Microbial Production of Long-chain Dicarboxylic Acids from n-Alkanes

Microorganisms which produced n-alkane ω,ω′-dicarboxylic acid (DC) from n-alkane were selected from natural sources. It was found that the best three producers thus obtained belonged to yeast. All of the stock cultures which are able to assimilate n-alkane and are belonged to genus Candida and Pichia were also found to produce DC from n-alkane.

Candida cloacae 310, a representative strain selected from natural source, was able to produce DCs having 5 to 16 carbon atoms from various n-alkanes. Among them, DCs with 5 to 9 carbon atoms were more heavily accumulated than those with more than 9, except those with the same number of carbon atoms as the substrates which were the main products from the substrates with less than 15 carbon atoms. It was also clearly demonstrated that DCs with odd carbons alone were produced from n-alkanes with odd carbons, while DCs with even carbons alone from n-alkanes with even carbons.

Then, cultural conditions of Candida cloacae 310 were studied for the production of DC-12 from n-dodecane (n-C₁₂) which showed the highest yield among the observed accumulation.

Under the optimum conditions, 2.28 g/liter of DC-12 was obtained together with 1.86 g/liter of DC-6 and 0.82 g/liter of DC-8 after 72 hr’ cultivation in a synthetic medium containing 100 ml of n-C₁₂ per liter.     

Microbial transformation of hydrocarbons. II. Growth constants and cell composition of microbial cells derived from n-alkanes

Cultivation of Norcardia sp., Mycobacterium phlei, and Candida lipolytica in inorganic salt solution containing n-alkanes C₁₀–C₂₀ as solo carbon and energy source was investigated. Generation times of 0.5–7.0 hr were typical during the exponential growth phase. The final cell concentrations (dry weight) were usually 9–26 g/l with n-alkane mixtures ranging from n-decane through n-eicosane. A linear dependence was found between the production of cell mass and the consumption of n-alkanes. The rest concentration of n-alkanes in the cell mass is in all experiments smaller than 0.5% (w/w). Cell yields were Y_sub 60–142% and for Y_e 50–97% based on n-alkane utilization. In one case, with the Nocardia NBZ 23, the substrate specifity on hydrocarbons and on a n-alkane mixture C₁₀-C₂₀ was studied. The cell mass recovered from the fermentations contained 47.8–57.7% carbon, 5.6–9.95% nitrogen, 7.2–9.4% hydrogen, 35–62% crude protein, and 6–36% lipid. Cellular protein and lipid synthesized by an organism is influenced by the type of nitrogen source. The amino acid, glucosamine, muramic acid, 2,6-diaminopimelinic acid, and fatty acid distribution in organisms grown on n-alkanes compared with a corresponding fermentation on glucose as sole carbon source were also estimated.     

Microbial Production of Long-chain Dicarboxylic Acids from n-Alkanes

Strain M-l which was derived from Candida cloacae 310 as a mutant unable to assimilate dicarboxylic acid (DC) produced large amount of DCs from n-alkanes, as expected. It produced DCs with the same number of carbon atoms as those of n-alkanes used (9 to 18 carbon atoms). Among DCs produced, n-tetradecane ω,φ′-dicarboxylic acid (DC-16) from n-hexadecane (n-C₁₆) was most abundantly accumulated and the highest level of DC-16, i.e., 29.3g/liter was obtained by resting cells.

On the other hand, since the growth rate of strain M-l on n-alkane markedly decreased in comparison with that of the parent strain, other carbon source which supported the growth of strain M-l was necessary for the production of DC from n-alkane by growing cells. When acetic acid was used as carbon source for the growth in DC-16 production from n-C₁₆, the highest level of DC-16, i.e., 21.8 g/liter was obtained ofter 3 days' cultivation.     

Production of Inosine from Hydrocarbons by Corynebacterium petrophilum

Adenine-auxotrophic mutants were derived from Corynebacterium petrophilum SB 4082 by ultraviolet irradiation. The isolation of this auxotroph was carried out as follows; the adenine-auxotrophic mutants which had been derived by the UV irradiation were concentrated with penicillin. The auxotrophs were raised in concentration by recycling procedure and were separated by the ordinary method. The resultant adenine-auxotroph was cultured in the hydrocarbon medium. From its filtrate was obtained a fermentation product as crystals by means of ion-exchange resin and identified as inosine from absorption spectra and other properties.

