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Use of polyphosphate accumulating organisms (pao) for treatment of phosphate sludgeSengupta, M. and Dalwani, R. (Editors). 2008
Proceedings of Taal2007: The 12th World Lake Conference: 918-922
Use of Polyphosphate Accumulating Organisms (Pao) For Treatment Of
Shyam S. Bajekal and Neelam S. Dharmadhikari
Department of Microbiology, Yashwantrao Chavan College of Science, Karad, Vidyanagar, KARAD – 415 124.
ABSTRACT Phosphate removal from wastewaters has been quite a problem over the years. The method largely employed these days is chemical precipitation although biological methods are regarded as preferable due to the fact that this phosphate can then be easily recovered in a relatively pure form. The role of Biological Phosphate Removal (BPR) in waste treatment often described as a `novel waste treatment tool’ has been known since some time. Microbial constituents of activated sludge remove phosphate by accumulating it as polyphosphate within their cells and are known as Polyphosphate Accumulating Organisms (PAO). Though this technology is presently available for phosphate removal from liquid wastes, there is no reason why it cannot be adapted and developed for the removal of phosphate from solid sludge generated by metal processing industries. This study reports precisely that kind of work done on phosphate removal from solid sludge generated from the painting department of a local industry. Microorganisms were enriched from garden soil and the solid sludge obtained from the industry using suitably amended nutrient media. Three isolates including two bacteria and a yeast showed tremendous potential in this respect in laboratory scale experiments. The mass of sludge was reduced by more than half (52 to 57%) and phosphate removal was to the extent of almost 78%. The removal of phosphate being due to intracellular accumulation by the organisms was confirmed by microscopic observation. The most efficient organism in this respect was found to be the yeast, confirmed by the microscopic observation and action of chitinase enzymes. Scale up studies for large-scale application that also include removal of Zn and Cu are in progress. and Quinn (2001) however, also showed that this uptake in many microorganisms was increased by Waste treatment technologies presently available for 50% to 143% when the growth pH was acidic and phosphorous stripping include physical, chemical, that too in solely aerobic conditions in the absence of and biological treatments. Among these, the greatest interest and most recent progress made have been in EBPR technology has been developed mainly the alternative biotechnological approach of for treatment of liquid wastes considering the Enhanced Biological Phosphate Removal (EBPR) removal of ‘P’ from domestic sewage being disposed used in activated sludge digestion processes. The into surface water. Several researchers have also EBPR has the potential to reduce ‘P’ down to very combined the removal of other waste materials with low levels at relatively lower costs (Strom, 2006). that of `P’ removal, such as metals, nitrates, nitrogen Under favourable conditions this removal from and carbon (Meknassi et al 2005). Chavez et al influent can be as high as 80 – 90%, achieving (2004) observed degradation of polychlorinated residual `P’ levels of less than 1mg/L in the effluent biphenyls simultaneously with massive accumulation of polyP in some microorganisms and concurrent This process involves a group of accumulation of polyhydroxyalkanoates with polyP microorganisms (that include a variety of bacteria was observed in Pseudomonas strains by Tobin et al and yeasts), called Polyphosphate Accumulating Organisms (PAOs) that actively take up soluble `P’ Several steel and metal processing industries from the system and accumulate it in the form of generate phosphate wastes as solid sludge. Present polyP granules (commonly known as `volutin’ or methods for the disposal of such wastes are dumping `metachromatic’ granules when observed by staining or burning followed by dumping. The idea of with the basic dye Toluidine Blue). This uptake, applying the EBPR concept to such solid wastes was known as the `luxury uptake’ is seen to be enhanced thus an attractive one. This paper reports results of when the organisms are alternated between a carbon- work done by us in this direction on the solid sludge rich anaerobic environment and carbon-poor aerobic generated by a local metal processing industry.
