Isolation and Biochemical Characterization of Bacteria Present In Roots of Water Hyacinth (Eichhornia crassipes (Mart) Solm.) In Robertson Lake Jabalpur.

Sadhana Jaiswal¹ and P. K. Singhal²

¹School of Studies in Life Sciences, Pt. Ravi Shankar Shukla University Raipur, (C G) 492010 India

²Department of Biological Sciences, Rani Durgawati University, Jabalpur, India.

ABSTRACT:

The present study was conducted to isolate bacteria from root of Eichhornia crassipes (water hyacinth). Bacteria were isolated in tryphtone glucose yeast extract agar (TGYEA) medium during different phases of growth of water hyacinth in Robertson lake and on basis of biochemical characteristics probable identification was done by using a PIB program, given by Bryant (1989). A total of 30 bacteria were isolated from roots of the growing water hyacinth plants in the Robertson lake Jabalpur. Out of 30 bacteria, 25 were gram - negative rods, comprising of 23 fermentative and 2 non - fermentative, 3 were gram - positive rods, and 2 were gram - positive cocci. Most of the bacteria were able to hydrolyze starch and glucose, and reduce NO₃ to NO₂, indicating their active role in transformation of C and N. The presence of gelatinase enzyme in most of the bacteria has indicated their role in active degradation of organic N.

KEYWORDS: Water hyacinth, bacteria, aquatic ecosystem, Robertson Lake, C and N transformation.

INTRODUCTION:

Water hyacinth is one of the fastest growing plants known to man and considered to be the most noxious aquatic weed (Gopal, 1987). Because of its high nutrient absorption potential, it has already been tried for treatment of large variety of wastewaters (Moorhead et al., 1988; Orth and Sapkota, 1988; Tripathi and Shukla, 1991; Casabian – Chassany et al., 1991; Jabahesan, 1997; Gupta and Sujatha, 1996; Krishanthi et al., 1991; Quazzani et al., 1995; Dhote, 2007). Such systems using water hyacinth or other aquatic plants have been appropriately named as living machines (Todd, 1994). It was reported that the total nutrient removal by water hyacinth systems far exceeds the plant uptake rate (Orth and Sapkota, 1988). Additional processes such as bacteria-mediated biochemical processes may be responsible for enhanced nutrient removal efficiency (Reddy and DeBusk, 1985; Moorhead et al., 1988). Its extensive root system creates an excellent habitat for bacterial populations. The transport of photosynthetic O₂ from water hyacinth shoots to its roots creates an oxygenic microenvironment in the root-water interface (Moorhead and Reddy, 1988). This oxygenic microenvironment plays a vital role in complete and faster oxidation of organic matter (Stowell et al., 1981; Wolverton, 1987; DeBusk and Reddy, 1987). The bacteria utilize O₂ as an electron acceptor in synthesis of their biomass along with oxidation of organic matter (Metcalf and Eddy, 1991).

In addition, the roots release some labile organic compounds that support the growth of heterotrophic bacteria (Silvester and Musgrave, 1991). The roots, therefore, can act as a trickling filter with thick bacterial biofilm in active degradation of organic matter (Stowell et al., 1981). These bacteria may produce metabolites for their as well as plant growth. These can also play a major role in removal of nutrients. The dynamic nature of such an aqueous rhizosphere may prove to be valuable in the management of wastewaters (Timberlake et al., 1988).

The above concepts about the processes operating and type of bacterial populations in the roots of water hyacinth are still in infancy. The knowledge about the bacteria present in the roots of water hyacinth can greatly help in management of the water bodies infested with water hyacinth and optimization of treatment designs based on water hyacinth. The present study has been planned to fill this void by gathering scientific information about the bacterial populations associated with water hyacinth roots.

MATERIAL AND METHOD:

Study area; The present study was performed in the actively growing water hyacinth plants collected from the Robertson lake, Jabalpur (23⁰ 6^' to 23⁰ 10^' North, 79⁰53^' to 80⁰6^' East, 397 AMSL). The lake is situated in the eastern part of Jabalpur and has the surface area of 0.7 km². The domestic wastes from neighbouring residential quarters and wastewater from the Vehicle factory are drained into the lake. The lake has dense stand of water hyacinth that covers nearly 75% of its surface area.

