Characterization of Aeromonas hydrophila strainsand their evaluation for biodegradation of night soil

Lokendra Singh*, M. Sai Ram1, M.K. Agarwal and S.I. AlamBiotechnology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India1Present address: Defence Institute of Physiology and Allied Sciences, Timarpur Lucknow Road, Delhi, India*Author for correspondence: Tel.: 0751 340354, Fax: 0751 341148

Received 10 March 2000; accepted 9 September 2000

Keywords: Aeromonas, biodegradation, protease, psychrotrophic

Summary

Among 67 psychrotrophic bacterial isolates of Leh, India screened for production of hydrolytic enzymes at 10 °C,four belonging to Aeromonas hydrophila were characterized and evaluated for biodegradation of night soil. Allstrains produced metalloproteases on a variety of carbon and nitrogen sources. Strains LA1 and LA15 alsoproduced a-amylase and PC5 both a- & b-amylase. No amylase was produced by PN7, however it produced lipase.Casein and glucose induced maximum enzyme activity (protease and amylase) in LA15 and PC5, respectively. InLA1, maximum induction of protease was observed with casein and of amylase with maltose. Corn oil/tributyrinserved as the best inducers for protease and lipase production by PN7. A. hydrophila strains were found to bepsychrotrophic with optimum growth and enzyme activity at 20 and 37 °C, respectively. Maximum biodegradationof night soil was observed by strain LA1 at 5±20 °C.

Introduction

In spite of the fact that 80% of the Earth's surfacepossesses extreme cold environments, our knowledgewith respect to the microorganisms of these areas isinadequate (Baross & Morita 1978). The study ofpsychrotrophic and psychrophilic microorganisms ofthese habitats is of ecological signi®cance and theirextracelluler hydrolytic enzymes are of immense potentialin the food, fermentation, pharmaceutical and detergentindustries. The low activity of mesophilic bacteria at lowtemperatures has a negative impact on the biodegrada-tion of organic wastes, which might eventually augmentthe level of pollution in these areas. The cold activebacteria of these habitats and their enzymes can thus be ofgreat use for bioremediation of organic pollutants.Undecomposed night soil causes aesthetic nuisance,

pollution of water bodies and is a potential source ofwater-borne diseases. Because their is no suitable methodfor biodegradation of night soil in low temperature areaslike Antarctica, it is either incinerated or physicallytransported to safer zones. In high altitude regions like theHimalayas the above methods are not feasible because ofhilly terrain and nonavailability of energy sources. In thiscontext, the biodegradation of waste by aerobic andanaerobic methods appears to be the viable solution. Wehave previously attempted biodegradation of night soilemploying a cold-adapted mixed bacterial consortium(Singh et al. 1993, 1995). This investigation aims at

isolation and selection of hydrolytic bacteria form Leh,India which is located at 4000 m altitude with an ambienttemperature of +25 to )25 °C. Among the severalbacterial isolates screened, four selected strains charac-terized as Aeromonas hydrophila have been studied forgrowth and enzyme production at di�erent temperaturesand evaluated for biodegradation of night soil.

Materials and Methods

Isolation of hydrolytic bacteria

Aerobic hydrolytic bacteria were isolated from samplesof water, soil and sediments from Leh, Ladakh (India).Ten-fold serial dilutions of samples were spread on thesurface of casein agar medium and plates were incubat-ed at 10 °C for 4 days. The basal medium consisted ofyeast extract, 1.5 g; beef extract, 1.5 g; NaCl, 5.0 g;casein, 2.0 g, agar, 20 g; distilled water, 1000 ml, pH7.4. Proteolytic characteristics of the isolates was ob-served by the method of Smith et al. (1954) by pouringsolution of mercuric chloride (15 g HgCl2 in 20 mlconcentrated HCl made up to 100 ml with distilledwater) on the culture plates.

