl sulfoxide, ethylene glycol (EG), 1,2-propanediol, 2,3-butanediol, acetamide and ethylene glycol monomethyl ether were chosen as the penetrating CPA components. Sugars including xylose, fructose, mannose, glucose, maltose, sucrose and trehalose were tested. Ficoll (MW 7 kDa), dextran (MW 7 kDa), chondroitin sulfate (CS, MW 18-30 kDa), bovine serum albumin (MW 68 kDa) and polyethylene glycol (MW 6 kDa, 10 kDa and 20 kDa) were chosen as the macromolecular compounds. CECs were also preserved by slow freezing as a control. The results showed that EG was the most suitable penetrating CPA component and glucose the most suitable sugar, and CS the most suitable macromolecule. The optimized concentrations for each component in the vitrification solution were 52% (w/w) EG, 8% (w/w) glucose and 3% (w/w) CS. The CEC survival rate of 89.4 ± 2.1% (mean ± SD) was obtained using this formula and established vitrification protocol which was comparable to that by slow freezing.     

Degradation of phenol and benzoic acid in a three-phase fluidized-bed reactor

Degradation of phenol and benzoic acid was studied in a fluidized-bed reactor (liquid volume 2.17 L) under nonsterile conditions with special emphasis on maximizing the flow through the reactor and investigating reactor performance at fluctuating feeds. Reactor response to substrate pulses was investigated by applying substrate square-wave inputs at a liquid flow of 1.00 L h(-1). A twofold increase of the phenol and benzoic acid feed concentrations for 2.5 h did not lead to accumulation and breakthrough. The cells were able to survive four to fivefold increases of the feed concentration for 1 h without loss of viability, although the phenol pulse lead to phenol accumulation in the reactor. Reactor performance at constantly fluctuating loads was investigated by varying the feed concentrations using sine wave functions. No accumulation of phenol or benzoic acid was observed. Influence of induction was studied using shift experiments. After 35 days of operation (369 hydrodynamic residence times) with phenol as sole substrate (carbon source) the reactor was able to mineralize benzoic acid without any adaptation or lag phase. The capability of phenol degradation, on the other hand, was lost by most cells after only 3 days operation with benzoic acid as the sole substrate. The experiments underline the importance of induction. In order to maximize the flow through the reactor, the liquid flow was increased stepwise while the feed concentrations were reduced correspondingly, keeping the volumetric conversion rates of phenol (0.24 g L(-1) h(-1)) and benzoic acid (0.17 g L(-1) h(-1)) constant. By this means, liquid flow could be increased up to 13.32 L h(-1), which was more than 20-fold higher than the maximum liquid flow achievable in a chemostat using the same conditions.     

Senescence marker protein 30 (SMP30) expression in eukaryotic cells: existence of multiple species and membrane localization

Senescence marker protein (SMP30), also known as regucalcin, is a 34 kDa cytosolic marker protein of aging which plays an important role in intracellular Ca(2+) homeostasis, ascorbic acid biosynthesis, oxidative stress, and detoxification of chemical warfare nerve agents. In our goal to investigate the activity of SMP30 for the detoxification of nerve agents, we have produced a recombinant adenovirus expressing human SMP30 as a fusion protein with a hemaglutinin tag (Ad-SMP30-HA). Ad-SMP30-HA transduced the expression of SMP30-HA and two additional forms of SMP30 with molecular sizes ～28 kDa and 24 kDa in HEK-293A and C3A liver cells in a dose and time-dependent manner. Intravenous administration of Ad-SMP30-HA in mice results in the expression of all the three forms of SMP30 in the liver and diaphragm. LC-MS/MS results confirmed that the lower molecular weight 28 kDa and 24 kDa proteins are related to the 34 kDa SMP30. The 28 kDa and 24 kDa SMP30 forms were also detected in normal rat liver and mice injected with Ad-SMP30-HA suggesting that SMP30 does exist in multiple forms under physiological conditions. Time course experiments in both cell lines suggest that the 28 kDa and 24 kDa SMP30 forms are likely generated from the 34 kDa SMP30. Interestingly, the 28 kDa and 24 kDa SMP30 forms appeared initially in the cytosol and shifted to the particulate fraction. Studies using small molecule inhibitors of proteolytic pathways revealed the potential involvement of β and γ-secretases but not calpains, lysosomal proteases, proteasome and caspases. This is the first report describing the existence of multiple forms of SMP30, their preferential distribution to membranes and their generation through proteolysis possibly mediated by secretase enzymes.     