The effects of cultural conditions on inosine production were investigated by the adenine auxotroph of Corynebacterium petrophilum SB 4082. That, the amount of adenine in the medium was very important on the inosine formation, was cleared. The addition of 10 mg of adenine and 0.5 g of yeast extract to 100 ml of medium was the best for the inosine formation. Ammonium chloride or ammonium sulphate was effective as nitrogen sources. As the carbon sources, n-C₁₀ to n-C₁₆ were utilized for the growth, but the hydrocarbons from n-C₁₂ to n-C₁₆ were the most suitable for the inosine formation. The inosine fermentation began at 24 hrs. after inoculation. The accumulation of inosine attained to the highest level after five days, the amount of which was 1.6 g per liter of the culture filtrate.     

Flocculant production from kerosene

Cultivation of Corynebacterium hydrocarboclastus, which is capable of synthesizing an extracellular polymer and utilized hydrocarbons, has been reported. Growth studies in shake flasks and fermenters were made to obtain maximum polymer production. Polymer formation was found to be growth associated. The highest level of polymer accumulation was attained after 50–60 hr cultivation in the fermenter and it amounted to approximately 5.5–6 g/liter of fermentation broth. The medium contained initially 2% (v/v) kerosene as a carbon source. The maximum yield obtained corresponds to 37–40% (w/w) of kerosene supplied. At the same time the cell concentration was 10–13 g/liter which represents the yield of 67–87% (w/w). The rate of polymer production in the exponential phase was 0.25 g/liter hr and cell production rate was 0.27 g/liter hr. Sodium nitrate, 0.5%, and yeast extract, 0.3%, (w/w) were the best nigrogen sources for polymer formation. The highest level of polymer produced in broth was 6 g/liter.     

Culture condition ofPseudomonas aeruginosa F722 for biosurfactant production

Pseudomonas aeruginosa F722 produces a biosurfactant (BS) during its degradation of carbon and hydrocarbon compounds. The culture conditions for upgrading the biosurfactant productivity were investigated. The concentration of the biosurfactant produced byP. aeruginosa F722 was 0.78 g/L in C-medium; however, this increased to 1.66 g/L in BS medium, which was experimentally adjusted to optimal conditions. NaNO₂ was found to be most effective for microbial growth, with an O.D_600nm of 1.18 for 0.1% NaNO₂. Microbial growths, according to the O.D_600nm were 2.53, 2.68, 2.89, and 2.87 for glucose, glycerol,n-C₁₀, andn-C₂₂, respectively. Clear zone diameters (cm), indicating biosurfactant activity, were 9.0, 8.8, 5.7, and 8.5 for glucose, glycerol,n-C₁₀, andn-C₂₂, respectively. Microbial growth was not consistent with the biosurfactant activity. The best biosurfactant activity was found with a C/N ratio of 20. Under optimal culture condition, the average surface tension decreased from 70 to 30 mN/m after 5 days. With aeration of 1.0 vvm, the biosurfactant produced increased to 1.94 g/L (up to 20%) compared to that of 1.66 g/L with no aeration. With aeration, the velocities of glucose degradation during both the log and stationary growth phases increased from 0.25 and 0.18 h⁻¹ to 0.33 and 0.29 h⁻¹, respectively, and the time for the culture to arrive at the maximum clear zone diameter became shorter, from 80 down to 60 h with no aeration.     

Production of L-glutamic acid from hydrocarbons: Incorporation of molecular oxygen

The production of L -glutamic acid from hydrocarbons by a newly isolated bacterium, which was identified as Corynebacterium, was investigated. The outstanding characteristic of this bacterium was found to be an accessory requirement of thiamine for growth. The optimum concentration of thiamine for growth was 50 μg./liter, while that for L -glutamic acid production was 3–5 μg./liter. n-Paraffins ranging from dodecane to heptadecane were best for L -glutamic acid production, and about 5 g. of L -glutamic acid were obtained from 30 g. of these individual n-paraffins. On the other hand, a tracer experiment using oxygen-18 revealed that molecular oxygen was incorporated into L -glutamic acid produced from dodecane. Based on the incorporation value of molecular oxygen in L -glutamic acid, a hypothetical pathway for the biosynthesis of L -glutamic acid from dodecane was discussed.     

