ABSTRACT. Azotobacter chroococcum was isolated from straw-amended soil and found to utilize 4-hydroxybenzoic acid, resor- cinol, pyrocatechol and vanillic ...
Folia Microbiol. 39 (1), 5 7 - 6 0 (1994)
Utilization of Some Phenolic Compounds by Azotobacter chroococcum and Their Effect on Growth and Nitrogenase Activity M.H. ABD-ALLA Department of Botany, Faculty of Science, Assiut University, Assiut 71514 Egypt Received lu~y 13, 1993 Revised version September 27, 1993
ABSTRACT. Azotobacter chroococcum was isolated from straw-amended soil and found to utilize 4-hydroxybenzoic acid, resorcinol, pyrocatechol and vanillic acid as sole carbon source. Growth and nitrogenase activity ofA. chroococcum were supported by 8, 6 and 4 mmol/L of 4-hydroxybeazoie acid, rcsorcinoi and pyrocatechol, respectively, The generation time of 1.71 h in 4-hydroxybenzoie acid did not significantly differ from the generation time of 1.64 h, observed when grown in mannitol. 4-Hydroxybenzoic acid was utilized rapidly. However, the decomposition of other tested phenolic compounds set in only slowly. It was concluded that this isolate has good potential to utilize some phenolic compounds released during biodegradation of plant wastes.
Plant residues which are low in nitrogen but rich in decomposable carbon offer the energy for dinitrogen fixation by diazotrophic bacteria (Gibson et al. 1988). This process can become particularly important in natural, low-input systems where dinitrogen fixation can make a major contribution to the overall nitrogen economy. Generally this requires the cooperation of a diazotroph with the second organism capable of degrading the cellulose and hemicellulose of plant residues (Abd-Alla et al. 1992). Phenolic compounds ( 1 - 3 %) are found in decaying plant residues, soil and roots (Whitehead et at. 1983; Hartley and Whitehead 1985; Dalton et al. 1989). They may also be synthesized by soil microorganisms (Flaig 1971) or formed during humification (Haider et aL 1975). Growth of Azotobacter in soil and its ability to fix nitrogen, could be influenced by the amount of available phenolic compounds. Data on the ability of members of genus Azotobacter to utilize phenolic compounds are limited (Hardisson et al. 1968; Groseclose and Ribbons 1981; Sala-Trepat and Evans 1971). In this study we report the isolation ofAzotobacter chroococcum from straw-amended soil that is able to degrade some phenolic compounds. Special emphasis will be given to growth and nitrogenase activity of this organism in the presence of phenolic compounds as sole source of carbon and energy.
MATERIALS AND METHODS Culture and culture conditions. Isolates of Azotobacter were isolated from straw-amended soil and identified as A. chroococcum (Tchan and New 1984; Rennie 1981). The cultures were maintained in mannitol nitrogen-free growth medium (Brown et al. 1962). Degradation of phenolic compounds. The ability of the isolates to utilize phenolic compounds (4-hydroxybenzoic acid, resorcinol, vanillic acid and pyrocatechol) was studied in nitrogen-free agar medium. The phenolic acid agars (10 retool/L) were prepared as by Henderson (1961). The degradation of phenolic compounds was assessed by adding 5 mL of 1.0 % (W/V) solution of ferric chloridepotassium ferricyanide (Sundman and Nase 1971). A blue color developed in uninoculated agar medium containing phenolic compounds. The presence of yellowish-green zone in agar medium was interpreted as evidence of degradation of these compounds. Growth conditions. For growth on phenolic substances the cells were cultivated in nitrogenfree medium to which falter-sterilized substances were added at Concentrations of 2, 4, 6, 8 and 10 mmol/L. After inoculation with 1 mL of bacterial suspension (ca 106 CFU/mL), the cultures were incubated at 30 *C on a rotatory shaker (2.1 Hz) for 5 d. Growth was measured turbidimetrically at 540 nm after 12, 24, 36, 48 and 60 h. To determine the generation time, cells were grown in nitrogenfree medium containing 10 mmol/L mannitol (control). Viable counts were made by plate counts in Brown's agar medium (Brown et al. 1962).