environment specially managed in an activated sludge digestion plant (Mino et al, 1998). McGrath The bacterial isolates were preserved at 40C on nutrient agar slants and the yeast on Sabouraud agar Collection of sludge
Sludge samples consisting of insoluble phosphatic Phosphate (PO4) removal studies
flakes and `sludge liquor’ were collected from the industry premises. The solid flakes were collected in Suspensions of the 48hr old isolates obtained on the plastic boxes and the liquor in plastic carboys that solidified medium were prepared in sterile normal were previously disinfected with isopropyl alcohol. saline and their optical densities adjusted to 1.0 at 660nm on a Systronics 105 Spectrophotometer. One Medium
milliliter each of these suspensions were inoculated into 100ml aliquots of amended Pikovskaya medium The Pikovskaya phosphate medium amended in two of pH 5.5 containing 5.0% v/v liquid waste (for the ways was used here for two different studies. The first experiment) and 5.0% w/v flakes (for the second basal medium (excluding tri-calcium phosphate) was experiment) taken in 250ml capacity Erlenmeyer prepared with the following composition of flasks that were shaken at 115rpm at 300C in a rotary ingredients per litre: (NH4)2SO4, 0.5g; KCl, 0.2g; shaker-incubator (LabHosp) for 7 days. Uninoculated MgSO4.7H2O, 0.1g; MnSO4, trace; FeSO4, trace; flasks were taken as controls in both the experiments. yeast extract, 0.5g. The pH was adjusted as required In the first experiment, 5ml aliquots of with 1N NaOH. It was sterilized by autoclaving at supernatant from each flask were withdrawn at 24hr 1210C for 15 min. Filter sterilized (Millipore intervals. They were subjected to centrifugation at membrane 0.22µm) glucose (1.0g/l) was then added 6000xg for 10’ and the soluble phosphorus (P) content in the supernatant measured by the In one modification used for the studies on chlorostannous reduced molybdophosphoric acid removal of soluble `P’, the basal medium was blue method described in Jackson (1973). amended with 5.0% sludge liquor, while for the In the second experiment, at the end of 7 days studies on removal of PO4 from the solid fraction, the incubation, the flasks were stood still for 15’ to allow sludge flakes were added in 5.0% w/v the flaky sludge to settle, leaving the cells in the concentrations. Agar agar in 25g/l quantities was supernatant that was then decanted. The residual included whenever a solidified medium was required. sludge was washed with distilled water five times and air-dried at 300C till constant weight, measured Enrichment and Isolation
on a Shimadzu model BL-220H electronic weighing balance. The phosphate content of this mass was also Enrichment of organisms was done using the determined by the same chlorostannous reduced Winogradsky column method described by molybdophosphoric acid blue method. The Veldkamp (1970). The sludge flakes were placed in accumulation of intracellular polyP by the quantities of 1.0, 5.0 and 10.0g in three separate glass microorganisms was determined by light microscopy measuring cylinders (Borosil) of 250ml capacity that after staining the cells in the supernatant by the were previously disinfected with isopropyl alcohol Albert’s Toluidine Blue staining technique described and washed with sterile distilled water. The Pikovskaya basal medium (initial pH 7.2 but was observed to be reduced to 5.0 after amendment with PO4) was added up to the 100ml mark in each cylinder. Two sets of enrichment were made, one for A total of five microbial isolates were obtained after organisms indigenous to the waste for which no enrichment and isolation. Only one isolate Pz, was exogenous inoculum was added and the other for indigenous to the sludge while the remaining four organisms from soil, for which 2g garden soil per were soil isolates. Based on colonial, staining and cylinder was used. After the contents were mixed morphological studies three of the isolates were thoroughly, the columns were covered with paper suspected to be yeasts and two were bacteria. and kept at ambient temperature (28-320C) till Confirmation was achieved with tests (results not biomass appeared at the surface and/or the interface shown here) of sensitivity to enzymes like chitinase and lysozyme and the anti-yeast antibiotic Isolation of microorganisms was achieved by Clotrimazole. Of the two bacterial isolates, one was streak inoculating the biomass onto solidified found to be a gram positive endospore forming rod amended Pikovskaya medium (containing 1.0, 5.0 and the other a gram negative non-spore forming rod. and 10.0% sludge respectively) in Petri-dishes that Phosphate removal studies using modified were incubated at ambient temperature till growth of Pikovskaya medium were carried out on all the microorganisms appeared (48 hours). Well-isolated isolates. Only three, Pz (yeast), P3 and N7 (the two colonies of the microorganisms were purified by re- bacteria) were found significantly effective and were streaking on solidified amended Pikovskaya medium. The soluble `P’ was removed from the liquid isolates. Microscopic examination of the three medium (pH 5.0) by isolating under aerobic isolates after staining by Albert’s method for conditions. By the end of seven days, the removal is intracellular polyP granules showed the presence of seen to be to the extent of 92 to 93% by all the three large granules in the yeast (Fig. 2) and bacteria. Table 1 shows the results of the action of plays a major role in removing PO4 from solid sludge. isolates on the solid flakes over a period of seven It could therefore be concluded that yeast indigenous days incubation. While all the three isolates to the phosphatic waste can be used to remove reduced the sludge mass by about half, when it phosphate from such wastes. Further work towards came to removal of PO4 only the yeast was found developing a technology for this purpose is currently to be most effective. A consortium of the three isolates that was tried showed also similar results. While it was observed that both the bacteria and yeast were equally efficient in removing soluble `P’ from the liquid waste, in removing PO4 The authors are thankful to Head, Department of from the solid flakes, the yeast alone was found to Microbiology and the Principal and Management of be more efficient. In the consortium also the role of Yashwantrao Chavan College of Science, Karad for the facilities provided for this work. Thanks are also due to Mr. N. R. Shaikh for his help in photography. Table 1 Characteristics of the sludge flakes after Chavez, F. P., Lunsdorf, H. and Jeref, C. A. (2004). Growth of polychlorinated biphenyl degrading bacteria in the presence of biphenyl and chlorobiphenyls generates oxidative stress and massive accumulation of inorganic polyphosphate. Appl. Environ. Microbiol. 70, 3064-3072.