Production of Dicarboxylic Acids by Candida cloacae Mutant Unable to Assimilate n-Alkane

Strain MR-12 which was derived from Candida cloacae M-l as a mutant unable to assimilate n-alkane showed marked increase in dicarboxylic acid (DC) productivity from n-alkane.

Resting cells of strain MR-12 produced 42.7g/liter of n-tetradecane 1,14-dicarboxylic acid (DC-16) from n-hexadecane (n-C₁₆) after 72 hr’ incubation. DC degradation activities of strain M-1 and MR-12 were found to be markedly reduced and their activities against DC-16 decreased to 40% and 10% of that of the parent strain, respectively.

Strain M-1 and MR-12 produced DC from the various oxidized derivatives of n-alkane such as alcohol, diol, aldehyde, fatty acid and methyl- or ethylester of fatty acid other than n-alkane.

The carbon balance in n-C₁₆ oxidation was determined by using resting cells of strain MR-12 and about 60% of utilized carbon was recovered as DC-16 and about 40% was recovered as CO₂.     

Production of Candida utilis protein from peat extracts

Sphagnum peat extracts or hydrolysates have been obtained and used as a culture medium for the production of Candida utilis biomass as single cell proteins. Acid hydrolysis of ground peat (4–60 mesh) in an autoclave operated under a set of conditions for acid strength (0.3-1.5 (v/v) H₂SO₄), holding time (1–4 hr), temperature (100–165°C), and weight ratio of dry peat to solution (3.3–16.7 g dry peat/100 g solution) yielded carbohydrate-rich extracts of different concentrations (1–34g/liter). The best yield (mg total carbohydrate/g dry peat) was obtained for a holding time of I hr and a temperature of 152°C. Low peat concentratio (4.1 g dry peat/100 g solution)resulted in high yield(280mg total carbohydrate/gdry peat) with a corresponding low carbohydrate content in hydrolysate (13 g/liter), while a lower yield with a higher carbohydrate content (34 g/liter)in hydrolysate were found when increasing peat concentration (16.7 g dry peat/100 g solution). Shake-fladk experiments using peat hydrolysates as the culture medium together with NH₄OH (～4.8 g/liter) and K₂HPO₄(5 g/liter) as nitrogen and phosphate supplement, respectively, gave a maximum biomass concentration of 7.5 g/liter after 60 hr at 30°C and 200rpm. Batch cultivation in a fermentor under controlled conditions for aeration (4.2 liter/min), agitation (500rpm), temperature (30°C), and pH (5.0) produced a maximum biomass of 10 g/liter after 20 hr with a specific growth rate of 0.13 hr^?1. For the continuous cultivation, a maximal biomass productivity of 1.24 g/gliter-he was obtained at a dilution rate of 0.125 hr ^?1. Monod's equation's equation has been used for the estimation of the coefficients μ_Max, K_s, and Y. It was found that the yield coefficient Y is not constant during the progress of batch cultivation.     

Differential protein expression during growth on linear versus branched alkanes in the obligate marine hydrocarbon-degrading bacterium Alcanivorax borkumensis SK2T

Alcanivorax borkumensis SK2^T is an important obligate hydrocarbonoclastic bacterium (OHCB) that can dominate microbial communities following marine oil spills. It possesses the ability to degrade branched alkanes which provides it a competitive advantage over many other marine alkane degraders that can only degrade linear alkanes. We used LC–MS/MS shotgun proteomics to identify proteins involved in aerobic alkane degradation during growth on linear (n-C₁₄) or branched (pristane) alkanes. During growth on n-C₁₄, A. borkumensis expressed a complete pathway for the terminal oxidation of n-alkanes to their corresponding acyl-CoA derivatives including AlkB and AlmA, two CYP153 cytochrome P450s, an alcohol dehydrogenase and an aldehyde dehydrogenase. In contrast, during growth on pristane, an alternative alkane degradation pathway was expressed including a different cytochrome P450, an alcohol oxidase and an alcohol dehydrogenase. A. borkumensis also expressed a different set of enzymes for β-oxidation of the resultant fatty acids depending on the growth substrate utilized. This study significantly enhances our understanding of the fundamental physiology of A. borkumensis SK2^T by identifying the key enzymes expressed and involved in terminal oxidation of both linear and branched alkanes. It has also highlights the differential expression of sets of β-oxidation proteins to overcome steric hinderance from branched substrates.     