Cholic acid modified 2 kDa polyethylenimine as efficient transfection agent

New lipopolymers were synthesized by conjugating cholic acid (ChA) to polyethylenimines (PEI; 2 and 25 kDa) and a polyallylamine (PAA; 15 kDa) via N‐acylation to develop effective gene delivery systems. The extent of ChA substitution linearly varied with the feed ratio during synthesis, indicating good control over grafting ratio. While ChA did not affect binding to plasmid DNA (pDNA) for higher molecular weight (MW) polymers, ChA substitution to 2 kDa PEI significantly affected the pDNA binding. Toxicity of the 2 kDa PEI was unaffected by ChA substitution, but it was improved for the higher MW polymers. Using immortal 293T cells and primary cord blood‐derived mesenchymal stem cells, low MW (2 kDa) PEI was shown to display much better transfection efficiency as a result of ChA substitution, unlike the higher MW polymers. We conclude that ChA could be a suitable substituent for non‐toxic (low MW) PEIs in order to improve their transfection efficiency. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1337–1341, 2013     

The impacts of cyanobacteria on pulp-and-paper wastewater toxicity and biodegradation of wastewater contaminants

This study investigated the effects of cyanobacteria from pulp-and-paper waste-treatment systems on biological toxicity removal and biodegradation of certain wastewater contaminants. In field and batch studies, using the Microtox assay, cyanobacterial biomass and final wastewater toxicity were significantly correlated. In softwood-based wastewater, a decrease in toxicity was negatively correlated with cyanobacterial biomass, but the correlation was positive in hardwood-based wastewater. In the softwood-based wastewater, toxicity remained higher in the light than it was in the dark, whereas in hardwood-based wastewater, toxicity was lower in the light than it was in the dark. All of these results were light-dependent, suggesting that the photosynthetic growth of cyanobacteria is required to induce significant effects. When grown in mixed cultures with bacterial degraders, cyanobacteria from pulp-and-paper waste-treatment systems generally impeded the biodegradation of the wastewater contaminants phenol and dichloroacetate (DCA). However, there was one case where the cyanobacterium Phormidium insigne improved the bacterial degradation of DCA. Doubling inorganic nutrient concentrations did not improve phenol or DCA biodegradation in the majority of cases, indicating that nutrient competition is not a major factor. These data suggest that cyanobacteria play an important role during the biological treatment of contaminants, and, hence, toxicity removal in pulp-and-paper waste-treatment systems.     

Phenylphosphate synthase: a new phosphotransferase catalyzing the first step in anaerobic phenol metabolism in Thauera aromatica

The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via para-carboxylation of phenol (biological Kolbe-Schmitt carboxylation). In the first step, phenol is converted to phenylphosphate which is then carboxylated to 4-hydroxybenzoate in the second step. Phenylphosphate formation is catalyzed by the novel enzyme phenylphosphate synthase, which was studied. Phenylphosphate synthase consists of three proteins whose genes are located adjacent to each other on the phenol operon and were overproduced in Escherichia coli. The promoter region and operon structure of the phenol gene cluster were investigated. Protein 1 (70 kDa) resembles the central part of classical phosphoenolpyruvate synthase which contains a conserved histidine residue. It catalyzes the exchange of free [(14)C]phenol and the phenol moiety of phenylphosphate but not the phosphorylation of phenol. Phosphorylation of phenol requires protein 1, MgATP, and another protein, protein 2 (40 kDa), which resembles the N-terminal part of phosphoenol pyruvate synthase. Proteins 1 and 2 catalyze the following reaction: phenol + MgATP + H(2)O-->phenylphosphate + MgAMP + orthophosphate. The phosphoryl group in phenylphosphate is derived from the beta-phosphate group of ATP. The free energy of ATP hydrolysis obviously favors the trapping of phenol (K(m), 0.04 mM), even at a low ambient substrate concentration. The reaction is stimulated severalfold by another protein, protein 3 (24 kDa), which contains two cystathionine-beta-synthase domains of unknown function but does not show significant overall similarity to known proteins. The molecular and catalytic features of phenylphosphate synthase resemble those of phosphoenolpyruvate synthase, albeit with interesting modifications.     

