Indian Journal of Biotechnology Vol 8, April 2009, pp 240-243 Microflora (fungal and bacterial) of selected industrial wastewaters, but mostly in the developed terrestrial and marshy species of rhizosphere countries1,2. However, their application in polishing organic rich wastewaters (treated) is not very in response to spent wash treatments encouraging3, possibly due to intolerance of plant P K Singh and K P Sharma* roots to the increased biological oxygen demand. Department of Botany, University of Rajasthan, Jaipur 302 004, Distillery waste/spent wash is one such wastewater India found toxic to both plants4 and microbes inhabiting their rhizosphere5-6. As molasses based distilleries are Received 10 July 2007; revised 3 October 2008; accepted 23 December 2008 the only source of ethanol for producing gasohol (petrol+ethanol) in India, to be commenced shortly on In the present study, 16 fungal species were recorded in the a larger-scale, it is likely that the existing distilleries rhizosphere of 4 terrestrial species [Acacia farnesiana (Linn.) will expand and new ones will be installed to fill the Willd, A. leucophloea (Robx.) Willd, A. nilotica (Linn.) Del. and gap between demand and supply and so also resulting A. raddiana Savi] and 4 marshy species (Arundo donax Linn., Phragmites karka Steud, Typha angustata Bory & Chaub and environmental problems. Scirpus tuberosus Derf.) grown in tap water (control) and spent Our objective was to identify the key players of wash treatments (COD = 750-12,000 ppm). Their species richness phytoremediation system, viz., plants and microbes was relatively higher (2-folds) in the spent wash treatments than tolerant to spent wash. We enumerated growth of their respective controls while their colony forming units were almost similar, exceeding in number to more than 300 units for a terrestrial and marshy plant species in the spent wash particular fungal species in the community. Aspergillus was the along with effects on their rhizosphere microflora. most dominant genus (4 species) followed by Fusarium (2 The present communication addresses only the effects species) while the remaining 10 genera had one species each. The of spent wash on microflora. bacteria were Gram (+) bacilli and cocci arranged singly and in chains (also in bunches in cocci). In comparison to control, their One young shoot (15-20 cm long) each of Arundo CFU values were significantly higher (2-10-folds) in spent wash donax Linn., Phragmites karka Steud. and Typha treatments, especially in the rhizosphere of marshy species. angustata Bory & Chaub was planted separately in 4 Keywords: Fungi, bacteria, rhizosphere, terrestrial and marshy L plastic jars (with a hole at bottom), each filled in plant species, spent wash with pebbles at bottom (0-4 cm thick layer) and overlaid with a mixture of coarse river sand and sandy The conventional biological wastewater treatments soil in equal proportions by volume (>4-25 cm) to such as trickling filter and activated sludge process facilitate good drainage. These jars, kept in bowls, are now becoming uneconomic due to ever growing were watered daily, adding 20% Hoagland (diluted energy prices. And therefore, globally, a need for with tap water) at weekly intervals, until the shoots sustainable eco-technologies for pollution abatement attained good growth (45-50 d for Arundo and Typha, has been felt. Environmental engineers looked back to and 90 d in Phragmites). Four-five shoots (15-20 cm ecological systems with diverse enzymatic pathways long) of Scirpus tuberosus Derf. growing under of microbial system, and complex surfaces for the flooded conditions (3-5 cm) were cultured separately exchange of gases and nutrients of plants, to design in 3 L bowls, in which water level was maintained technologies with the potential of several orders of daily with tap water and with 20% Hoagland (diluted magnitude and greater efficiency than contemporary with tap water) at weekly intervals for 45 d. This mechanical and chemical technologies. As evident, treatment continued in the control sets was, however, the success of these systems relies on tolerance of replaced with spent wash (at weekly intervals) of their key players (plants and microbes) to pollutants. varying dilutions (3000-12,000 ppm in terms of COD Such systems are in use in polishing municipal and values) with tap water (@ 20mm/m2/week) in others, with the exception of Scirpus grown at still higher —————— dilutions (750-6,000 ppm). The evapotranspiration *Author for correspondence: Tel: 91-141-2711546; Fax: 91-141-2711546 losses in the spent wash treatments