Rhamnolipid production by a novel thermophilic hydrocarbon-degrading Pseudomonas aeruginosa AP02-1

Thermophilic bacterial cultures were isolated from a hot spring environment on hydrocarbon containing mineral salts media. One strain identified as Pseudomonas aeruginosa AP02-1 was tested for the ability to utilize a range of hydrocarbons both n-alkanes and polycyclic aromatic hydrocarbons as sole carbon source. Strain AP02-1 had an optimum growth temperature of 45°C and degraded 99% of crude oil 1% (v/v) and diesel oil 2% (v/v) when added to a basal mineral medium within 7 days of incubation. Surface activity measurements indicated that biosurfactants, mainly glycolipid in nature, were produced during the microbial growth on hydrocarbons as well as on both water-soluble and insoluble substrates. Mass spectrometry analysis showed different types of rhamnolipid production depending on the carbon substrate and culture conditions. Grown on glycerol, P. aeruginosa AP02-1 produced a mixture of ten rhamnolipid homologues, of which Rha-Rha-C₁₀-C₁₀ and Rha-C₁₀-C₁₀ were predominant. Rhamnolipid-containing culture broths reduced the surface tension to ≈28 mN and gave stable emulsions with a number of hydrocarbons and remained effective after sterilization. Microscopic observations of the emulsions suggested that hydrophobic cells acted as emulsion-stabilizing agents.     

Effect of charge density of cationic polyelectrolytes on complex formation with DNA

The interaction between DNA and ionen polymers, -[N⁺(CH₃)₂(CH₂)_mN⁺(CH₃)₂(CH₂)_n], with m-n of 3–3, 6–6, and 6–10 were examined in order to know how the binding behavior of cationic polymers with DNA depends on the charge density of polycation. The ionen polymer has no bulky side chain and the binding forces with DNA would be attributed mainly to electrostatic interaction. When 3–3 ionen polymers were added to DNA solution, precipitable complexes with the ratio of cationic residue to DNA phosphate (+/?) of 1/1 and the free DNA molecules were segregated, while 6–6 and 6–10 ionen polymers formed soluble complexes with DNA molecules up to (+/?) = 0.5. This suggests that 3–3 ionen polymers bind cooperatively with DNA while 6–6 and 6–10 ionen polymers bind noncooperatively. The cooperative binding of 3–3 ionen polymer and the noncooperative binding of 6–6 ionen polymer were also supported by the thermal melting and recooling profiles from the midpoint between first and second meltings. It was concluded that the charge density of DNA phosphate is a critical value determining whether the ionen polymers bind to DNA by a cooperative or by a noncooperative binding, since the distance between successive cationic charges of 3–3 ionen polymer is shorter than that between successive phosphate charges on DNA double helix and those of 6–6 and 6–10 ionen polymers are longer.     

Authigenic mineralization and detrital clay binding by freshwater biofilms: The Brahmani river,India

This study sought to understand the origin and fate of one of the bitumen mounds found on the bottom of Lake Baikal. These mounds are located at a depth of 900 m beneath oil spots detected on the surface of Lake Baikal (53° 18′24^″, 108° 23′20^″). The two mounds were sampled with a manipulator from a “MIR” deep-water manned submersible. Mature mound No. 8 was subjected to chemical and microbiological studies. Mound No. 3 was subjected only to chemical studies; we failed to perform microbiological analyses of this mound for logistic reasons. Oil spots collected from the water surface, samples of mound No. 3 and No. 8, were subjected to GC/MS analysis. The water contained aliphatic hydrocarbons with chains between C₈ and C₂₃, with the most abundant chain length being C₁₈. Mound No. 3 with the most abundant chain length being C₁₈ actively released oil droplets into the water. It contained 770 mg/g of C₁₃-C₃₂ n-alkanes, with a maximum at C₂₃ (160 mg/g). Mound No. 8 was inactive and contained 148 mg/g of aliphatic C₂₂-C₃₄ n-alkanes, with a maximum at C₂₅. Mound No. 8 also consisted of 3% inorganic matter, 48% unresolved complex mixture (UCM) and less than 1% other compounds (polyaromatic hydrocarbons, isoprenoids, carotenoids, and hopanes). The core of this sample used as inoculate, yielded Rhodococci when cultivated on oil as the only source of carbon. Cultivation of the sample on agar-containing Raymond inorganic medium with crude West Siberian oil as the only source of carbon revealed colonies of these bacteria, which all appeared identical. PCR was performed with DNA isolated from 5 colonies, using primers for 16S rRNA genes. Comparison of the sequences of the 5 PCR products over a length of 714 bp revealed that they were almost identical. Phylogenetic analysis of these homologous sequences showed that they were similar to the corresponding sequences of the genus Rhodococcus. Substrate demands, the morphology of the colonies, and SEM and TEM data confirmed that the isolates obtained could indeed be Rhodococci. All of the isolates could grow in bulk cultures with inorganic medium supplemented with crude oil. Moreover, all of the isolates degraded aliphatic hydrocarbons with lengths between C₁₁ and C₂₉. C₂₃-C₂₉ hydrocarbons were degraded completely. The isolates could grow at 4–37°C. The most unexpected finding was that of the many microorganisms capable of consuming oil, only Rhodococci exhibited this ability in the inactive bitumen mound. The possible mechanisms of how crude oil is transformed into bitumen mounds and mature bitumen are discussed.     