58
M.H. ABD-ALLA
Vol. 39
Nitrogenase activity. Nitrogenase activity was determined in a closer system as described previously (Abd-Alla et aL 1992) spectrophotometrically (LaRue and Kurz 1973). Determination of protein content. Total protein of the bacterial biomass was determined by the Lowry method. Determination of residual phenolic substances. The determination of residual phenolic substances were carried out by centrifugation of growth medium at 3000g for 15 min. Following centrifugation the supernatant was allowed to stand for 24 h at 15 ~ for extracellular protein precipitation, and thereafter the precipitate was removed by centrifugation at 3000 g for 15 min. Residual phenol was determined using 1 mL aliquots of the supernatant mixed with 5 mL of double-distilled water, followed by the addition of 1 mol/L Folin-Ciocalteau reagent and immediate mixing. Three min later, 0.5 mL of a saturated Na2CO3 solution was added and mixed thoroughly (Swain and Hillis 1959). The resulting blue color was read 2 h later at 660 nm using Spectronic 2000 Bausch and Lomb spectrophotometer. All the results of growth and assays are the means of three separate experiments. Statistical analysis. Experimental data were subjected to analysis of variance. Means were separated by Duncan's multiple range test.
RESULTS A N D DISCUSSION Ability of isolates to utilize phenolic compounds
Isolates of Azotobacter chroococcum were tested for their ability to utilize phenolic compounds. Isolate MH1 decomposed all the tested phenolic compounds while other isolates utilized 4-hydroxybenzoic acid only and failed to oxidize the others (Table I). Therefore, isolate MH1 was used for further studies. Table 1. Activity ofA. chroococcum isolated M1 - M H 4 on phenolic compound agarsa
Isolate MH1 MH2 MH3 MH4
4-Hydroxybenzoic acid
Resorcinol
+ + + +
+ (+) -
Pyrocatechol
Vanillic acid
+ (+) -
(+) -
a+ complete degradation, ( + ) partial degradation, - no degradation.
Growth on phenolic compounds
Table II. Generation time ofA. chroococcum in phenolic compounds
Substance
Concentration a mmol/L
Mannitol c 4-Hydroxybenzoic acid Resorcinol Pyrocatechol Vanillie acid
Generation time, h b
10
1.64 d
8 6 4 2
1.71 d 3.87 c 5.11 b 10.67 a
aphenolic substrates at their optimum concentrations. beach value represents the mean of three replicates. Means without a different letter are significantly different at 5 % level according to Duncan's multiple range test. CControl.
Isolate MH1 was grown in liquid Brown's medium (without mannitol) containing 4-hydroxybenzoic acid, resorcinol, pyrocatechol and vanillic acid as sole carbon source. At 8 mmol/L, 4-hydroxybenzoic acid supported maximum growth (Fig. 1A). Increase in concentration to 10 mmol/L inhibited growth. Concentration of 6 mmol/L resorcinol yielded a higher cell density (Fig. 1B). Substrate concentrations above 6, 4, 2 mmol/L inhibited growth on resorcinol, catechol and vaniUic acid, respectively (Fig. 1B- D). The mean generation times for the selected isolate for growth on phenolic substances ranges from approximately 1.71 h to more than 10.6 h. The generation time of 1.71 h in 4-hydroxybenzoic acid did not significantly differ from the generation time of 1.64 h, observed during growth in mannitol (Table II). The utilization of phenolic compounds was determined during different growth periods. It was apparent that the isolate degraded 4-hydroxybenzoic acid, and this compound was depleted after 72 h. However, the utilization of other phenolic compounds set in only slowly. About 85 % of 4-hydroxybenzoic acid was utilized after 60 h. However, 47, 33 and 30 % of resorcinol, pyrocatechol and vanillic acid were degraded after 60 h, respectively (Table III).
1994
UTILIZATION OF PHENOLIC COMPOUNDS
1.2
As~o
I A
59
i A540
B
0.6 0.8
%
0/.
0.1, 0.2
8 12 I
0 0.6
D
I
A.