were compensated E-mail: email@example.com by adding tap water in the evening. SHORT COMMUNICATIONS 241 One-month-old shoots of Acacia species [A. A total of 16 fungal species were present in the farnesiana (Linn.) Willd, A. leucophloea (Robx.) rhizosphere of marshy and terrestrial plants, their Willd., A. nilotica (Linn.) Del. and A. raddiana Savi] richness being higher in the former (3-8 species) than raised in plastic jars similar to marshy species were the latter (2-4 species, Table 1). Further, spent wash also exposed to spent wash (3,000-10,000 ppm) as treatments had a little higher diversity (almost 2- described earlier. For examining fungal and bacterial folds) than their respective controls. The relatively flora in the rhizosphere of these plant species, soil more diverse Aspergillus (4 species) and Fusarium (2 samples collected from replicates of control and spent species) genera also had wide ecological amplitude wash treatments were pooled separately, and 1 g of it since they were present in the rhizosphere of marshy was mixed in 25 mL sterilized distilled water. This and terrestrial species, whereas the remaining 14 soil suspension was serially diluted and finally genera were monotypic. inoculated over potato dextrose agar (PDA) and The colony forming units (CFU) of Aspergillus nutrient agar (NAA) media to characterize fungi and niger, Curvularia lunata, Penicillium chrysogenum bacteria, respectively. PDA made in diluted (50%) and Trichoderma harzianum were > 300; i.e. “Too spent wash (COD = about 18,000 ppm) was similarly Numerous To Count” (TNTC), more particularly in inoculated and the cultures were designated as on the spent wash treatments rich in biodegradable modified PDA hereafter in the text. organic matter. Other species had low-moderate (3- Initially, fungal colonies were identified, based on 50) colony counts, except for Rhizopus (125-200 their colour and then counted. Their identity was later colonies) in Phragmites rhizosphere. confirmed by slide study and referring standard refer- Interestingly, when soil suspension of marshy ence books7-11. Bacterial colonies were also counted, species rhizosphere was inoculated on modified PDA and their colour and shape were noted. The Gram (made in 50% spent wash), only Fusarium oxysporum stained slides were studied microscopically for the colonies (<10) developed suggesting it to be relatively shape of bacteria. As microbial diversity in the more tolerant to toxic compound/s in the spent wash rhizosphere of Acacia species differed little, the data while the remaining 15 susceptible species grew in were pooled and mean values have been reported here. their absence on normal PDA (made in distilled Table 1–Fungal diversity and their CFU counts in the rhizosphere of terrestrial and marshy plant species growing in control (tap water) and spent wash treatments Fungi Arundo donax Phragmites karka Scirpus tuberosus Typha angustata Acacia sp Control SW Control SW Control SW Control SW Control SW Aspergillus flavus Link ex. Fr. Abs. 25±5 27±23 7±12 5±6 A. fumigatus Fresnius Abs. Abs. 13±23 50±23 A. niger Van Tiegh TNTC TNTC TNTC 35±27 TNTC TNTC TNTC TNTC A ochracious Wilhelm Abs. 13±3 Chaetomium spinosum Kunze ex. Fries Abs. 25±37 Cladosporium sphaerospermum Penz. Abs. 20±9 Curvularia lunata (Wakker) Boedij 87±31 93±76 70±15 TNTC 13±3 Drechslera tetramera (Mckinney) 62±53 210±25 TNTC Subramanian & Jain Fusarium oxysporum Schlecht. ex. Fr. TNTC TNTC TNTC 113±147 TNTC 53±25 Nil- TNTC F. moniliforme var. Mucor sp. 3±5 Papulospora sp. Preuss. 5±3 Penicillium chrysogenum Thom. TNTC TNTC TNTC 5±6 33±35 TNTC TNTC Rhizopus nigricans Ehrenberg. 200±10 125±138 20±1 25±22 Trichoderma harzianum Rafai TNTC 13±23 15±25 20±6 10± Verticillium alboatrum Reinke & Berth 50±92 Total species 3 8 7 6 3 7 4 8 2 4 TNTC = Too Numerous to Count; Abs. = Absent 242 INDIAN J BIOTECHNOL, APRIL 2009 water). It is likely that degradation of toxic occurrence of rich microflora on the normal PDA principle/s, most probably by bacteria, allowed (made in distilled water), even better than controls, occurrence of susceptible species in the rhizosphere of following inoculation of soil suspension of spent wash spent wash treatments. treatments is only possible when toxic pollutants of The majority of bacterial colonies were off-white spent wash are degraded in the rhizosphere12. The in colour while a few were white. A few off-white increased availability of soft carbon (reducing sugars, coloured colonies, changed into orange colour after protein) and minerals (N, P, K) in the spent wash 48 h, were flat, round and mucoid