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Bacterial suppression of chlorella by hydroxylamine production
Water Research Vol. 13, pp. 267 to 273 Pergamon Press Ltd 1979. Printed in Great Britain.
BACTERIAL SUPPRESSION OF CHLORELLA BY HYDROXYLAMINE PRODUCTION* PAUL S. BERGER, JINNQUE RHO and HAIM B. GUNNERt Department of Environmental Sciences, University of Massachusetts, Amherst, MA 01003, U.S.A.
(Received 2 October 1978) Abstract--An actively nitrifying Arthrobacter sp. isolated from a lake inhibited the growth of Chlorella vulgaris. This was found to be due to hydroxylamine released by the bacterium during the oxidation of ammonium or other reduced nitrogen compounds. Plates containing Plate Count Agar streaked with Arthrobacter accumulated 5/~gml - t hydroxylamine-N. Chlorella was sensitive to less than 0.24/~g ml - 1 hydroxylamine-N. Non-nitrifying bacterial isolates of lacustrine origin did not demonstrate inhibiting activity. Our results indicate that nitrifying microorganisms may thus affect the population dynamics of algae in eutrophic lakes.
INTRODUCTION Algal growth has been shown to be stimulated or repressed by bacteria, depending upon species and environmental factors. F o r example, bacteria-mediated algal inhibition may be due to cell-wall lysis upon direct contact with several types of myxobacteria (Shilo, 1970; G r o m o v et al., 1972), unsuccessful competition for limiting nutrients (Rhee, 1972; Coughlan, 1977), phage transfer (Zavarzina, 1964) and toxic substances released by bacteria into the surrounding medium (Berland et al., 1972; Reim et al., 1974). Several investigators have found that low molecular weight c o m p o u n d s liberated by bacteria suppress algal growth (Granhall & Berg, 1972; Shiaris & Morrison, 1976), but seldom have been identified. This investigation was undertaken to identify the mechanism and causative agent by which a lake bacterium strongly inhibited the growth of Chlorella vulgaris. MATERIALS AND METHODS
Culture and media The bacterium in this study was originally isolated by standard dilution plate techniques from water in the littoral zone of Quabbin Reservoir in central Massachusetts. Its biochemical and morphological characteristics were most consistent with those for the genus Arthrobacter (Bergey's Manual, 1974), and henceforth is referred to as Arthrobacter sp. Q I. Other lacustrine bacteria were also isolated. A strain of Chlorella vulgaris (IUCC no. 398) was obtained from the Carolina Biological Supply Co. Confirmation of cellular purity was achieved by successive streaking on Plate Count Agar (PCA) (Difco). * This work was supported in part by the Massachusetts Agricultural Experiment Station Project NE 39 and Department of the Interior, Water Resources Research Grant WRB 058. t Please forward all correspondence to Dr. Haim B. Gunner, Principal Investigator, Department of Environmental Sciences, University of Massachusetts, Amherst, MA 01003, U.S.A.
A stock culture of Chlorella was maintained on a rotary shaker at room temperature under two 40-W cool-white fluorescent lights in Bold's Basal Medium (BBM) (Nichols and Bold, 1965). Fresh cultures were prepared as required. Most interactions between the Arthrobacter and Chlorella were studied in Petri dishes containing PCA. Pour plates of Chlorella were prepared, incubated at 26°C in the dark for 1 day, then streaked once with the bacterial culture and reincubated in the dark. In one experiment, plates were incubated at 20°C under 40-W cool-white fluorescent light. Algal colonies appeared within 3-4 days. Other media were used as indicated.
Chemical tests Chemical tests were performed on slices of agar medium to determine the presence of nitrogenous compounds. To prepare agar for these tests, a total of 4 g agar medium taken in equal aliquots from three different plates was used in all tests. Agar was sliced from the plates and transferred to a tissue grinder (glass tube and glass pestle). Deionized, distilled water was then added, the agar macerated, and the resultant slurry transferred quantitatively to a test tube. A total of 10 ml water was used. The.test tube containing the agar slurry was mixed thoroughly on a Vortex mixer and the sample subjected to immediate chemical testing. Hydroxylamine was measured by the 8-hydroxyquinoline method (Magee & Burris, 1954). Nitrite was determined by the sulfanilamide procedure (Standard Methods, 1975). After color development, the reaction mixtures were centrifuged and the optical density of the clarified supernatant was determined spectrophotometrically. No significant amounts of hydroxylamine or nitrite adhered to the centrifuge-formed pellet. Chlorophyll-a determinations Chlorophyll-a determinations were performed by a modification of the trichromatic method (Standard Methods, 1975) with the following modifications. Four grams of Chlorella-inoculated agar from three plates were transferred to a tissue grinder and 6--8 ml 90% acetone added. The agar was macerated and quantitatively transferred to a screw-cap test tube. The total volume of the mixture was brought to 10ml with 90% acetone. The mixture stood overnight at 4°C in the dark and was then centrifuged. The volume of the clarified extract was measured and the optical density determined at wavelengths of 663, 645 and 630 nm. Chlorophyll-a concentrations were calculated by the equation presented in Standard Methods. 267
268
PA~L ~. Bi]~(ilR, JINNQI I RHO and HAIM B. (}t NNIR !