Desai, J. D. and Desai, A. J. (1980). Methods in Microbiology: Microscopy and Staining. Prashant Jackson M. L. (1973). Soil chemical analysis, Prentice Hall a Initial mass = 5.01g and initial PO4 content McGrath, J. W., Cleary, A., Mullan A. and Quinn J. P. Results are means of at least three determinations. (2001). Acid-stimulated phosphate uptake by activated sludge microorganisms under aerobic laboratory conditions. Wat. Res. 35(18), 4317-4322.
McGrath, J.W., and Quinn, J. P. (2000). Intracellular accumulation of polyphosphate by the yeast Candida Accumulation of intracellular polyP granules by humicola G-1 in response to acid pH. Appl. Environ. yeasts was first shown in Saccharomyces Microbiol. 66, 4068-4073.
cerevisiae by Liebermann in 1888 (Chavez et al McGrath, J. W., and Quinn J. P. (2003). Microbial 2004). McGrath and Quinn (2000) isolated a yeast phosphate removal and polyphosphate production Candida humicola, from an activated sludge from wastewaters. Adv. Appl. Microbiol. 52, 75-100.
inoculum that accumulated 10.5 times (or 55%) Meknassi, Y. F., Auriol, M. A., Tyagi, R. D., Comeau Y., more phosphate under acidic conditions at pH 5.5. and Surampalli, R. Y. (2005). Phosphorous co- The most important aspect of this observation was precipitation in the biological treatment of slaughter house waste water in a sequencing batch reactor. that it neither required the anaerobic phase of Pract. Period. Haz. Toxic and Radioact. Wst. Mgmt. growth nor any stress conditions like phosphate 9, 179-192.
starvation or nutrient limitation, etc. Melasniemi Melasniemi, H., and Hernesmaa, A. (2000). Yeast spores and Hernesmaa (2000) also showed a prominent seem to be involved in biological phosphate removal: role of a yeast-like morphotype in phosphate a microscopic in situ case study. Microbiology. 146,
removal in an activated sludge process. Similarly, McGrath et al (2001) showed a 50% enhancement Mino, T. (2000). Microbial selection of polyphosphate in uptake of phosphate from sewage by an accumulating bacteria in activated sludge waste water activated sludge inoculum grown at pH 5.5 with treatment processes for enhanced biological
phosphate removal. Biochemistry. 65, 405-413.
glucose as a carbon source and in only aerobic Strom, P. E. (2006). Technologies to remove phosphorous conditions. Microscopic examination of the Neisser stained culture showed not just yeasts, but also Tobin, K. M., McGrath J. W., Mullan A., Quinn J. P., and bacteria and fungi accumulating huge quantities of O’Connor, K. E. (2007). Polyphosphate accumulation polyP granules intracellularly. This paved the way by Pseudomonas putida CA-3 and other medium- for the development of their concept of the `one chain-length polyhydroxyalkanoate-accumulating bacteria under aerobic growth conditions. Appl. Our study has also shown that while bacteria Environ. Microbiol. 73, 1383-1387.
and yeast both are involved in significant removal Veldkamp, H. (1970). Enrichment cultures of prokaryotic organisms. In, ‘Methods in Microbiology’ Vol. 3A of `P’ from liquid waste, it is only the yeast that (J.R. Norris and D.W. Ribbons eds), pp: 305-361. Academic Press, London.
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