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     

Biodegradation of p-nitrophenol by Pseudomonas aeruginosa HS-D38 and analysis of metabolites with HPLC–ESI/MS

Pseudomonas aeruginosa strain HS-D38 was capable of mineralizing p-nitrophenol (PNP) as the sole source of carbon, nitrogen and energy. Degradation of 200 mg L^?1 PNP was examined in different media including: (i) MSM (mineral salts medium, no carbon and nitrogen source); (ii) addition of 1% ammonium chloride as additional nitrogen source (ANM); and (iii) addition of 1% glucose as a carbon source (ACM). Complete degradation of 200 mg L^?1 PNP was achieved in 12 h in MSM. Additional ammonium chloride accelerated the PNP degradation, but additional glucose inhibited this process. This strain metabolized as high concentration as 300 and 500 mg L^?1 of PNP in 14 h and 24 h, respectively, in MSM. The degradation was accompanied by release of stoichiometric amount of nitrate from PNP. During the bacterial growth on PNP, hydroquinone and 1,2,4-benzenetriol were observed as the key degradation intermediates by using a combination of techniques, including HPLC–DAD and LC–ESI/MS compared with the authentic standards. These results indicated that PNP was degraded via a hydroquinone pathway.     

Isolation and characterization of Arthrobacter sp. HY2 capable of degrading a high concentration of p-nitrophenol

A soil bacterium strain, capable of using p-nitrophenol (PNP) as its sole source of carbon and energy, was isolated by enrichment on minimal salt medium (MSM). On the basis of a phylogenetic analysis of 16S rRNA gene sequences the bacterium is a species of Arthrobacter, closely related to Arthrobacter ureafaciens DSM 20126. This strain has an unusually high substrate tolerance for PNP degradation in MSM. Greatest degradation of PNP was observed at 30 °C and under slightly alkaline pH (pH 7–9) conditions. Effective degradation rates slowed as the concentration of PNP was increased. Addition of glucose from 0.1% to 0.5% generally enhanced the degradation of PNP at high concentration (400 mg/l) although acidification as a result of glucose metabolism had a negative effect on PNP depletion. Biodegradation of PNP at high concentration was greatly accelerated by β-cyclodextrin at a concentration of 0.5%, indicating that β-cyclodextrin could be a promising addictive for effective PNP bioremediation.     

Enhancement of phenol biodegradation by Ochrobactrum sp. isolated from industrial wastewaters

Ochrobactrum sp., was tested with regard to its phenol degradation capacity at different pH levels, and with different carbon sources (mineral salt medium with glucose (MSG) and the same medium with 0.5%, 1%, and 2% (v/v) molasses (MSM)) and phenol concentrations. The highest degradation was in mineral salt medium with 1% (v/v) molasses (45.9%), while degradation was 21.1% in mineral salt medium with 5 g l⁻¹ glucose. These data show that the addition of molasses to mineral salt medium enhanced phenol degradation by Ochrobactrum sp. The bacterium can be used effectively to treat wastewaters containing phenol.     

氧化胁迫诱导苍白杆菌JP1形成VBNC状态及其特征

石油降解菌在各种有害环境因素作用下会进入活的非可培养(viable but non-culturable, VBNC)状态，从而影响其生长及石油降解率。为了研究有害环境因素对石油降解菌生长及石油降解率的影响，采用分光光度法、荧光染色-激光共聚焦显微镜观测H₂O₂胁迫下苍白杆菌(Ochrobactrum sp.)JP1细胞的生长及VBNC状态形成情况。结果表明，不同浓度H₂O₂对其生长有一定抑制作用，当培养液中H₂O₂浓度为75.0 mmol/L时，可有效抑制苍白杆菌JP1生长，处理12 h后苍白杆菌JP1进入VBNC状态。VBNC状态的苍白杆菌JP1细胞缩小变成球体，周质间隙增大；在适宜条件下，VBNC状态苍白杆菌JP1能够复苏为可培养状态，添加丙酮酸钠能够促进VBNC状态细菌细胞的复苏。复苏后的苍白杆菌RJP1具有良好的环境适应性和石油降解能力，为石油污染生物修复的菌种筛选及应用提供了新的策略。     

