Effects of p-Nitrophenol and Organophosphorous Nitroaromatic Insecticides on the Respiratory Activity of Free and Immobilized Cells of Strains C-11 and BA-11 of Pseudomonas putida

The possibility of using the respiratory activity (RA) of microbial cells (of strains C-11 and BA-11 of Pseudomonas putida) as an instrument for quantitative determination of organophosphorous nitroaromatic insecticides, metaphos and Sumithion, and their hydrolysis product, p-nitrophenol (PNP), has been explored. The dependences of RA on the concentrations of the three compounds were linear within the range 0.5–2.5 mM. The cells of the strain BA-11 exhibited maximum selectivity in the determination of the compounds. The RAs of microbial cells differing in the modes of immobilization (adsorption to carrier surfaces vs. incorporation into gels) have been compared. Prospects of development of the microbial cell-based sensor system for determining metaphos, Sumithion, and PNP in aqueous media are discussed.     

Determination of organophosphorus aromatic nitro insecticides and p-nitrophenol by microbial-cell respiratory activity

We examined the possibility of measuring the organophosphorus aromatic nitro insecticides metaphos and sumithion as well as their hydrolysis product p-nitrophenol (PNP) by the specific respiratory activity (SRA) of Pseudomonas putida C-11, P. putida BA-11, and Acinetobacter calcoaceticum A-122. The plots of cellular SRA against the two insecticides and PNP were linear over the ranges of 0.5-2.5 microM for P. putida C-11 and BA-11 and 0.5-1.0 microM for A. calcoaceticum A-122. P. putida BA-11 showed the greatest respiratory-response selectivity in the determination of the test substrates. We made comparison studies of the SRA of cells immobilised by two methods: carrier-surface adsorption and inclusion in various gels. We discuss the feasibility of developing a microbial sensor system for the determination of metaphos, sumithion, and PNP in aqueous media.     

An investigation into the electro-physical characteristics of microbial cells during the metabolism of toxic compounds under conditions of limited O2 availability

The purpose of the work reported here was to experimentally clarify the interconnection between changes in the electro-physical characteristics of microbial suspensions and processes of the metabolism of certain toxic compounds (acrylamide and p-nitrophenol (PNP)) in cells containing enzyme systems of the initial metabolism of these compounds. In this work, we used cells of two strains, Acinetobacter calcoaceticum A-122 and Brevibacterium sp. 13PA, which are capable of utilising PNP and acrylamide, respectively, as the sole source of carbon. Suspensions of these cells exhibited appreciable decreases in the magnitude of the electro-optical signal when the microbial metabolism of PNP and acrylamide was inhibited under conditions of limited O2 supply. This attests to a relationship between the electro-physical characteristics of microbial suspensions and cellular metabolic reactions and also to the negligibly small effect that the non-specific interaction of substrate with the cells has on the suspensions' electro-optical properties.     

One carbon metabolism in SAR11 pelagic marine bacteria

The SAR11 Alphaproteobacteria are the most abundant heterotrophs in the oceans and are believed to play a major role in mineralizing marine dissolved organic carbon. Their genomes are among the smallest known for free-living heterotrophic cells, raising questions about how they successfully utilize complex organic matter with a limited metabolic repertoire. Here we show that conserved genes in SAR11 subgroup Ia (Candidatus Pelagibacter ubique) genomes encode pathways for the oxidation of a variety of one-carbon compounds and methyl functional groups from methylated compounds. These pathways were predicted to produce energy by tetrahydrofolate (THF)-mediated oxidation, but not to support the net assimilation of biomass from C1 compounds. Measurements of cellular ATP content and the oxidation of (14)C-labeled compounds to (14)CO(2) indicated that methanol, formaldehyde, methylamine, and methyl groups from glycine betaine (GBT), trimethylamine (TMA), trimethylamine N-oxide (TMAO), and dimethylsulfoniopropionate (DMSP) were oxidized by axenic cultures of the SAR11 strain Ca. P. ubique HTCC1062. Analyses of metagenomic data showed that genes for C1 metabolism occur at a high frequency in natural SAR11 populations. In short term incubations, natural communities of Sargasso Sea microbial plankton expressed a potential for the oxidation of (14)C-labeled formate, formaldehyde, methanol and TMAO that was similar to cultured SAR11 cells and, like cultured SAR11 cells, incorporated a much larger percentage of pyruvate and glucose (27-35%) than of C1 compounds (2-6%) into biomass. Collectively, these genomic, cellular and environmental data show a surprising capacity for demethylation and C1 oxidation in SAR11 cultures and in natural microbial communities dominated by SAR11, and support the conclusion that C1 oxidation might be a significant conduit by which dissolved organic carbon is recycled to CO(2) in the upper ocean.     