Screening for production of amylase and lipase

The isolates showing proteolysis were evaluated forhydrolysis of starch and tributyrin. The starch agar

World Journal of Microbiology & Biotechnology 16: 625±630, 2000. 625Ó 2001 Kluwer Academic Publishers. Printed in the Netherlands.

medium was similar to casein agar but casein wasreplaced with soluble starch. Lipolysis was observed ontributyrin agar comprising of peptone, 5.0 g; yeastextract, 3.0 g; tributyrin, 10 ml; agar, 20 g; distilledwater, 1000 ml; pH 7.4. Inoculated plates were incubat-ed for 4 days at 10 °C. Amylolytic property wasexhibited by exposing the plates to iodine vapours andlipolysis by direct observation of a clear zone.

Culture identi®cation

The selected bacterial strains were subjected to mor-phological studies and biochemical reactions (API Kit)and were further con®rmed at Institute of MicrobialTechnology, Chandigarh, India.

E�ect of carbon sources

E�ect of carbon sources on growth and enzyme pro-duction by A. hydrophila LA1, PC5 and LA15 wasobserved in basal media containing KH2PO4, 1.5 g;K2HPO4, 1.0 g; (NH4)2SO4, 2.0 g, MgSO4, 0.8 g; CaCl2,0.1 g; yeast extract, 2.0 g; sodium citrate, 1.0 g; distilledwater 1000 ml and was added with either glucose,maltose, starch or casein (4 g/L). For studies on A.hydrophila PN7, the broth contained peptone, 1.0 g;soluble starch, 1.0 g; K2HPO4, 0.2 g; MgSO4, 0.1 g;CaCO3, 0.5 g and 2.0 g/L of soyabean meal, tributyrin,corn oil, casein or starch.Exponentially growing cultures (2 ml) were inoculated

into 250 ml Erlenmeyer ¯asks containing 100 ml ofspeci®c broth. The broth was incubated at 20 °C on ashaker (120 rev/min) and growth was monitored byabsorbance at 520 nm. The enzyme assay was per-formed from broth supernatant (7000 rev/min, 30 min)after 3 days of incubation.

E�ect of temperature

Casein-containing basal broth was inoculated withexponentially growing cultures (10 °C) and ¯asks wereincubated at 5±35 °C under continuous agitation. Theoptical density and enzyme assay were performed at theend of 4 days. Optimum temperature for enzyme activ-ity was estimated by incubating the culture supernatantand substrate mixture (as described under enzyme assay)at various temperatures between 5 and 50 °C.

Enzyme assays

ProteaseA total of 1 ml of the culture supernatant was added to1 ml of Hammarsten casein (1%) and 2 ml of Tris HClbu�er (0.1 M, pH 8.0) and incubated at 37 °C for 1 h.The reaction was stopped by adding 2 ml of trichloro-acetic acid (20%) and centrifuged at 5000 rev/min for20 min. The end products in the supernatant weredetermined by the Lowry method. One unit of enzymeactivity was de®ned as the amount of enzyme required

to release 1 lmol of tyrosine equivalents under assayconditions.

AmylaseAmylase was measured by adding 1 ml of supernatantto 1 ml of starch (1%) and 2 ml of Tris HCl bu�er(0.1 M, pH 7.0). After incubation for 30 min at 37 °C,0.5 ml of sample was transferred to a test tube contain-ing 1 ml HCl (1 M) followed by addition of 5 ml ofiodine reagent. The amount of enzyme required todecrease the optical density of blue colour of mixture by0.01/min (equivalent to 12.5 lg starch degraded) wastaken as one unit. Reducing sugars in the mixture weremeasured by the method of Miller (1959).

LipaseLipase was estimated by modi®ed method of Liu et al.(1972). A total of 1 ml of the culture supernatant wasincubated at 37 °C for 30 min with 5 ml tributyrin(emulsion with polyvinyl alcohol) and 4 ml of 0.05 M

Tris buffer, pH 7.0. The enzyme reaction was terminatedby the addition of 20 ml of acetone±ethanol mixture(1:1). Released fatty acids were titrated with 0.05 M

NaOH, using thymolphthalein as indicator. One unit oflipase was de®ned as the amount of enzyme required toliberate 1 lmol of free fatty acids (n-butyric acid) perminute.

Biodegradation studiesExponentially growing cultures in broth containing0.5% peptone and 0.1% yeast extract at 10 °C wereused as inocula. Diluted night soil (10% w/v) in ¯askswas incubated at 5, 10 and 20 °C on shaker (120 rev/min) and Biochemical Oxygen Demand (BOD) andChemical Oxygen Demand (COD) were measured as perstandard methods (APHA 1985). All the above exper-iments have been carried out in duplicate and datapresented are average values.