Enumeration and isolation of bacteria from the roots of water hyacinth:

The density of bacterial population was enumerated during different phases of active growth of water hyacinth in the lake. Fresh water hyacinth plants from the lake were harvested in its different phases of growth (i.e. about 25 g, 50 g, 100 g, 200 g, 500 g, and 1000 g fresh weight) in sterile polythene bags. The bags were sealed immediately at the site and brought to the laboratory under ice. The root and shoot portions of the plants were weighed separately to determine the root:shoot ratio. Thereafter, the roots were washed with sterile distilled water and 5 grams root pieces were thoroughly macerated in a sterilized pastle and mortar. The homogenized mixture was diluted up to 50 ml with sterile distilled water, and shaken vigorously. One ml of this homogenate was taken and diluted appropriately by serial dilution technique. The diluted samples were plated in TGYEA medium. The whole process was completed under aseptic conditions in an inoculation chamber. The plates were incubated at 37 ± 1°C for 24 to 48 h, and the bacterial colonies appeared were counted with the help of a digital colony counter. The segregated colonies were picked up and purified by sub-culturing and maintained on agar slants.

Biochemical analysis of isolated strains of bacteria:

The bacteria isolated from roots of water hyacinth were subjected to various tests. Gram positive or negative nature and the shape of bacteria were determined by Gram staining, motility by gelatin stab method, and other characteristics by various biochemical tests as Indole test, Methyl red test, Vogus proskauer test, Citrate test, Triple Sugar Iron agar test , H₂S test and Oxidation-Fermentation test were performed by following standard methods. The enzyme activities like Catalase, Oxidase, Amylase, Gelatinase and Nitrate-Reductase were determined by following the standard methods. The growth of bacterial isolates was also measured in XLD, MacConkey agar, 1% NaCl, and 5% NaCl at room temperature and at 37⁰C.

Identification of Bacterial Isolates:

On the basis of biochemical tests, the bacterial isolates were identified by using a PIB program (Bryant, 1989). The identification routine used in PIB is based on Willcox’s implementation of Baye’s theorem for use with bacteria (Willcox et al., 1973, 1980). An isolate was considered as identified if the identification score for one taxon equaled the identification threshold (0.98), though accepted range for this value was from 0.95 to 0.999. An identification score of 0.6 or less did not confirm the identification of the isolate but specified its most likely taxon.

Table 1: Change in bacterial density on roots of water hyacinth during its different phases of growth in Roberson Lake. (Values are mean ± standard deviation).

Weight of plant parts			Bacterial density on root (log cfu g dw ^-1)
Whole Plant g fw (g dw)	Shoots g dw (%)	Roots g dw (%)
25 ± 0.57 (1.21 ± 0.02)	0.717 ± 0.26 (59%)	0.491 ± 0.28 (41%)	8.5 ± 0.076
50.2 ± 0.49 (2.15 ± 0.30)	1.308 ± 0.44 (60.8%)	0.836 ± 0.23 (38.8%)	8.63 ± 0.021
99.4 ± 3.05 (4.59 ± 0.21)	3.068 ± 0.12 (66.8%)	1.52 ± 0.1 (33.1%)	8.92 ± 0.004
190 ± 4.18 (9.37± 0.23)	6.66 ± 0.45 (71%)	2.71 ± 0.09 (28.9%)	9.02 ± 0.014
486 ± 7.48 (22.65± 0.36)	14.65 ± 0.22 (64.5%)	8.00 ± 1.20 (35.4%)	9.66 ± 0.021
964 ± 14.37 (45.77± 0.57)	33.06 ± 0.58 (72.2%)	12.7 ± 1.66 (27.7%)	9.93 ± 0.006

fw, fresh weight; dw, dry weight; cfu, colony forming unit.

Table 2 : Biochemical characteristics of bacteria isolated from root of water hyacinth plants growing in Robertson lake.