A SEAWATER MEDIUM FOR DINOFLAGELLATES AND THE NUTRITION OF CACHONINA NIEI1

Two media have been devised: an enriched seawater medium for culture of dinoflagellates and a defined medium for rapid growth of the dinoflagellate Cachonina niei. A wide range in salinity (10.23–42.38 g/liter NaCl) is tolerated by C. niei. Below 0.6 g/liter MgSO₄, 0.19 g/liter KCl, and 0.22 g/liter CaCl₂, the generation time greatly increases. Increase in MgSO₄ to 7.22 g/liter, KCl to 1.12 g/liter or CaCl₂ to 2.22 g/liter has little effect on generation time. The temperature optimum is 19–23 C. Saturating light intensity for growth is 1000 ft-c and for photosynthesis (determined manometrically) is slightly less than 2000 ft-c. Cachonina niei requires B₁₂ and thiamin. Neither silicate nor its competitive inhibitor germanate affects generation time or cell yield indicating silicon is not required. Of a variety of buffers tested, Tris is the best. Optimal growth occurs at pHs of 7.5–8.3. Glycerol is inhibitory and does not support dark growth.     

Comparative Characterization of Extracellular and Intracellular Hydrocarbons of Clostridium pasteurianum

Extracellular and intracellular hydrocarbons produced by Clostridium pasteurianum VKM 1774 during cultivation on glucose-containing media in an argon atmosphere or in the presence of carbon dioxide and molecular hydrogen were analyzed by gas-liquid chromatography. Intracellular hydrocarbons were 50-55% (C₂₅-C₃₅) n-alkanes. Carbon dioxide and molecular hydrogen stimulated synthesis of extracellular hydrocarbons, which comprised 90-95% (C₁₁-C₂₄) n-alkanes.     

Tetradecane 1,14-Dicarboxylic Acid Production from n-Hexadecane by Candida cloacae

The better condition of cultivation for tetradecane 1,14-dicarboxylic acid (DC-16) production from n-hexadecane (n-C₁₆) by Candida cloacae MR-12 was investigated by using acetic acid as carbon source for the growth. In general, the condition suitable for the growth was also favorable for the production of DC-16. The change of pH during cultivation, the use of NaOH solution as pH controlling agent after pH-change and the addition of antifoam stimulated the production of DC-16.

Under the optimum conditions where the culture medium contained 15% (v/v) n-C₁₆, 1.4% (w/v) acetic acid, inorganic salts and growth factors, and pH was changed from 6.5 to 7.75 at 16 hr after the inoculation, the highest level of DC-16 production was attained after about 72 hr cultivation and the amount of the product accumulated was 61.5 g per liter of the medium.

When a mixture of various n-alkanes was used as starting material, DCs corresponding to the respective n-alkanes were produced as mixture.     