2
0
' 0.3
0.~, 0.2
0.2
0.1
0
2~
48 h Fig. 1. Growth (absorbanceA540) ofA. c h r o o c o c c u m on 4-hydroxybenzoic acid (A), resoreinol (B), pyrocatechol (C) and vanillic acid (D); n u m b e r a t c u r v e s - concentration in mmol/L. h
,~8
Table III. Utilization of phenolic compounds during growth ofA. c h r o o c o c c u m MH1
Substancea
Remaining phenolb, mmol/L h after inoculation 0
4-Hydroxy benzoic acid Resorcinol Pyrocatechol Vanillic acid
8 6 4 2
24
5.0 5.5 3.9 1.8
48
3.8 4.6 2.9 1.6
0
24
Table IV. Effect of phenolic compounds on nitrogenase activity of MH1 a
A. chroococcurn
Nitrogenase activityc Substance
Concentrationb mmol/L
24 h
48 h
10
1.08 a
6.6 a
8 6 4 2
1.06 a 0.62 b 0.30 c 0.00 d
3.2 a 0.98 b 0.89 c 0.00 d
60
1.2 3.2 2.7 1.4
aphenolic substrates at their optimum concentrations. bin the growth medium.
Bacteria utilizing some phenolic compounds seem to be limited to a small
Mannitolcl 4-Hydroxybenzoic acid Resorcinol Pyrocatechol Vanillic acid
aEach value represents the mean of three replicates. Values in the same column followed by the same letter are not significantly different at the 5 % level by Duncan's multiple range test. bphenolic substrates at their optimum concentrations. ~ m o l ethylene per mg protein per h. dControi.
number of organisms which include Mycobacterium species (Brune and Schink 1990), Pelobacter acidigallici (Samain et al. 1986) and Rhodococcus species (Armstrong and Patel 1992). Nitrogenase activity
Nitrogenase activity was detected when A. chroococcum MH1 was cultured in a medium supplemented with 4-hydroxybenzoic acid, resorcinol and pyrocatechol. No nitrogenase activity was detected in a medium containing vanillic acid (Table IV). Apparently, A. chroococcum MH1 isolated from decomposing straw was more effective in the decay of a wide range of phenolic substances as sole carbon sources. The ability of the isolate to utilize phenolic substances indicates the potential of the isolate to readily adapt to these substances. Reduction of acetylene to ethylene confirmed that the cell possessed nitrogenase activity and the biological activity of the bacterium remained unaffected.
60
M.H. ABD-ALLA
Vol. 39
Wu et al. (1987) failed to detect nitrogenase activity dependent on phenolic compounds in Azotobacter vinelandii. Cells were cultured on soil extracts and growth was supported by phenolic compounds. They did not test the effect of phenolic compounds on nitrogenase activity ofA. vinelandii in a defined medium or soil extract poor in nitrogen. The absence of detectable nitrogenase activity was ascribed to the presence of combined nitrogen in the soil and repression of nitrogen fixation (Buhler et al. 1987; Klugkist and Haaker 1984). If so, it would suggest that this type of nitrogen f~ation byAzotobacter in a natural environment may be limited to soils poor in nitrogen. Results of the present work indicate that some phenolic compounds would be expected to fuel nitrogen fixation. Such results are confirmed by the findings of Peterson and Peterson (1988) who reported that 4-hydroxybenzoate supported nitrogenase activity ofA. vinelandii. The results presented in this paper show that Azotobacter isolate has a good potential to utilize some phenolic compounds released during the biodegradation of plant residues (Hartley and Whitehead 1985; Abd-Alla et aL 1992). I am very grateful to an unknown referee for many important comments.