Degradation of p-nitrophenol by Achromobacter xylosoxidans Ns isolated from wetland sediment

Achromobacter xylosoxidans Ns strain, capable of utilizing p-nitrophenol (PNP) as the sole source of carbon, energy, and nitrogen, was isolated from wetland sediment and confirmed based on 16S rRNA gene sequence. The strain Ns could tolerate concentrations of PNP up to 1.8 mM, and degradation of PNP was achieved in 7 d at 30 °C in the dark under aerobic conditions. Biodegradation of PNP occurred quickly at an optimal pH of 7.0 and higher, and at ⩽0.5% salt (NaCl) contents. During bacterial growth on PNP, 4-nitrocatechol was observed as a key degradation intermediate using a combination of techniques, including HPLC, UV–visible spectra, and comparison with the authentic standard. In a similar way, a second degradation intermediate was identified to be 1,2,4-benzenetriol. Moreover, A. xylosoxidans Ns could also degrade 3-nitrophenol as the sole source of carbon, nitrogen, and energy, but 2-nitrophenol could not. The experimental results showed that bacteria indigenous to the wetland sediment are capable of degradading PNP and chemicals with similar structures.     

Characterization of a para-nitrophenol catabolic cluster in Pseudomonas sp. strain NyZ402 and construction of an engineered strain capable of simultaneously mineralizing both para- and ortho-nitrophenols

Pseudomonas sp. strain NyZ402 was isolated for its ability to grow on para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy, and was shown to degrade PNP via an oxidization pathway. This strain was also capable of growing on hydroquinone or catechol. A 15, 818 bp DNA fragment extending from a 800-bp DNA fragment of hydroxyquinol 1,2-dioxygenase gene (pnpG) was obtained by genome walking. Sequence analysis indicated that the PNP catabolic gene cluster (pnpABCDEFG) in this fragment shared significant similarities with a recently reported gene cluster responsible for PNP degradation from Pseudomonas sp. strain WBC-3. PnpA is PNP 4-monooxygenase converting PNP to hydroquinone via benzoquinone in the presence of NADPH, and genetic analysis indicated that pnpA plays a key role in PNP degradation. pnpA1 present in the upstream of the cluster (absent in the cluster from strain WBC-3) encodes a protein sharing as high as 55% identity with PnpA, but was not involved in PNP degradation by either in vitro or in vivo analyses. Furthermore, an engineered strain capable of growing on PNP and ortho-nitrophenol (ONP) was constructed by introducing onpAB (encoding ONP monooxygenase and ortho-benzoquinone reductase which catalyzed the transformation of ONP to catechol) from Alcaligenes sp. strain NyZ215 into strain NyZ402.     

Statistical medium optimization and biodegradative capacity of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> toward <Emphasis Type="Italic">p</Emphasis>-nitrophenol

The effect of p-nitrophenol (PNP) concentration with or without glucose and yeast extract on the growth and biodegradative capacity of Ralstonia eutropha was examined. The chemical constituents of the culture medium were modeled using a response surface methodology. The experiments were performed according to the central composite design arrangement considering PNP, glucose and yeast extract as the selected variables whose influences on the degradation was evaluated (shaking in reciprocal mode, temperature of 30°C, pH 7 and test time of about 9 h). Quadratic polynomial regression equations were used to quantitatively explain variations between and within the models (responses: the biodegradation capacity and the biomass formation). The coefficient of determination was high (R _adjusted² = 0.9783), indicating the constructed polynomial model for PNP biodegradative capacity explains the variation between the regressors fairly well. A PNP removal efficiency of 74.5% occurred within 9 h (15 mg/L as the initial concentration of PNP with use of yeast extract at 0.5 g/L).     