Results and Discussion

A total of 67 psychrotrophic hydrolytic bacteria, capa-ble of growing at 10 °C on casein, starch and tributyrinwere isolated from soil, water and sediment samples.Based on qualitative screening for hydrolysis of di�erentsubstrates, four bacterial isolates, viz. PC5, LA1, PN7and LA15 were selected. Hydrolytic zones on di�erentsubstrates by the isolates are shown in Table 1. Allisolates indicated production of protease, amylase andlipase simultaneously, although the zone of hydrolysisvaried with the substrate and isolate. PC5, LA1 andLA15 produced bigger zones of hydrolysis for proteinand starch and a poor zone for tributyrin. On the otherhand, PN7 e�ciently hydrolysed lipid substrate com-pared to other substrates. All these cultures were foundto be Gram-negative rods of size varying from 0.7 to1.5 lm and were actively motile. These cultures wereidenti®ed as A. hydrophila.

626 L. Singh et al.

A. hydrophila PC5, LA1 and LA15 produced signif-icant amounts of amylases and proteases and strain PN7produced proteases and lipases in broth cultures. Lipasecould not be detected in the former three strains noramylase in PN7. Other workers have also reported thesecretion of proteases (Rivero et al. 1991; Ibanez et al.1992), amylases (Gobius & Pemberton 1988) and lipases(Upton & Buckley 1995) from A. hydrophila.

Enzyme production on di�erent substrates

Table 2 shows growth and enzyme production by strainsPC5, LA1 and LA15 in presence of glucose, maltose,starch and casein. Maximum growth of strains PC5 wasobserved in the presence of casein, which served as apoor carbon source for growth of LA1 and LA15.Glucose has been the best carbon source for LA15 asagainst maltose and starch for LA1. In general, proteaseand amylase production were in accordance with thegrowth of the isolates, excepting protease production byA. hydrophila LA15 which has been highest (2.73 units/ml) in presence of maltose as compared to glucose. Thusenzyme induction by di�erent carbon sources variedwith the strains. The enzymes appear to be partiallyconstitutive since substantial levels were recorded withall substrates. In general the induction of protease andamylase was maximal in the presence of their respectivepolymeric substrates, i.e. maltose and casein, howeverA. hydrophila PC5 showed the unique characteristic ofexhibiting the highest induction of both the enzymes inthe presence of glucose. In a similar ®nding O'Reilly &Day (1983) have reported better protease induction byA. hydrophila in the presence of sucrose. In anotherstudy protease production by Vibrio sp. has beensuggested to be under an inducer catabolite repression

that is actually re¯ected by the growth rate and energystatus of the cell (Wiersma & Harder 1978). Thisbacterium when under stress grew at a lower rate andproduced abundant amounts of protease. The reportedstrain also showed comparatively poor growth in thepresence of glucose and possibly could be undernutritional stress and thus might have induced higherlevel of enzymes.A. hydrophila PN7 showed better growth in the

presence of casein as compared to starch, maltose andglucose (data not shown). The e�ect of di�erentinducers for protease and lipase production is shownin Table 3. Fat-containing substrates elicited highertitres of protease as compared to protein and starchsubstrates. Titres obtained with tributyrin and corn oilwere 4.70 and 4.10 units/ml, respectively. which even-tually increased to 4.95 units/ml when the bacteriumwas grown on the mixture of two substrates. Surpris-ingly the protease induction was higher in the presenceof fat-containing substrates which may be due tonutritional stress and slower growth of bacteria onthese poorly utilizable carbon sources. Lower inductionin presence of casein can be explained on the line ofobservations made by Wiersma & Harder (1978) whohypothesized that the presence of excess amino acidsand peptides in the substrate which would repressprotease synthesis. O'Reilly & Day (1983), while ob-serving the e�ect of di�erent nitrogen sources onprotease induction, recorded the repression of enzymein the presence of casein. The lipase titre was maximum

Table 1. Qualitative assay for hydrolytic enzymes of A. hydrophila.