(a) gram- negative fermentative rods

Isolate no. Name (Identification score)	Motility	Growth on				Enzyme assay				In dole	MR	VP	Citrate	Fermentation			NR
		NaCl		McConkey	XLD	Amylase	Gelatinase	Oxidase	Catalase					Glucose	Lactose	Sucrose
		1%	5%
R1 Aeromonas hydrophila (0.93)	-	+	-	-	-	+	+	+	+	+	+	-	-	+	-	-	-
R2 Salmonella pullorum (0.81)	-	+	+	-	-	+	+	-	+	-	+	-	-	+	-	-	-
R3 Providencia stuartii (0.89)	-	+	-	-	-	+	+	-	+	+	+	-	-	+	-	-	+
R4 Vibrio vulnificus (0.98)	+	+	-	-	-	+	+	+	+	-	-	-	-	+	-	-	+
R5 Kl.pneumoniae (0.87)	-	+	-	-	-	+	-	-	+	-	+	-	-	+	-	-	+
R6 Proteus penneri (0.87)	-	+	-	-	-	-	+	-	+	-	-	-	-	+	+	+	+
R7 Kl.pneumoniae (0.85)	+	+	-	-	-	+	+	-	+	-	+	-	-	+	+	+	+
R8 Kl.pneumoniae (0.85)	-	+	-	+	+	+	+	-	+	-	+	-	+	+	+	+	+
R9 Erwinia herbicola (0.92)	-	+	-	-	-	+	+	-	+	-	+	-	-	+	-	-	-
R10 Providencia stuartii (0.94)	-	+	+	+	+	+	+	-	+	+	+	-	+	+	-	-	+
R11 Erwinia herbicola (0.89)	-	+	-	-	-	+	+	-	+	-	+	-	-	+	-	-	+
R12 Vibrio vulnificus (0.67)	+	+	-	-	-	+	+	-	+	-	+	-	-	+	-	-	+
R13 Kl. pneumoniae (0.90)	-	+	-	-	-	+	+	-	+	-	+	-	-	+	-	-	+
R14 Shigella sp. (0.83)	-	+	+	-	-	+	+	-	+	+	+	-	-	+	-	-	+
R15 Salmonella gellinarum (0.88)	-	+	+	-	-	+	+	-	+	-	+	-	-	+	-	-	+
R16 Salmonella pullorum (0.88)	-	+	+	-	-	+	+	-	+	-	+	-	-	+	-	-	+
R17 Vibrio metschnikovii (0.98)	-	+	+	-	-	+	+	-	+	-	-	-	-	+	-	-	+
R18 Aeromonas salmonicida (0.90)	-	+	+	-	-	-	-	-	+	-	+	-	-	+	-	-	-
R19 Unidentified	+	+	-	+	+	-	-	-	+	-	-	-	+	+	+	+	-
R20 Unidentified	-	+	+	-	-	+	+	-	+	-	+	-	-	+	+	+	+
R21 Unidentified	+	+	+	-	-	+	+	-	+	-	+	-	-	+	-	-	+
R22 Unidentified	-	+	+	-	-	+	+	-	+	-	+	-	-	+	-	-	+
R23 Unidentified	-	+	+	-	-	-	+	-	-	-	+	-	-	+	-	-	+

(b). Gram - negative non - fermentative rods.

Isolate no. Name (Identification score)

Motility

Growth on

Enzyme assay

In dole

MR

VP

Citrate

Fermentation

NR

NaCl

McConkey

XLD

Amylase

Gelatinase

Oxidase

Catalase

Glucose

Lactose

Sucrose

1%

5%

R24 Pseudomonas pseudoalcaligens (0.88)

+

+

+

-

-

+

-

-

+

-

-

-

-

-

-

-

-

R25 Alcaligens faecalis (0.67)

+

+

+

+

+

-

-

-

+

+

-

-

+

-

-

-

+

(c). Gram - positive fermentative rods.

R26 Erysipelothrix rhusiopathiae

-

+

-

-

-

-

+

-

-

-

+

-

-

+

-

-

+

R27 Bacillus alvei

+

+

+

-

-

+

+

-

+

-

-

-

-

+

-

-

+

R28 Brochothrix sp.

-

+

+

-

-

+

+

-

+

+

+

+

-

+

-

-

+

(d). Gram - positive cocci

R29 Unidentified

-

+

+

-

-

+

+

-

+

-

+

-

-

+

-

-

+

R30 Unidentified

-

+

+

-

-

+

+

-

+

-

+

-

+

+

-

-

+

MR, Methyl red; VP, Voges proskauer ; NR, Nitrate reductase; XLD, Xylose Lysine Deoxycholate

RESULTS:

During the growth by water hyacinth plants, the proportion of roots to the total biomass decreased and that of the shoots increased. The proportion of roots to total biomass decreased from 41% in young plants to 28% in mature plants (Table 1). The proportion of shoots to total biomass increased from 59% in the young plants to 72% in the mature plants. During the growth by plants, the bacterial density on the roots increased from 3.2´10⁸ cfu g dw^-1 in young plants to 8.5´10⁹ cfu g dw^-1 in mature plants (Table 1). The increase in bacterial density was explained well by a simple linear curve (r²= 0.99). The bacterial density in the roots has increased linearly despite a decrease in the relative proportion of roots in the total plant biomass. As a result, total bacterial density in the roots had increased considerably from 8.2´10⁹ cfu plant^-1 in young plants to 1.1´10¹¹ cfu plant^-1 in mature plants.