type and had possibly favoured microbial growth. regular margins. They were Gram (+) bacilli found We have now standardized optimal conditions for singly as well as in long chains. The remaining off- growing both marshy and terrestrial plant species in white colonies, which turned bluish, were similar to the spent wash, even at higher concentrations (7,500- off-white in their shape, surface and margins and were 35,000 ppm)13, while occurrence of rich microflora in also mucoid type. However, these gram (+) cocci, their rhizosphere, as documented in the present study, were found either single or arranged in pairs, chains suggests good scope of phytoremediation for spent and also in bunches. The white coloured colonies wash. were flat, non-mucoid and spread irregularly (rhizoidal). These were also Gram (+) cocci arranged Acknowledgement as described earlier. Thus, bacterial diversity was The authors are thankful to the Department of relatively poor in comparison to that of fungi in the Biotechnology and CSIR, New Delhi, for financial spent wash treatments, but their CFU values were assistance to undertake this study, and the Head, several folds higher, more particularly in the distillery Department of Botany, University of Rajasthan, treatments (Table 2). Jaipur for laboratory facilities. In contrast to findings of the present study, most workers reported adverse effects of spent wash on References rhizosphere microflora. The total microbial 1 Urbanc B O & Bulc T, Integrated constructed wetlands for communities, in Proc Int Conf- Constructed Wetlands population was reduced significantly in soil samples in Water Pollution Control, Guangzhou, China, 1994, 138- collected from the effluent channels (COD = 7930- 146. 8212 ppm) which was maximum in bacteria and 2 Yirong C & Puetpaiboon U, Performance of constructed minimum in actinomycetes populations5. Juwarkar wetlands treating waste water from seafood industry, Water and Dutta6 also reported greater toxicity of raw Sci Technol, 49 (2004) 289-294. distillery wastewater (COD = 72,792-1,37,697 ppm) 3 Billore S K, Singh N, Ram H K, Sharma J K, Singh V P et al, Treatment of a molasses based distillery effluent in a to soil microflora (bacteria, fungi, actinomycetes and constructed wetland in Central India., Water Sci Technol, 44 nitrogen fixing bacteria), which was minimized after (2001) 441-448. mixing stabilization pond effluent (1:1). During the 4 Singh P K, Sharma K P, Kumar S, Sharma S & Sharma S, present study also, spent wash had toxic effects on Assessment of environmental contamination potential of fungal flora when soil suspension of rhizosphere was distillery effluent using plant and animal bioassays, Nature, Environ Pollut Technol, 6 (2007) 63-74. inoculated on modified PDA (made in 50% spent 5 Kumar A, Singh Y, Josh B D & Rai J P N, Effect of distillery wash having COD = about 18,000 ppm). However, spent wash on some characteristics of soil and water, Indian J Ecol, 30 (2003) 7-12. Table 2–Colony forming units (CFU; × 104/g soil) of bacteria in the rhizosphere of terrestrial and marshy plant species growing in 6 Juwarkar A & Dutta S A, Impact of distillery effluent control and distillery treatments application to land on soil microflora, Environ Monit Assess, 15 (2003) 201-210. Plant species Control Distillery treatments 7 Gilman J C, A manual of soil fungi, 2nd edn (The Iowa State College Press-Ames, Iowa, USA) 1957. Acacia species 4.5±1.8 10.4±0.3-40.3±28.3 8 Ainsworth G C, Dictionary of the fungi, 5th edn Arundo donax 7.3±2.0 13.1±4.3-43.8±6.2 (Commonwealth Mycological Institute. Kew), 1961, 1-547. 9 Barron G L, The genera of hyphomycetes from soil (Robert Phragmites karka 4.89±0.35- 10.7±3.8- E. Krieger Publishing Co.,) 1972, 1-363. 8.7±1.2 43.8±3.5 10 Tandon R N & Chandra S, Supplement to the list of Indian Typha angustata 7.02±2.9 6.33±12.83- fungi (1957-1962) (Bishen Singh Mahendra Pal Singh, 37.48±17.36 Dehradun) 1976, 1-246. SHORT COMMUNICATIONS 243 11 Mehrotra R S & Aneja K R, An introduction to mycology Schliden. Bull Environ Contam Toxicol, 73 (2004) 903-910. (New \Age International Publishers Limited, New Delhi) 13 Sharma K P & Sharma S, Ecotechnology for polishing 1999. distillery waste and its reuse: Bench and pilot scale studies. 12 Kumar P & Chandra R, Detoxification of distillery effluent Final technical report submitted to the Department of through Bacillus thuringiensis (MTCC 4714) enhanced Biotechnology, Ministry of Science & Technology, New phytoremediation potential of Spirodela polyrrhiza (L.) Delhi, March 2007.
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