REFERENCES ABD-ALLAM.H., OMAR S.A., ABDEL-WAABA.M.: The role of cellulose-decomposing fungi in nitrogenase activity of Azotobacter chroococcum. Folia MicrobioL 37, 215- 218 (1992). ARMS'rrUNG S., PATEL T.IL: Utilization of 1,3,5-trihydroxybenzene (phloroglucinol) by a soil isolate, Rhodococcus species B P G - 8. ZBasic MicrobioL 32, 3-11 (1992). BROWNM.E., BURLINGHAMS.IC, JACKSONR.M.: Studies onAzotobacter species in soil. I. Comparison of media and techniques for countingAzotobacter in soil. Plant & Soil 17, 309-319 (1962). BRUNEA., SCHINKB.: Pyrogallol-to-phloroglucinol conversion and other hydroxy-hydroxyl-transfer reaction catalyzed by the cell extract of Pelobacter acidigallici, l.BacterioL 172, 1070-1076 (1990). BUHLERT., SANN R., MONTERU., DINGLERC., KUHLAJ., OELZEJ.: Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii. ArcltMicrobiol. 148, 247- 251 (1987). DALTONB.R., BLUMU., WEED S.B.: Plant phenolic acid in soils: Sorption of ferulic acid by soil and soil components sterilized by different techniques. SoilBiol.Biochem. 21, 1011 - 1018 (1989). FI~m W.: Organic compounds in soil. Soil Sci. 111, 19-33 (1971). GmsoN A.H., I - I A ~ D.M., Roper M.M.: Nitrogen fixation associated with residue breakdown, pp. 753-758 in H. Bothe, F.N. DeBruijn, W.E. Newton (Eds): Nitrogen Fixation - 100 Years After. Gustav Fischer, Stuttgart 1988. GROSECL(~E E.E., RIBBONS D.W.: Metabolism of resorcinylie compounds by bacteria: New pathway for resorcinoi catabolism in Azotobacter vinelandii. J.Bact. 146, 460-466 (1981). HAIDER K., [~IARTINJ.P., FILIP Z.: Humus biochemistry, pp. 195-244 in E.A. Paul, A.D. McLarren (Eds): Soil Biochemistry. Marcel Dekker, New York 1975. HARDISSONC., SALA-TREPATJ.M., STANIERR.Y.: Pathways for the oxidation of aromatic compounds by Azotobacter chroococcum. ZGen.MicrobioL 58, 1 - 110 (1969). ~ T L E Y ILD., WHrrEHFADD.C.: Phenolic acid in soil and their influence on plant growth and soil microbial processes, pp. 109-199 in D. Vaughan, R.E. Malcolm (Eds): Soil Organic Matter and Biological Activity. Martinus Nijhoff, Dordreeht 1985. HENDERSONM.E.IC: Isolation, identification and growth of some soil Hyphomycetes and yeast-like fungi which utilise aromatic compounds related to lignin. ZGen.Microbiol. 26, 149-154 (1961). KLUGgaSTJ., HAAKER H.: Inhibition of nitrogenase activity by ammonium chloride in Azotobacter vinelandii, l.Bact. 157, 148-151 (1984). LARUE T.A., KURZ W.G.W.: Estimation of nitrogenase using a colorimetr/c determination for ethylene. Plant PRysiol. 51,
i074-1075 (1973). PErEgSON J.B.,PE'mRSON A.L: para-Hydroxybenzoate supported nitrogen fLxation in Azotobacter vinelandiistrain OP (13705). Can,12~icrobioL 34, 1271 - 1275 (1988). RENNIE R.J.:A single medium for the isolation of acetylene-reducing (d/nitrogen-fixing)bacteria from soils.Can~.MicrobioL 27, 8-14 (1981). S^L^-TREPAT J.M., EVANS W.C.: The meta cleavage of catechol byAzotobacter sp. Eur.l.Biochem.20, 400-413 (1971). SAMAIN E., ALBAGNACE G., BUBOURGUIER H.-C.: Initialsteps of catabolism of trihydroxybenzenes in Pelobacter acidigallici. Arcl~MicrobioL 144, 242-244 (1986). SUNDM.*~',~V., NASE L.: A simple plate test for direct visualisationof biological lignin degradation. Paperjija puu 53, 67-71 (1971). SWAIN T., HILl.ISW.E.." The phenolic constituents of domestica. I.The quantitative analysis of phenolic constituents.ZSci.Food dgric. 10, 63-68 (1959). TCrL~NT.T., NEW P.B.: Azotobacteriaceae, pp. 220-229 in N.R. Krieg, J.G. Holt (Eds): Bergey's Manual o f Systematic Bacteriology, Vol. 1. William & Wilkins, Baltimore-London 1984. WHITEHEADD.C., DIBBH., HAR'ILEYR.D.: Bound phenolic compounds in water extract of soils, plant roots and leaf litter. Soil Biol.Biochem. 15, 133-136 (1983). Wu F.J., MOREr~OJ., VELAG.IL: Growth of Azotobacter on soil nutrients. Appl.Environ.Microbiol. 53, 489-494 (1987).