Explanations for the acclimation period preceding the mineralization of organic chemicals in aquatic environments

A study was conducted of possible reasons for acclimation of microbial communities to the mineralization of organic compounds in lake water and sewage. The acclimation period for the mineralization of 2 ng of p-nitrophenol (PNP) or 2,4-dichlorophenoxyacetic acid per ml of sewage was eliminated when the sewage was incubated for 9 or 16 days, respectively, with no added substrate. The acclimation period for the mineralization of 2 ng but not 200 ng or 2 micrograms of PNP per ml was eliminated when the compound was added to lake water that had been first incubated in the laboratory. Mineralization of PNP by Flavobacterium sp. was detected within 7 h at concentrations of 20 ng/ml to 2 micrograms/ml but only after 25 h at 2 ng/ml. PNP-utilizing organisms began to multiply logarithmically after 1 day in lake water amended with 2 micrograms of PNP per ml, but substrate disappearance was only detected at 8 days, at which time the numbers were approaching 10(5) cells per ml. The addition of inorganic nutrients reduced the length of the acclimation period from 6 to 3 days in sewage and from 6 days to 1 day in lake water. The prior degradation of natural organic materials in the sewage and lake water had no effect on the acclimation period for the mineralization of PNP, and naturally occurring inhibitors that might delay the mineralization were not present. The length of the acclimation phase for the mineralization of 2 ng of PNP per ml was shortened when the protozoa in sewage were suppressed by eucaryotic inhibitors, but it was unaffected or increased if the inhibitors were added to lake water.(ABSTRACT TRUNCATED AT 250 WORDS)     

Explanations for the acclimation period preceding the mineralization of organic chemicals in aquatic environments.

A study was conducted of possible reasons for acclimation of microbial communities to the mineralization of organic compounds in lake water and sewage. The acclimation period for the mineralization of 2 ng of p-nitrophenol (PNP) or 2,4-dichlorophenoxyacetic acid per ml of sewage was eliminated when the sewage was incubated for 9 or 16 days, respectively, with no added substrate. The acclimation period for the mineralization of 2 ng but not 200 ng or 2 micrograms of PNP per ml was eliminated when the compound was added to lake water that had been first incubated in the laboratory. Mineralization of PNP by Flavobacterium sp. was detected within 7 h at concentrations of 20 ng/ml to 2 micrograms/ml but only after 25 h at 2 ng/ml. PNP-utilizing organisms began to multiply logarithmically after 1 day in lake water amended with 2 micrograms of PNP per ml, but substrate disappearance was only detected at 8 days, at which time the numbers were approaching 10(5) cells per ml. The addition of inorganic nutrients reduced the length of the acclimation period from 6 to 3 days in sewage and from 6 days to 1 day in lake water. The prior degradation of natural organic materials in the sewage and lake water had no effect on the acclimation period for the mineralization of PNP, and naturally occurring inhibitors that might delay the mineralization were not present. The length of the acclimation phase for the mineralization of 2 ng of PNP per ml was shortened when the protozoa in sewage were suppressed by eucaryotic inhibitors, but it was unaffected or increased if the inhibitors were added to lake water.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between the splotch mutation and retinoic acid in mouse neural tube defects in vitro

The interaction between the splotch gene (Sp) and all-trans retinoic acid (RA) was investigated using cytogenetically marked Sp/+ and +/+ mouse embryos cultured in the presence of RA. Retinoic acid retarded the development of and had a teratogenic effect on mouse embryos in culture. In particular, RA had seemingly opposite effects on the posterior neural tube, inducing abnormally early fusion in some embryos and causing a dose-dependent delay in others. When the effects of RA on identified Sp/+ and +/+ embryos were compared, the only observed difference in their responses was in the degree of the delay in posterior neuropore (PNP) closure. At the end of the culture period, among the untreated control embryos, the Sp heterozygotes showed retardation of PNP closure compared to +/+ embryos. In addition, the RA treatment was found to have induced a greater delay in posterior neural tube closure in Sp/+ than in +/+ embryos. The basis for this difference in response to RA is presumed to be the retardation of PNP closure that is caused by the Sp gene in heterozygous form. The effects of the gene and the teratogen are additive and the gene carriers thus have greater mean PNP lengths at the end of culture. Since the length of the PNP is an indication of an embryo's likelihood of developing spina bifida, this provides an explanation for the observation that Sp/+ embryos are more sensitive to the spina bifida-causing effects of RA than are +/+ embryos.     