Monitoring bioremediation in creosote-contaminated soils using chemical analysis and toxicity tests

Three soils with a history of creosote contamination (designated NB, TI and AC) were treated in bench-scale microcosms using conditions (nutrient amendment, moisture content and temperature) which had promoted mineralization of ¹⁴C-pyrene in a preliminary study. Bioremediation was monitored using the solid-phase Microtox test, seed germination and earthworm survival assays, SOS-chromotest, Toxi-chromotest and a red blood cell (RBC) haemolysis assay. Contaminant concentrations in the AC soil did not change after 150 days. Polycyclic aromatic hydrocarbon (PAH) concentrations decreased in the NB soil, and toxicity decreased overall according to the earthworm, seed germination and Microtox tests. Although total petroleum hydrocarbons (TPHs) in the TI soil were reduced following treatment, results of the earthworm, seed germination, RBC and Microtox tests suggested an initial increase in toxicity indicating that toxic intermediary metabolites may have formed during biodegradation. Toxicity testing results did not always correlate with contaminant concentrations, nor were the trends indicated by each test consistent for any one soil. Each test demonstrated a different capacity to detect reductions in soil contamination. Journal of Industrial Microbiology & Biotechnology (2000) 24, 132–139. Received 14 June 1999/ Accepted in revised form 12 November 1999     

Energy substrate utilization in the common brushtailed possum (Trichosurus vulpecula) using intravenous tolerance tests

The aim of this study was to investigate the energy substrate requirements of the common brushtailed possum (Trichosurus vulpecula) using intravenous tolerance tests for glucose, alanine, and propionate in five adult male and female animals under standardized conditions. Significant differences (p<0.01) were observed for fasting blood glucose values between males (6.3±0.16 mmol L(-1)) and females (4.8±0.13 mmol L(-1)), and males had a significantly (p<0.001) increased response to glucose. All animals returned to fasting glucose levels within 120 min after the glucose challenge. No significant change in blood glucose levels was observed for either the alanine or propionate tolerance tests (p>0.05). However, following propionate administration, there was a highly significant (p<0.001) decrease in blood lactate concentrations over 120 min. There was no evidence of ketone formation using β-hydroxybutyrate as a biomarker during any of the tests, indicating that there was no significant switch to lipolysis. In conclusion, the study provides new information on energy substrate utilization in this species and has identified that a gluconeogenic response normally identified in other species is not apparent in the common brushtailed possum.     

Predicting Ligand Binding Affinity with Alchemical Free Energy Methods in a Polar Model Binding Site

We present a combined experimental and modeling study of organic ligand molecules binding to a slightly polar engineered cavity site in T4 lysozyme (L99A/M102Q). For modeling, we computed alchemical absolute binding free energies. These were blind tests performed prospectively on 13 diverse, previously untested candidate ligand molecules. We predicted that eight compounds would bind to the cavity and five would not; 11 of 13 predictions were correct at this level. The RMS error to the measurable absolute binding energies was 1.8 kcal/mol. In addition, we computed “relative” binding free energies for six phenol derivatives starting from two known ligands: phenol and catechol. The average RMS error in the relative free energy prediction was 2.5 kcal/mol (phenol) and 1.1 kcal/mol (catechol). To understand these results at atomic resolution, we obtained x-ray co-complex structures for nine of the diverse ligands and for all six phenol analogs. The average RMSD of the predicted pose to the experiment was 2.0 Å (diverse set), 1.8 Å (phenol-derived predictions), and 1.2 Å (catechol-derived predictions). We found that predicting accurate affinities and rank-orderings required near-native starting orientations of the ligand in the binding site. Unanticipated binding modes, multiple ligand binding, and protein conformational change all proved challenging for the free energy methods. We believe that these results can help guide future improvements in physics-based absolute binding free energy methods.