Degradation of p-nitrophenol and pentachlorophenol mixtures by Sphingomonas sp. UG30 in soil perfusion bioreactors

The degradation of mixtures of pentachlorophenol (PCP) and p-nitrophenol (PNP) were evaluated in pure cultures of Sphingomonas sp. UG30, statically incubated soils (60% water-holding capacity) and soil perfusion bioreactors where encapsulated cells of UG30 were used as a soil inoculant. In pure-culture studies, conditions were optimized for mineralization of PCP and PNP mixtures at concentrations of 30 mg l⁻¹ each. Optimum in vitro mineralization of PCP and PNP mixtures by UG30 was facilitated using ammonium phosphate as a nitrogen source, while inhibition was observed with ammonium nitrate. The bioreactor system used columns containing soil treated with mixtures of 100, 225 or 500 mg kg⁻¹ of PCP and PNP. Rapid dissipation of both substrates was observed at the 100 mg kg⁻¹ level. Inoculation with UG30 enhanced PCP degradation at the 100 mg kg⁻¹ level in bioreactors but not in static soil microcosms. At higher PCP and PNP concentrations (225 mg kg⁻¹), occasional complete degradation of PNP was observed, and PCP degradation was about 80% compared to about 25% in statically incubated soils after 20 days at 22°C. There was no additional degradation of the PCP and PNP mixtures attributable to inoculation with encapsulated cells of UG30 in either soil system at concentrations of 225 or 500 mg kg⁻¹. Journal of Industrial Microbiology & Biotechnology (2000) 25, 93–99. Received 25 February 2000/ Accepted in revised form 07 June 2000     

Optimization of activated carbon fiber preparation from Kenaf using K2HPO4 as chemical activator for adsorption of phenolic compounds

The present work reports the preparation of activated carbon fiber (ACF) from Kenaf natural fibers. Taguchi experimental design method was used to optimize the preparation of ACF using K₂HPO₄. Optimized conditions were: carbonization at 300 °C, impregnation with 30% w/v K₂HPO₄ solution and activation at 700 °C for 2 h with the rate of achieving the activation temperature equal to 2 °C min⁻¹. The surface characteristics of the ACF prepared at optimized conditions were also studied using pore structure analysis, scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR) spectroscopy. Pore structure analysis shows that micropores constitute the most of the porosity of the prepared ACF. The ability of the ACF prepared at optimized conditions to adsorb phenol and p-nitrophenol from aqueous solution was also investigated. The equilibrium data of phenol and p-nitrophenol adsorption on the prepared ACF were well fitted to the Langmuir isotherm. The maximum adsorption capacities of phenol and p-nitrophenol on the prepared ACF are 140.84 and 136.99 mg g⁻¹, respectively. The adsorption process follows the pseudo-first-order kinetic model.     

Staphylococcus sp. KW-07 contains nahH gene encoding catechol 2,3-dioxygenase for phenanthrene degradation and a test in soil microcosm

We isolated three species of phenanthrene-degrading bacteria from oil-contaminated soils and marine sediment, and assessed the potential use of these bacteria for bioremediation of soils contaminated by polycyclic aromatic hydrocarbons (PAHs). Based on 16S rDNA sequences, these bacteria were Staphylococcus sp. KW-07 and Pseudomonas sp. CH-11 from soil, and Ochrobactrum sp. CH-19 from the marine sediment. By PCR amplification, catechol 2,3-dioxygenase genes (nahH genes) mediating PAH degradation in the chromosome of Staphylococcus sp. KW-07 and Ochrobactrum sp. CH-19, and in plasmid DNA of Pseudomonas sp. CH-11 were detected. All isolates had a similar optimal growth temperature (25 °C) and optimal growth pH (7.0) in a minimal salt medium (MSM) with 0.1% (w/v) phenanthrene as the sole source of carbon and energy. Pseudomonas sp. CH-11 and Staphylococcus sp. KW-07 degraded 90% of added phenanthrene in 3 days and Ochrobactrum sp. CH-19 degraded 90% of the phenanthrene in 7 days under laboratory batch culture conditions. However, Staphylococcus sp. KW-07 was the most effective among the three strains in degradation of phenanthrene in soil. After inoculation of 1 × 10¹¹ cells of Staphylococcus sp. KW-07, over 90% degradation of 0.1% phenanthrene (0.1 g/100 g soil) was achieved after 1 month at 25 °C. The results collectively suggest that the Staphylococcus sp. KW-07 strain isolated may be useful in bioremediation of PAH-contaminated soils.     