Solid-phase microextraction and cuticular hydrocarbon differences related to reproductive activity in juniper bark borer Semanotus bifasciatus Motschulsky

Cuticular hydrocarbons of Cerambycidae species can function as signals for sex recognition. Little is known about the copulatory signals of the juniper bark borer Semanotus bifasciatus, a major economic threat to Platycladus orientalis Franco in China. Here, we investigated the cuticular hydrocarbons of both sexes of S. bifasciatus to determine the chemically mediated mating signals using the solid-phase microextraction (SPME) technique with carbowax/divinylbenzene fibers (CAR/DVB) and then analyzed by coupled gas chromatography-mass spectrometry (GC-MS). A series of aliphatic saturated straight-chain n-alkanes (n-C₂₃ to n-C₂₈), internally branched monomethylalkanes at carbons 3, 11, or 13, and dimethylalkanes were identified, which showed no qualitative differences in either sex and were similar in the samples with SPME fiber extraction and those with hexane extraction. The bioassay showed that 11-methylpentacosane (11-MeC₂₅), 11-methylhexacosane (11-MeC₂₆), and 11-methylheptacosane (11-MeC₂₇) have sex-specific recognition functions that triggered more mating attempts at a female-specific ratio of 100:4:60 than at a male-specific ratio of 100:85:50. In addition, the female-specific ratio of 11-methylalkanes can elicit about 70% of male mating attempts within about 60 s, whereas live females elicit about 98% of male mating attempts within 25 s. The discrepancy in the initiation of mating attempts by synthetic mixtures and live females suggests that the methyl isomers 3-MeC₂₅, 3-MeC₂₇, and/or 11,15-diMeC₂₇ may also be involved in the mating behavior of S. bifasciatus. These results suggest that 11-MeC₂₅, 11-MeC₂₆, and 11-MeC₂₇ constitute the contact sex pheromone of S. bifasciatus, with the presence or absence of 11-MeC₂₆ in particular playing an important role in mate recognition by males.     

Resorcinol and m-guaiacol alkylated derivatives and asymmetrical secondary alcohols in the leaves from Tamarix canariensis

We have discovered that leaves from the halotolerant plant saltcedar (Tamarix canariensis [Willd.]) are a source of resorcinols and guaiacols. Specifically, the waxes of the saltcedar leaves contained high amounts of 5-n-alkylresorcinols (ARs; 17 g/kg dry weight), 5-n-alkyl-m-guaiacols (AGs; 14 g/kg dw) and secondary alcohols (44 g/kg dw). Herein we provide the report of 5-n-alkylresorcinols with long side-alkyl chains as natural compounds in Tamaricaceae. These compounds are homologous to ones previously reported almost exclusively in cereals. The ARs span the formulas n-C₁₄ to n-C₂₇, the most abundant of which is n-C₂₁. Although the odd-C-numbered compounds dominate, there are non-negligible amounts of the even-numbered homologs. We also provide, the first-ever report of 11 homologs of 5-n-alkyl-m-guaiacols (AGs) as natural compounds from the sample plant, which we characterized as the corresponding trimethylsilyl (TMS)-ether derivatives. The AGs contain a hydroxyl group at carbon 1 of the phenolic nucleus, a methoxy group at position 3, and a (predominantly odd-C-numbered) linear alkyl chain linked to the benzene ring at position 5. They span the formulas n-C₁₃ to n-C₂₇, the most abundant of which is n-C₂₁. Finally, we also isolated from saltcedar a series of eight asymmetric secondary alcohols, whose formulas range from n-C₂₅ to n-C₃₅ and whose major homolog is n-hentriacontan-12-ol.     

Catalyses by polymer complexes. VII. Enhanced esterolytic reactivity of polymer-coenzyme A,glutathione complexes

The association of coenzyme A(CoASH) and glutathione (GSH) with the water-soluble polymers and their esterolytic reactivities were evaluated through the reaction with p-nitrophenyl acetate in the presence of cationic polymer micelles: partially laurylated poly(2-ethyl-1-vinylimidazole) and poly(4-vinylpyridine). The polymer micelles with high lauryl-group content (more than 12 mol%) markedly accelerated the reaction at very low concentrations of the polymer. Other polymers with no or small lauryl-group content only slightly enhanced the association and the reaction rate. From the rate-polymer concentration profiles, the association constants (K) and the rate constants for thiol coenzymes bound to the polymer (k′_a,bound) were determined: for polymers with more than 12 mol % lauryl-group content, K_CoASH = 1110–2270 M^?1, K_GSH = 170–503M^?1, k′_a,bound at pH 8.65 = 142–341M^?1 sec^?1. k′_a,bound were 20–340 times larger than that observed in the absence of the polymer. The logarithm of k′_a,bound was found to be correlated well with the polymer hydrophobicity, indicating that the hydrophobic environment of the polymer activated the bound thiol anions. On the other hand, the polymer hydrophobicity did not correlate with the association constant.