Strain Protease Amylase Lipase

PC5 +++ +++ +

LA1 +++ +++ +

LA15 +++ ++ +

PN7 ++ + +++

Zone of hydrolysis (diameter) after 4 days at 10 °C: + = <10 mm,

++ = 10±20 mm, +++ = >20 mm.

Table 2. Protease and amylase production on di�erent carbon substratesa.

Strain Glucose Maltose Starch Casein

A. hydrophila PC5 Growth (5´) 0.79 0.84 0.83 1.18

Protease (units/ml) 5.70 (2.65) 4.93 (2.15) 5.31 (2.35) 7.93 (2.46)

Amylase (units/ml) 3.10 (1.44) 2.42 (1.06) 2.63 (1.16) 3.71 (1.15)

A. hydrophila LA1 Growth (5´) 0.78 0.82 0.82 0.49

Protease (units/ml) 4.61 (2.16) 5.22 (2.33) 4.70 (2.10) 3.81 (2.84)

Amylase (units/ml) 1.91 (0.90) 2.73 (1.22) 2.00 (0.89) 1.33 (0.99)

A. hydrophila LA15 Growth (5´) 0.96 0.51 0.40 0.41

Protease (units/ml) 1.98 (0.75) 2.73 (1.96) 2.20 (2.02) 2.43 (2.17)

Amylase (units/ml) 1.37 (0.52) 1.20 (0.86) 0.94 (0.86) 1.04 (0.93)

a Values in parentheses indicate enzyme units per milligram of cell protein.

Table 3. Production of protease and lipase by A. hydrophila PN7 on

di�erent carbon substrates.

Substrates Protease (units/ml) Lipase (units/ml)

Tributyrin 4.70 (4.27) 0.75 (0.68)

Corn oil 4.10 (4.55) 0.75 (0.83)

Casein 3.43 (1.14) 2.10 (0.70)

Starch 2.32 (1.05) 1.00 (0.45)

Soyabean 2.82 (1.17) 1.50 (0.62)

Casein + Tributyrin 3.00 (1.07) 2.33 (0.83)

Casein + Corn oil 3.50 (1.30) 1.83 (0.68)

Corn oil + Tributyrin 4.95 (4.17) 1.60 (1.33)

Soyabean + Tributyrin 2.94 (1.47) 1.33 (0.66)

a Values in parentheses indicate enzyme units per milligram of cell

protein.

Night soil biodegradation by Aeromonas 627

on casein-containing medium (2.10 units/ml), howeverits induction (as shown by speci®c activity) has beenbetter on fat-containing media.

E�ect of temperature on growthand enzyme production

The e�ect of temperature on A. hydrophila strains isshown in Figures 1±4. The cultures showed growth inthe range of 5±35 °C with the optimum at 20 °C. In asimilar study Shivaji et al. (1989a, b) have reported thegrowth of psychrotrophic Antarctic isolates in thetemperature range of 4±30 °C with the optimum at20 °C. Amylase and protease production by PC5 corre-lated with the growth, though no protease activity wasdetected at 5 and 35 °C. Other strains produced detect-able levels of enzymes at 5 °C however, the productionat 35 °C varied with the strain and enzyme. A. hydro-phila LA1 and LA15 could not produce protease at35 °C whereas culture supernatant of PN7 containeddetectable levels of protease. At the same temperature,amylase activity was not exhibited by strain LA15compared to 0.48 units/ml of amylase produced bystrain LA1. Lipase production by PN7 was observed at5±35 °C. All of the enzymes were produced maximallyat the optimum temperature of growth, i.e. 20 °C.Burini et al. (1994) have reported that in the psychro-trophic bacterium Pseudomonas ¯uorescens, productionof several enzymes was regulated by the growth tem-perature. The present ®nding is in agreement with thisobservation.It has been observed that production of amylase,

protease and lipase correlates with the growth ofdi�erent strains of A. hydrophila. Although optimumtemperature for growth and enzyme production hasalways been 20 °C, all enzymes were found to beoptimally active at 37 °C. Similar higher temperatureoptima for protease activity than its production hasbeen reported in Candida humicola isolated from

Antartica (Ray et al. 1992). Amylase and lipase of A.hydrophila strains have been most active at neutral pH(7.0) and proteases in the alkaline range (8.0±9.0). Theproteases of these strains were inhibited by EDTA andstimulated in the presence of Fe2+ and thus theseenzymes have been characterized as metalloproteases.Based on blue value reduction and the production ofreducing sugars, PC5 was observed to produce a- andb-amylases (Singh et al. 1994). Amylases of LA1 andLA15 showed fast blue value reduction with a slowincrease in reducing power, hence were characterised asa amylase.