A total of 30 bacteria were isolated from roots of the growing water hyacinth plants in the lake. Out of 30 bacteria, 25 were gram-negative rods comprising of 23 fermentative and 2 non – fermentative strains, 3 were gram-positive rods, and 2 were gram-positive cocci (Table 2). Out of 23 gram-negative rods, two isolates scored the maximum identification score of 0.98 and were identified as Vibrio vulnificus (R₄) and Vibrio metschnikovii (R₁₇). Thirteen bacterial isolates showed the identification score of about 0.9, and were identified as Aeromonas hydrophila (R₁), Providencia stuartii (R₃ and R₁₀), Erwinia herbicola (R₉ and R₁₁), Klebsiella pneumoniae (R₁₃), Salmonella gellinarum (R₁₅), Salmonella pullorum (R₁₆) and Aeromonas salmonicida (R₁₈). Two isolates, viz. Salmonella pullorum (R₂) and Shigella sp. (R₁₄), obtained an identification score of about 0.8. One isolate (R₁₂) was identified as Vibrio vulnificus with identification score of 0.7. The remaining 5 isolates (R₁₉, R₂₀, R₂₁, R₂₂ and R₂₃) showed an identification score of less than 0.7 and were not identified (Table 2 a).

The two gram-negative non-fermentative rods were identified as Pseudomonas pseudoalcaligens (R₂₄) and Alcaligens faecalis (R₂₅) with identification score of 0.9 and 0.7, respectively (Table 2 b). The three gram-positive fermentative rod were identified as Erysipelothrix rhusiopathiae (R₂₆), Bacillus alvei (R₂₇) and Brochothrix sp. (R₂₈) (Table 2 c). The two gram-positive cocci (R₂₉ and R₃₀), and could not be identified (Table 2 d).

The motility was present only in 8 bacteria, i.e. isolate no. R₄, R₇, R₁₂, R₁₉, R_21, R_24, R₂₅ and R₂₇, while the remaining 22 isolates were non-motile. All the isolates showed flourishing growth in 1% NaCl, but only 17 isolates could grow in 5% NaCl. Kl. pneumoniae (R₈), Providencia stuartii (R₁₀), an unidentified strain (R₁₉) and Alcaligens faecalis were the only ones recording growth on McConkey and XLD agar. Amylase activity was detected in all the isolates, except for Proteus penneri (R₆), Aeromonas salmonicida (R₁₈) and two unidentified strains (R₁₉ and R₂₃). Many of the isolates were able to liquefy gelatine, except for only 5 isolates. The activity of oxidase enzyme was detected in only two isolates (R₁ and R₄), while the catalase activity was shown by all the bacterial isolates except for R₂₃ and R_26. Only six isolates, viz. R₁, R_3, R₁₀, R₁₄, R₂₅ and R₂₈, were capable of producing indole from tryptophan medium. Most of the isolates showed the positive Methyl red test. Brochothrix sp. (R₂₈) was the only isolate that showed the positive Voges Proskauer test. Kl. pneumoniae (R₈), Providencia stuartii (R₁₀), Alcaligens faecalis (R₂₅) and two unidentified bacteria (R₁₉ and R₂₅₎ were able to use citrate as the sole source of carbon. Most of the strains were able to ferment glucose anaerobically, whereas only few strains like R₆, R₇, R₈, R₁₉ and R₂₀ were capable of fermenting all the three sugars of TSI medium both anaerobically and aerobically. All the isolates were able to reduce NO₃ to NO₂, except for 6 isolates, R₁, R₂, R₉, R₁₈, R₂₄ and R₁₉.

DISCUSSION:

The buffered anaerobic and organic environment in the root-water interface has favored the proliferation of facultative anaerobic bacteria on the roots. The bacterial density per unit root biomass has increased with the maturation of plants, obviously due to a rapid increase in the root’s surface area and release of labile organic compounds therein (Silvester and Musgrave, 1981). Majority of the bacteria isolated from the roots were able to hydrolyze starch and glucose and reduce NO₃ to NO₂, indicating their active role in transformation of C and N. The presence of gelatinase enzyme in most of the bacteria has indicated their role in active degradation of organic N. Some of them, like Aeromonas hydrophila, Alcaligens faecalis, Brochothrix sp. Providencia stuartii, Pseudomonas stutzeri and Shigella sp., were able to degrade proteins under anaerobic conditions by producing indole through deamination of tryptophan. The others like Klebsiella pneumoniae, Micrococcus agilis, Proteus penneri and 3 unidentified bacteria could ferment glucose, lactose and/or sucrose, highlighting their role in degradation of monomeric as well as oligomeric carbohydrates. Alcaligens faecalis has been reported to play an important role in simultaneous nitrification and denitrification (Robertson and Kuenen, 1989). Therefore, the abundant growth of bacteria on the extensive root system of water hyacinth has helped in rapid transformations of C, N and P in the root - water interface.

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Received on 10.05.2011

Modified on 13.05.2011

Accepted on 17.05.2011

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Research J. Science and Tech. 3(3): May-June. 2011: 164-168