ATP hydrolysis promotes interactions between the extracellular ends of transmembrane segments 1 and 11 of human multidrug resistance P-glycoprotein

P-glycoprotein (P-gp, ABCB1) actively pumps a broad range of structurally unrelated cytotoxic compounds out of the cell. It has two homologous halves that are joined by a linker region. Each half has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD). Cross-linking studies have shown that the drug-binding pocket is at the interface between the TM domains. The two NBDs interact to form the ATP-binding sites. Coupling of ATP hydrolysis to drug efflux has been postulated to occur by conversion of the binding pocket from a high-affinity to a low-affinity state through alterations in the packing of the TM segments. TM 11 has also been reported to be important for drug binding. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for changes in the packing of TM 11 during ATP hydrolysis. We generated 350 double cysteine mutants that contained one cysteine at the extracellular end of TM11 and another cysteine at the extracellular ends of TMs 1, 3, 4, 5, or 6. The mutants were expressed in HEK293 cells and treated with oxidant in the absence or presence of ATP. Cross-linked product was not detected in SDS-PAGE gels in the absence of ATP. By contrast, cross-linked product was detected in mutants M68C(TM1)/Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/A954C(TM11), M69C(TM1)/A954C(TM11), and M69C(TM1)/ F957C(TM11) in the presence of ATP but not with ADP or AMP.PNP. These results indicate that rearrangement of TM11 may contribute to the release of drug substrate during ATP hydrolysis.     

Retinoic acid reduces p11 protein levels in bronchial epithelial cells by a posttranslational mechanism

p11 is a member of the S100 family of proteins, is the cellular ligand of annexin II, and interacts with the carboxyl region of 85-kDa cytosolic phospholipase A(2) (cPLA(2)), inhibiting cPLA(2) activity and arachidonic acid (AA) release. We studied the effect of retinoic acid (RA) on PLA(2) activity in human bronchial epithelial cells and whether p11 contributes to these effects. The addition of 10(-6) M RA resulted in reduced p11 protein levels at 4 days, with the greatest effect observed on days 6 and 7. This effect was dose related (10(-6) to 10(-9) M). RA treatment (10(-6) M) had no effect on cPLA(2) protein levels. p11 mRNA levels were unchanged at 6 and 8 days of treatment (correlating with maximum p11 protein reduction). Treatment with RA reduced p11 levels in control cells and in cells transfected with a p11 expression vector, suggesting a posttranslational mechanism. Lactacystin (10(-6) M), an inhibitor of the human 26S proteasome, blocked the decrease in p11 observed with RA treatment. Compared with control cells (n = 3), RA-treated cells (n = 3) had significantly increased AA release after treatment with the calcium ionophore A-23187 (P = 0.006). Therefore, RA reduces p11 protein expression and increases PLA(2) activity and AA release.     

Studies on the Mode of Action of Organophosphorus Compounds

In order to further elucidate the mechanism of metabolic difference between sumithion and methylparathion, distribution of sumithion and methylparathion into several tissues, activation, that is, conversion into more toxic oxygen analogs, and degradation into non-toxic compounds were examined in vivo following the intravenous administration of the phosphorothioates to Guinea pigs and white rats. Sumioxon and methylparaoxon were detected in all tissues tested, among which lung and liver were richest in them. More sumioxon than methylparaoxon was found. Chese organophosphorus compounds were found to be decomposed to non-toxic desmethyl compounds and dimethyl phosphorothioic acid mainly in liver and kidney. From these results it seems improbable that the lower toxicity of sumithion than that of methylparathion results from the different in their rate of metabolism.     