Nuclear Magnetic Resonance Spectroscopic Studies on the Microbial Degradation of Mononitrophenol Isomers

The biochemical pathways followed by a mixed bacterial culture and one of its constituent strains, Sarcina maxima, MTCC 5216 (hitherto unreported) during the degradation of mononitrophenol isomers was studied using extensive nuclear magnetic resonance (NMR) spectroscopy (One- and Two-Dimensional Heteronuclear Multiple Quantum Coherence Transfer-2D HMQCT NMR). NMR investigations revealed that o-nitrophenol (ONP) could be degraded by the consortium to metabolites such as catechol, cis, cis-muconic acid, γ-hydroxymuconic semialdehyde, maleylacetate and β-ketoadipate. The spectra of ONP reaction mixture degraded by S.␣maxima showed that formation of maleylacetate from γ-hydroxymuconic semialdehyde should go through a new metabolite γ-hydroxymaleylacetate, hitherto unreported. The consortium could breakdown m-nitrophenol (MNP) to 4-aminocatechol indicating that it came from 3-hydroxyaminophenol. However, S. maxima MTCC 5216, could convert MNP to hitherto unreported 2-nitrohydroquinone and the subsequent 2-hydroxylaminohydroquinone to 1,2,4-benzenetriol along with γ-hydroxymuconic semialdehyde, muconolactone and maleylacetate. The pathway followed by the consortium during p-nitrophenol (PNP) degradation was by the formation of 4-nitrocatechol, maleylacetate and β-ketoadipate. PNP reaction mixture of S.␣maxima, MTCC 5216 on the other hand, showed that the pathway could proceed through the formation of p-hydroquinone as the initial metabolite. The present study conclusively established the nitrophenol-degrading ability of both the consortium and S. maxima MTCC 5216, including exhibiting slight deviations from the pathways followed by the other reported microorganisms.     

Biodegradation of p-cresol by immobilized cells of Bacillus sp. strain PHN 1

The Bacillus sp. strain PHN 1 capable of degrading p-cresol was immobilized in various matrices namely, polyurethane foam (PUF), polyacrylamide, alginate and agar. The degradation rates of 20 and 40 mM p-cresol by the freely suspended cells and immobilized cells in batches and semi-continuous with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 20 and 40 mM p-cresol than freely suspended cells and the cells immobilized in polyacrylamide, alginate and agar. The PUF- immobilized cells could be reused for more than 35 cycles, without losing any degradation capacity and showed more tolerance to pH and temperature changes than free cells. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for degradation of p-cresol.     

Screening Botryosphaeria species for lipases: Production of lipase by Botryosphaeria ribis EC-01 grown on soybean oil and other carbon sources

Nine isolates of Botryosphaeria spp. were screened for lipases when cultivated on eight different plant seed oils and glycerol, and all produced lipases. Botryosphaeria ribis EC-01 produced highest lipase titres on soybean oil and glycerol, while eight isolates of Botryosphaeria rhodina produced significantly lower enzyme titres. B. ribis EC-01 produced lipase when grown on different fatty acids, surfactants, carbohydrates and triacylglycerols, with highest enzyme titres produced on Triton X-100-emulsified stearic (316.7 U/mL), palmitic (283.5 U/mL) and oleic (247.4 U/mg) acids, and soybean oil (105.6 U/mL), as well as castor oil (191.2 U/mg); an enhancement of 9-fold over soybean oil-grown cultures. Glycerol was also a good substrate for lipase production. The crude lipase extract was optimally active at pH 8.0 and 55 °C, stable between 30 and 55 °C and pH 1–10, and tolerant to 50% (v/v) glycerol, methanol and ethanol. The crude lipase showed affinity for substrates of short, average and long-chain fatty acids (different esters of p-nitrophenol and triacylglycerols). Zymograms developed with 4-methylumbelliferyl-butyrate showed two bands of lipolytic activity at 45 and 15 kDa. This is the first report on the production of lipases by B. ribis grown on these different carbon sources.     

