*Corresponding Author E-mail: hemantdrd@hotmail.com © 2002 Association of Applied Biologists Ann. appl. Biol. (2002), 140:159-162 Printed in Great Britain 159 Isolation of bacterial consortia for degradation of p-nitrophenol from agricultural soil By ASIFA A QURESHI and HEMANT J PUROHIT* National Environmental Engineering Research Institute, Nehru Marg, Nagpur 4400 20, India (Accepted 7 December 2001; Received 16 May 2001) Summary A bacterial consortium has been isolated containing Pseudomonas spp. strains S1 and S2, which was able to degrade p-nitrophenol (PNP). The strains were isolated from agricultural soil contaminated with organophosphorus pesticides. Pseudomonas spp. strain S2 could convert p-nitrophenol to 4- nitrocatechol (4NC) after pre-exposure to phenol, when PNP was used as the only carbon source in the medium. Pseudomonas spp. strain S2, when mixed with strain S1 in the ratio 1:5 respectively, decolorised PNP completely. Key words: p-nitrophenol, 4-nitrocatechol, phenol, Pseudomonas spp. Introduction We are working on developing a culture collection from the environmental niches contaminated with phenolic and substituted phenolic waste. We have isolated cultures for treatment of PNP (Qureshi et al., 2001), chlorophenols (Atuanya et al., 2000), aminophenols (Kutty et al., 2000) and are developing strategies to track phenols in the environment (Kapley & Purohit, 2000). We have reported degradation of p-nitrophenol (PNP) using Pseudomonas SF1, isolated from an industrial dumpsite (Qureshi et al., 2001). The strain utilises PNP, via the intermediate 1,4-benzoquinone, as also reported by other groups (Spain, 1995; Zeyer & Kocher, 1988). The present study reports degradation of PNP with two different soil isolates in co-culture (S1 and S2) from agricultural land contaminated with organophosphorus pesticides. Pseudomonas strain S2 converted PNP to 4- nitrocatechol (4NC); followed by further degradation by Pseudomonas strain S1. Furthermore, it has been demonstrated that a minimal ratio of the two cultures is required for PNP utilisation. Experimental Procedures Media and culture conditions The mineral medium had the following composition: 36.9 mM Na 2 HPO 4 , 20.3 mM KH 2 PO 4 , 4.67 mM NH 4 Cl, 0.68 mM CaCl 2 , 0.811 mM MgSO 4 , 0.0035 mM FeSO 4 ; and was used for both the enrichment and growth studies. The carbon source used in the cultivation medium was phenol (1 mM) or PNP (0.07 mM). The culture temperature was kept at 30°C with shaking at 150 rpm. Enrichment and isolation The enrichment approach was used as described earlier (Qureshi et al. , 2001). The soil sample derived from the contaminated agricultural field was suspended as slurry in Na-K phosphate buffer pH7 and used as an inoculum for the enrichment of microorganisms. The minimal media amended with 0.07 mM PNP was inoculated with the soil slurry and incubated for 3 wk in suspension culture in the shaking incubator with regular spiking with the same amount of PNP after every 24 h. In a 24 h period, the yellow coloured growth medium turned colourless due to PNP utilisation. The sample from the reactor was diluted and streaked on a minimal media plate amended with PNP (0.07 mM). After 3 days of incubation, the colonies that showed colorless zones were picked and tested for their ability to transform PNP in the liquid cultures. Apparently, the colony which showed the colourless zone appeared to be a co-culture when it was streaked onto an LB plate, with two different colony morphologies. According to the Bergeys Manual of Systematic Bacteriology both the strains were classified and identified as pseudomonads (Palleroni, 1984). Neither culture grown separately could utilise PNP as sole source of carbon. Transformation of PNP to 4-nitrocatechol by Pseudomonas strain S2 Cells were grown in LB medium overnight and harvested by centrifugation (6000 X g) and were then washed with phosphate buffer (pH 7). The cell pellet was resuspended in minimal medium with 1 mM phenol as a sole source of carbon and incubated for 48 h at 30°C for pre-adaptation. The phenol grown cells were pelleted and inoculated into the