Chromosome 11 rearrangements in various hematological neoplasias

Chromosome 11 abnormalities in leukemic bone marrow cells were observed in 14.0% of the cases of acute lymphoblastic leukemia (ALL), in 18.7% of acute myeloid leukemia (AML) cases, and in 16.7% of refractory anemia (RA) cases. Bands 11pl3, 11pl4, 11pl5 on the short arm and 11ql4, 11q21, 11q23 on the long arm of chromosome 11 were involved in these rearrangements. Rearrangements of band 11q23 were detected most often. Reciprocal translocations were found with the highest frequency, while para-and pericentic inversions and deletions, both terminal and interstitial, occurred less often. In RA cases only deletions were observed. Comparison of clinical features showed no correlation with age and major hematological indexes such as the number of blast cells in the initial period. These results show that the prognosis is poor in cases of abnormalities at both 11q21 and 11q23 in acute leukemia (AL) as well as in 11pl3 and 11pl5 in AML. This is the first observation of these phenomena.     

Expression of adipogenesis markers in a murine stromal cell line treated with 15-deoxy Delta(12,14)-prostaglandin J2, interleukin-11, 9-cis retinoic acid and vitamin K2.

Recent studies have demonstrated that bone marrow stromal cells can undergo adipogenesis or osteoblastogenesis in vivo, and in vitro, and that peroxisome proliferator-activated receptor gamma (PPAR gamma) plays a central role in the control of adipocyte differentiation. In the present study, we treated a murine stromal cell line (TMS-14) with a cocktail of dexamethasone, insulin and glucose (DIG cocktail), which caused the cells to convert to fat-laden cells with adipocyte-like morphology. We also exposed TMS-14 cells to DIG cocktail followed by 15-deoxy Delta(12,14)-prostaglandin J2 (15d-PGJ2), a ligand of PPAR gamma, interleukin- 11 (IL-11), 9-cis retinoic acid (9-cis RA) and vitamin K2. 15d-PGJ2 enhanced DIG cocktail-induced adipogenesis, whereas IL-11, 9-cis RA and vitamin K2 each inhibited adipogenesis induced by DIG cocktail. The gene expressions of four adipogenesis markers, PPAR gamma 2, adipocyte P2 (aP2), adipocyte determination and differentiation factor 1 (ADD1), and fatty acid synthase (FAS) were enhanced by DIG cocktail and these expressions were more enhanced by 15d-PGJ2, in contrast they were attenuated by 9-cis RA. IL-11 also attenuated the adipogenesis markers except ADD1. Western blotting showed that 15d-PGJ2 enhanced the levels of PPAR gamma, C/EBP alpha and RXR alpha proteins, while IL-11 and 9-cis RA decreased the level of PPAR gamma protein, but not C/EBP alpha protein and vitamin K2 decreased the level of C/EBP alpha protein. We also tested the effect of 15d-PGJ2 on osteoblastogenesis, using TMS-12 cells, another stromal cell clone from the same mouse, which differentiate into osteoblasts spontaneously. 15d-PGJ2 did not affect osteoblastogenesis, as detected by von Kossa staining and Cbfa-1 gene expression. These data indicate that 15d-PGJ2 enhances the expression of both PPAR gamma and C/EBP alpha and as a result it stimulates adipogenesis in murine bone marrow cells.     

Gender-specific metabolism of benz[a]anthracene in hepatic microsomes from Long-Evans and Hooded Lister rats

The cytochrome P450 isoforms responsible for the regio-selective metabolism of benz[a]anthracene (BA) are poorly defined but as with other polycyclic aromatic hydrocarbons (PAHs) may include members of the CYP2C sub-family. Since the expression of some of these is regulated in a gender-specific manner and may be altered by age, rat strain or by phenobarbital treatment, the effects of these variables on metabolism of BA to diols was investigated. These studies used hepatic, microsomal membranes from immature and adult Long-Evans rats and adult Hooded Lister rats. BA-diols were resolved by normal phase HPLC into three discrete peaks identified as benz[a]anthracene-5,6-diol (BA-5,6-diol), benz[a]anthracene-10, 11-diol (BA-10,11-diol) and a mixture of benz[a]anthracene-3,4- and -8,9-diols (BA-3,4-diol and BA-8,9-diol and termed Peak(3/8)). Significant gender-related differences were found in the rates of diol formation in adults of both the Long-Evans and Hooded Lister rat strains. Formation of BA-10,11-diol and to a lesser extent the components of Peak(3/8) were greater in the male compared to female animals by factors of at least 14 and two, respectively. An age-dependent effect is also observed in the Long-Evans rat since these differences are still apparent in prepubertal animals but to a lesser extent (gender ratio male:female BA-10,11-diol 9X; Peak(3/8) 1.4X). In contrast BA-5,6-diol was formed at similar rates by membranes from female and male rats whether mature (Long-Evans and Hooded Lister) or immature (Long-Evans). Phenobarbital treatment of the adult Long-Evans rats resulted in a moderate increase in the formation of each diol other than at the 10,11-position and the induction was not gender specific. The rate of formation of BA-10, 11-diol was decreased in phenobarbital-treated male rats suggesting modulation of a male specific isoform. Measurement of microsomal epoxide hydrolase revealed no gender or age differences and suggests that this enzyme is not rate limiting in BA-diol formation and thus is not responsible for the differences in BA-diol formation observed. The results suggest that CYP2C11 along with a male-specific isoenzyme not regulated by age are important in the formation of BA-10,11-diol and a component(s) of Peak(3/8) in males. CYPs 2B2 and/or 2C6 appear to be involved in formation of BA-5,6-diol in male and female. Identification of the CYPs involved in the regio-selective metabolism of BA may lead to an explanation of the lower carcinogenic potency of this PAH compared to dimethylbenz[a]anthracene and this study provides novel clues concerning the identities of the CYPs, which are important.     