Nitrogen source effects on the growth and toxicity of two strains of the ciguatera-causing dinoflagellate Gambierdiscus toxicus

Two Caribbean strains (1651 and 1655) of the ciguatera-causing dinoflagellate Gambierdiscus toxicus were grown in xenic, batch culture under defined, measured nutrient conditions with nitrate, ammonium, urea, a mix of free amino acids (FAA), or putrescine as the nitrogen source. Cultures were maintained at 27 °C, salinity 35, 110 μmol m⁻² s⁻¹ (12 h:12 h light:dark cycle) on L2 medium at an initial nitrogen concentration of 50 μM N. Toxicity was determined using a ouabain/veratridine-dependent cytotoxicity assay (N2A assay) standardized to a ciguatoxin standard. Nitrate, ammonium, FAA, and putrescine supported growth, but urea did not. The appearance of ammonium in the organic nitrogen cultures indicated that G. toxicus and/or associated bacteria remineralized the available organic nitrogen. Both strains were exposed to nitrogen-limiting conditions as evidenced by chlorophyll a content per cell, nitrogen content, and nitrogen (N) to phosphorus (P) (N:P) ratio significantly declining once nitrogen was no longer available in the medium and cells entered stationary phase. Strain 1651 grew significantly faster than strain 1655 when nitrate, FAA, and putrescine was the nitrogen source, but not ammonium. Nitrogen source had no effect on growth rate (0.14 d⁻¹) in strain 1651. The growth rate of strain 1655 (0.10–0.13 d⁻¹) was significantly faster on ammonium than the other nitrogen sources. Strain 1655 was significantly more toxic (10-fold) than strain 1651 except when growing on ammonium at exponential phase. Toxicity ranged from 1.3 to 8.7 fg C-CTX1-Eq cell⁻¹ in strain 1651 and from 30.7 to 54.3 fg C-CTX1-Eq cell⁻¹ in strain 1655. Nitrogen source had no significant affect on toxicity. Toxicity was greater in stationary versus exponential phase cells for strain 1651 when grown on nitrate and strain 1655 regardless of nitrogen source. The difference in toxicity between growth phases may result from an increase in ciguatoxin and/or maitotoxin. Our results suggest that some strains of G. toxicus when associated with bacteria are able to take advantage of organic as well as inorganic nitrogen sources on short time scales to support future growth. The uncoupling of total nitrogen and phosphorus pools from conditions in the water column suggest that instantaneous growth rates can be supported by nutrients acquired hours to days earlier.     

Biodegradation of methyl parathion and <Emphasis Type="Italic">p</Emphasis>-nitrophenol: evidence for the presence of a <Emphasis Type="Italic">p</Emphasis>-nitrophenol 2-hydroxylase in a Gram-negative <Emphasis Type="Italic">Serratia</Emphasis> sp. strain DS001

A soil bacterium capable of utilizing methyl parathion as sole carbon and energy source was isolated by selective enrichment on minimal medium containing methyl parathion. The strain was identified as belonging to the genus Serratia based on a phylogram constructed using the complete sequence of the 16S rRNA. Serratia sp. strain DS001 utilized methyl parathion, p-nitrophenol, 4-nitrocatechol, and 1,2,4-benzenetriol as sole carbon and energy sources but could not grow using hydroquinone as a source of carbon. p-Nitrophenol and dimethylthiophosphoric acid were found to be the major degradation products of methyl parathion. Growth on p-nitrophenol led to release of stoichiometric amounts of nitrite and to the formation of 4-nitrocatechol and benzenetriol. When these catabolic intermediates of p-nitrophenol were added to resting cells of Serratia sp. strain DS001 oxygen consumption was detected whereas no oxygen consumption was apparent when hydroquinone was added to the resting cells suggesting that it is not part of the p-nitrophenol degradation pathway. Key enzymes involved in degradation of methyl parathion and in conversion of p-nitrophenol to 4-nitrocatechol, namely parathion hydrolase and p-nitrophenol hydroxylase component “A” were detected in the proteomes of the methyl parathion and p-nitrophenol grown cultures, respectively. These studies report for the first time the existence of a p-nitrophenol hydroxylase component “A”, typically found in Gram-positive bacteria, in a Gram-negative strain of the genus Serratia. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Bacillus sp. mutant for improved biodegradation of Congo red: Random mutagenesis approach

