Microflora (fungal and bacterial) of selected terrestrial and marshy by jwt67394


									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: sharmakp_in@yahoo.co.in                                      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-
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
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                                                SHORT COMMUNICATIONS                                                        243

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   phytoremediation potential of Spirodela polyrrhiza (L.)         Delhi, March 2007.

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