Studies on the Mode of Action of Organophosphorus Compounds

For the purpose of distinguihsing sumithion from methylparathion in the mammalian metabolism, phosphorus³² labeled compounds were administered to Guinea pig and white rat. Both compounds were found to be absorbed readily, and phosphorus containing metabolites excreted chiefly into urine. By chromatographic separation and identification of the metabolites, the decomposition of sumithion was observed to proceed presumably more easily than methylparathion. From these results lower toxicity of the former toward mammals than the latter was discussed. In addition, compounds remaining in rice plant and German cockroach were also analysed.     

Chromosome 11 rearrangements in the different haematological neoplasias

The cases of chromosome 11 abnormalities in leukemic bone marrow cells have constituted 14.0% in acute lymphoblastic leukemia (ALL), 18.7% in acute myeloid leukemia (AML), and 16.7% in refractory anemia (RA). The bands of the short arms 11p13, 11p14, llp15 and the long arms 11q14, 11q21, 11q23 were involved in chromosome rearrangements. The rearrangements of the band 11q23 were detected more often. Reciprocal translocations were found with the highest frequency, while para- and pericentic invertions, terminal and intestitial deletions occured with the lower incidence. Deletions were found in RA cases only. Comparison with the clinical features showed no correlation with the age and the main haematological indexes including the amount of blast cells in the initial period. The results have showed the poor prognosis of the abnormalities not only of 11q21, 11q23 in acute leukemia (AL), but of 11p13, 11p15 in AML as well, while not enough data on this subject is availalbe in the literature.     

A method of acoustic analysis for detection of bacteriophage-infected microbial cells

The dependence of the changes of physical parameters of the suspension of Azospirillum brasilense Sp7 cells infected by FAb-Sp7 bacteriophage on their number and exposure time was studied using a biological sensor based on a piezoelectric resonator with a lateral electric field. The change in the value of the analytical signal was recorded at 1 minute from the beginning of the infection of the cells by bacteriophage. The selectivity of the action of the FAb-Sp7 bacteriophage was studied for Azospirillum brasilense (strains Cd, Sp107, Sp245, Jm6B2, Br14, KR77, S17, S27, SR55, and SR75), A. lipoferum (strains Sp59b, SR65, and RG20a), A. halopraeferans Au4, Nitrospirillum amazonense Am14, Niveispirillum irakense (strains KBC1 and KA3) bacteria, as well as for heterologous bacteria of the genera Escherichia coli (strains XL-1 and B-878), Pseudomonas putida (strains C-11 and BA-11), and Acinetobacter calcoaceticum A-122. The limit of the reliable determination of the concentration of microbial cells during bacteriophage infection process was found: ~10⁴ cells/mL. At the same time, the presence of heterologous cell cultures (E. coli XL-1 cells) did not complicate the detection. It was shown that the method of electroacoustical analysis of cell suspensions can be used for the detection of microbial cells of Azospirillum infected by the FAb-Sp7 bacteriophage. The results are promising for the development of methods for determining and controlling the number of soil microorganisms.     