Biodegradation studies

The diluted night soil, subjected to study the e�ciency ofbiodegradation, contained a BOD of 11125 mg O2/l andCOD of 13240 mg O2/l. The BOD reduction of A.hydrophila strains at di�erent temperatures is shown inFigure 5. During the course of 20 days study, it wasobserved that all selected strains of A. hydrophilareduced the BOD of night soil proportionally tothe temperature (5±20 °C). The biodegradation at 5 °Cwas comparatively slow and continued for 20 days.

Figure 1. E�ect of temperature on growth and enzyme production by

A. hydrophila PC5.

Figure 2. E�ect of temperature on enzyme production by A. hydro-

phila LA1.

Figure 3. E�ect of temperature on growth and enzyme production by

A. hydrophila LA15.

628 L. Singh et al.

However, at higher temperatures most of the organicmatter was degraded during 10 days of incubation. Thepercent BOD reduction at the end of 20 days variedfrom 44±70 (5 °C), 59±78 (10 °C), and 62±80 (20 °C).Very little di�erence in biodegradation was observed at10 and 20 °C by strains LA1 and LA15.A. hydrophila LA1 has been observed as the most

e�cient bacterium for biodegradation of night soil atlow temperature, since it reduced the BOD of night soilin the range of 34±77% (COD 30.5±59%) compared to26.5±72.4% (COD 23±56.8%) reduction by other iso-lates during the period of 10 days. The results ofbiodegradation are in accordance with the productionof hydrolytic enzymes as LA1 produces higher level ofamylases and proteases compared to other strains. OtherGram-negative bacteria such as Vibrio, Aeromonas andPseudomonas have been reported to exist in decompos-ing seaweeds and other marine habitats and to secreteagarases and similar enzymes (Hamamoto et al. 1994).Odagami et al. (1993) reported cold active extracellularproteases from A. haloplanctis which were found to beresponsible for the decomposition of ®sh meat at lowtemperature. The dominance of proteases in the present

study appears to be more relevant for biodegradation ofnight soil as it is mainly composed of proteins.This study shows that psychrotrophic bacteria isolat-

ed from low temperature areas have great potential forbiodegradation of organic matter. Psychrotrophicstrains of A. hydrophila play the desired role due toproduction of signi®cant amounts of proteases as well asother hydrolytic enzymes at low temperature in presenceof a variety of carbon sources.A. hydrophila has been reported as pathogenic to ®sh

and other aquatic fauna (Dahle 1971; Allen & Stevenson1981). The pathogenicity was attributed to the produc-tion of extracellular haemolysin and proteases (Ambor-ski et al. 1984). It was reported that pathogenic strainsof A. hydrophila produce three type of proteases: a heat-labile serine protease, a heat-stable metalloprotease andan elastase (Rivero et al. 1991). Chiou & Chang (1994)also demonstrated the lipase and protease gene ofA. hydrophila and deduced that the lipase contributesinsigni®cantly to the virulence of the bacterium whereasthe protease does not act as the sole virulence factor.However, contrary to these reports, all strains ofpsychrotrophic. A. hydrophila reported in this studydid not show any pathogenicity to ®sh (Rasboradaniconius) and snail (Indoplanorbis exustus) when testedin the laboratory. The non-pathogenicity may be due todi�erence in temperature optima, type of enzymes andtheir characteristics compared to reported strains. How-ever, further investigations are required to understandthe exact reason. These strains not only have importancefor biodegradation of wastes at low temperature, but theenzymes may also ®nd applications in the detergent,leather, beer and food industries.

Acknowledgement

The authors are grateful to Dr R.V. Swamy, Director,DRDE, Gwalior for his guidance and continued interestin the study.

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