This study presents the improved biodegradation of Congo red, a toxic azo dye, using mutant Bacillus sp. obtained by random mutagenesis of wild Bacillus sp. using UV and ethidium bromide. The mutants obtained were screened based on their decolorization performance and best mutants were selected for further studies. Better decolorization was observed in the initial Congo red concentration range 100–1000 mg/l for wild species whereas mutant strain was found to offer better decolorization up to 3000 mg/l. Mutant strain offered 12–30% reduction in time required for the complete decolorization by wild strain. The optimum pH and temperature were found to be 7.0 and 37 °C, respectively. Two efficient strains such as Bacillus sp. ACT 1 and Bacillus sp. ACT 2 were isolated from the various mutants obtained. Bacillus sp. ACT 2 showed improved enzymatic production and Bacillus sp. ACT 1 showed improved growth compared to wild strain. The enzyme responsible for the degradation was found to be azoreductase by SDS–PAGE and about 53% increased production of enzyme was achieved with mutant species. The experimental data were modeled using growth and substrate inhibition models.     

Biodegradation of methyl parathion and p-nitrophenol by a newly isolated Agrobacterium sp. strain Yw12

Strain Yw12, isolated from activated sludge, could completely degrade and utilize methyl parathion as the sole carbon, phosphorus and energy sources for growth in the basic salt media. It could also completely degrade and utilize p-nitrophenol as the sole carbon and energy sources for growth in the minimal salt media. Phenotypic features, physiological and biochemical characteristics, and phylogenetic analysis of 16S rRNA sequence showed that this strain belongs to the genus of Agrobacterium sp. Response surface methodology was used to optimize degradation conditions. Under its optimal degradation conditions, 50 mg l⁻¹ MP was completely degraded within 2 h by strain Yw12 and the degradation product PNP was also completely degraded within 6 h. Furthermore, strain Yw12 could also degrade phoxim, methamidophos, chlorpyrifos, carbofuran, deltamethrin and atrazine when provided as the sole carbon and energy sources. Enzymatic analysis revealed that the MP degrading enzyme of strain Yw12 is an intracellular enzyme and is expressed constitutively. These results indicated that strain Yw12 might be used as a potential and effective organophosphate pesticides degrader for bioremediation of contaminated sites.     

Biodegradation of p-nitrophenol by microalgae

A study was made on the use of a mixed microalgal consortium to degrade p-nitrophenol. The consortium was obtained from a microbial community in a waste container fed with the remains and by-products of medium culture containing substituted aromatic pollutants (nitrophenols, chlorophenols, fluorobenzene). After selective enrichment with p-nitrophenol (p-NP), followed by an antibiotic treatment, an axenic microalgal consortium was recovered, which was able to degrade p-nitrophenol. At a concentration of 50 mg L^–1, total degradation occurred within 5 days. Two species, Chlorella vulgaris var. vulgaris f. minuscula and Coenochloris pyrenoidosa, were isolated from the microalgal consortium. The species were able to accomplish p-NP biodegradation when cultured separately, although Coenochloris pyrenoidosa was more efficient, achieving the same degradation rate as the original axenic microalgal consortium. When Coenochloris pyrenoidosa was associated with Chlorella vulgaris in a 3:1 ratio, complete removal of the nitro-aromatic compound occurred within three days. This is apparently the first report on the degradation of a nitro-aromatic compound by microalgae.     

Monitoring ofgfp-taggedMoraxella sp. under starvation condition

A PNP(p-nitrophenol)-degradingMoraxella sp. was genetically marked bygfp gene for monitoring. Stable chromosomal integration of the introducedgfp gene was confirmed by examining the transformants under epifluorescent microscope. The survival ofgfp-taggedMoraxella sp. cells during long-term storage under starvation condition was examined by viable cell counting and direct fluorescence microscopic counting. The number of green fluorescent cells obtained by direct microscopic counting was approximately 10 times greater than viable cell counts by plating. The number of cells from both counting methods was higher at lower temperature (4°C), although the drop of cell number after 8 weeks of starvation was comparable (approximately 100 fold drop from initial counts). Results obtained by two different methods correlated well with each other indicating that thegfp markedMoraxella sp. can be directly monitored following environmental release using epifluorescence microscopy.