A novel biosensor based on Lactobacillus acidophilus for determination of phenolic compounds in milk products and wastewater

Different branches of industry need to use phenolic compounds (PCs) in their production, so determination of PCs sensitively, accurately, rapidly, and economically is very important. For the sensitive determination of PCs, some biosensors based on pure polyphenol oxidase, plant tissue and microorganisms were developed before. But there has been no study to develop a microbial phenolic compounds biosensor based on Lactobacillus species, which contain polyphenol oxidase enzyme. In this study, we used different forms of Lactobacillus species as enzyme sources of biosensor and compared biosensor performances of these forms for determination of PCs. For this purpose, we used lyophilized Lactobacillus cells (containing L. bulgaricus, L. acidophilus, Streptococcus thermophilus), pure L. acidophilus, pure L. bulgaricus, and L. acidophilus- and L. bulgaricus adapted to catechol in Lactobacilli MRS Broth. The most suitable form was determined and optimization studies of the biosensor were carried out by using this form. For preparing the bioactive layer of the biosensor, the Lactobacillus cells were immobilized in gelatin by using glutaraldehyde. In the study, we used catechol as a substrate. Phenolic compound determination is based on the assay of the differences on the respiration activity of the cells on the oxygen meter in the absence and the presence of catechol. The microbial biosensor response depends directly on catechol concentration between 0.5 and 5.0 mM with 18 min response time. In the optimization studies of the microbial biosensor the most suitable microorganism amount was found to be 10 mg, and also phosphate buffer (pH 8.0; 50 mM) and 37.5 °C were obtained as the optimum working conditions. In the characterization studies of the microbial biosensor some parameters such as substrate specificity on the biosensor response and operational and storage stability were examine. Furthermore, the determination of PC levels in synthetic wastewater, industrial wastewater, and milk products was investigated by using the developed biosensor under optimum conditions.     

Comparison of p-Nitrophenol Biodegradation in Field and Laboratory Test Systems

Acclimation of microbial communities exposed to p-nitrophenol (PNP) was measured in laboratory test systems and in a freshwater pond. Laboratory tests were conducted in shake flasks with water, shake flasks with water and sediment, eco-cores, and two sizes of microcosm. The sediment and water samples used in the laboratory experiments were obtained from the pond. After a 6-day acclimation period, PNP was biodegraded rapidly in the pond. When the pond was treated with PNP a second time, biodegradation began immediately. The acclimation periods in laboratory test systems that contained sediment were similar to that in the pond. The acclimation period was threefold longer in shake flasks without sediment. PNP was biodegraded more slowly by microbial communities acclimated in the laboratory than it was in the pond, and the rate of biodegradation varied with the type of test. The number of bacteria able to mineralize PNP increased by 3 orders of magnitude in the pond during the acclimation period. Similar increases accompanied acclimation in the laboratory systems.     

Effects of organic compounds on the degradation ofp-nitrophenol in lake and industrial wastewater by inoculated bacteria

Many microorganisms fail to degrade pollutants when introduced in different natural environments. This is a problem in selecting inocula for bioremediation of polluted sites. Thus, a study was conducted to determine the success of four inoculants to degradep-nitrophenol (PNP) in lake and industrial wastewater and the effects of organic compounds on the degradation of high and low concentrations of PNP in these environments.Corynebacterium strain Z4 when inoculated into the lake and wastewater samples containing 20 µg/ml of PNP degraded 90% of PNP in one day. Addition of 100 µg/ml of glucose as a second substrate did not enhance the degradation of PNP and the bacterium utilized the two substrates simultaneously. Glucose used at the same concentration (100 µg/ml), inhibited degradation of 20 µg of PNP in wastewater byPseudomonas strain MS. However, glucose increased the extent of degradation of PNP byPseudomonas strain GR. Phenol also enhanced the degradation of PNP in wastewater byPseudomonas strain GR, but had no effect on the degradation of PNP byCorynebacterium strain Z4.Addition of 100 µg/ml of glucose as a second substrate into the lake water samples containing low concentration of PNP (26 ng/ml) enhanced the degradation of PNP and the growth ofCorynebacterium strain Z4. In the presence of glucose, it grew from 2×10⁴ to 4×10⁴ cells/ml in 3 days and degraded 70% of PNP as compared to samples without glucose in which the bacterium declined in cell number from 2×10⁴ to 8×10³ cells/ml and degraded only 30% PNP. The results suggest that in inoculation to enhance biodegradation, depending on the inoculant, second organic substrate many play an important role in controlling the rate and extent of biodegradation of organic compounds.Abbreviations PNP p-nitrophenol