Fig. 1. Pour plates of Chlorella in Plate Count Agar streaked once with a bacterial isolate. Zone of inhibition is evident around Arthrohacter streak (center plate) but not around Flavobacterium (upper plate) or Nocardia (lower platc). RESULTS
In order to investigate in depth our preliminary observation that Arthrobacter sp. Q1 colonies were hindering the growth of Chlorella t~ulgaris on PCA plates, pour plates of PCA containing this alga were streaked once with selected bacterial isolates of lacus-
trine origine. Our results are indicated in Fig. 1. The wide zone of inhibition of Chlorella by Arthrobacter sp. Q1 on PCA failed to appear in the presence of two other bacterial species--Flavobacterium sp. and Nocardia sp. The zone of inhibition always occurred with Arthrobacter regardless of whether the plates
Bacterial suppression of Chlorella
269
robacter cultures never rose above 7.4 in m-Plate Count Broth. Earlier experiments by our group had revealed that Arthrobacter sp. Q1 and several other lake isolates were heterotrophic nitrifiers (Rho et al., 1978). To ascertain whether any correlation existed between nitrification and algal inhibition on agar plates, both nitrifiers and non-nitrifiers were streaked once onto two types of Chlorella-inoculated agar plates. The results are shown in Table 1. Ability to nitrify in a particular medium was indicated by the production of hydroxylamine and nitrite. It can readily be observed that for both PCA and Nutrient Agar (with 0.5% glucose added to permit algal growth) a strong correlation existed between algal inhibition and nitrification. Interestingly, Arthrobacter sp. Q1 failed to produce a zone of inhibition in Nutrient Agar. The lack of hydroxylamine or nitrite in this medium was a further indication of a link between nitrification and Chlorella inhibition and indicated that a nitrogenous compound might be the cause of the inhibition. Besides hydroxylamine and nitrite, other nitrogenous intermediates may also be produced and released by Arthrobacter sp. QI. An examination was made of a number of possible intermediates to determine whether any were toxic to Chlorella. All tests were conducted by using PCA pour plates containing Chlorella and 10pgm1-1 sample-N. As can be observed in Fig. 2, ammonium, nitrite, nitrate, and the nitro compound proved nontoxic at this concentration. Only free hydroxylamine and the oxime (a type of bound hydroxylamine) proved toxic, with no algal growth at all noted even after 5 days incubation with the former. One other group of nitrogenous compounds was tested indirectly for inhibitory effect. Fe (as FeC13) was added in the preparation of Chlorella-inoculated agar pour plates such that its final concentration was 2 pg ml-l. The magnitude of the zones of inhibition around the Arthrobacter streak did not depart from that previously observed. Thus the possible class of nitrification intermediates known as hydroxamic acids, which is produced in an irondeficient medium, was unlikely as the causative agent
were incubated in the light or dark, and persisted over time. In peripheral zones of the plates, Chlorella grew well. Undoubtedly the alga was using the glucose in the medium as a carbon source for heterotrophic growth. Strips of agar transferred from the zone of inhibition (1, 2, 3 and 6 day old plates) to fresh Chlorellaincubated pour plates showed no inhibitory activity. Indeed colonies of the alga appeared within the transferred strips. Thus, the growth-restraining factor did not kill the algal cells, but merely inhibited their growth. Later evidence suggested that this phenomenon was not due to a dilution of an inhibitory substance by diffusion. To investigate the mechanism of inhibition more closely, Arthrobacter sp. QI was subjected to various treatments and then placed in penicylinders on freshly prepared pour plates containing Chlorella. Bacteria cells which had been treated with chloroform or boiled for 5 min, and cell filtrates from actively growing or disrupted cells failed to inhibit the alga. Only actively growing cells inhibited Chlorella. Nutrient enrichment studies which involved the addition of different organic and inorganic compounds to the PCA failed to permit algal growth in the zone of inhibition. In addition, Chlorella in agar strips from zones of inhibition (6 day old plates) grew on plates containing an uninoculated, highly purified preparation of agar (Noble Agar). No growth resulted when washed Chlorella cells were streaked across this agar, precluding the possibility that the Noble Agar was providing the necessary ingredients for growth. The results of these experiments indicated that competition for nutrients was probably not a factor in the algal inhibition observed. Studies using ultrafiltration procedures indicated that stable high molecular weight (> 1000) bacterial products such as enzymes were not directly responsible for Chlorella inhibition, even when concentrated up to 20 times. The pH of the agar media was also apparently not a factor in algal inhibition. Chlorella grew well with agar pH values between 3.0 and 9.2. Moreover, the pH of exponential of stationary Arth-
Table 1. Inhibition of Chlorella by intermediates of heterotrophic nitrification Plate count agar Organism* Nocardia sp. Flavobacterium sp. Q1 Q2 Q3 P1 P2 P3 P4
Zone of inhibition + . . + + + .
NH2OH-N . . + . .
NO~-N . .
. .
. .
+ . .
. .
+ + + .
Nutrient agart Zone of inhibition NHzOH NO~-N
. .
+ + + .
.
. .
+ + + .
-
-
+ + +
+ + +
. .
.
* "Q" strains isolated from Quabbin Reservoir, "P" strains isolated from Pontoosuc Lake. t 0.5% glucose added to permit Chlorella growth. :~Chemical tests performed three days after bacterial streak.
270
PAt I. S. BIl~{iJ~, .lIxN{)t I RH{}and [tAIM 13. (h NXI ~,' 120%
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~-
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cE 0 0 7
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§
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016 0.20 024 0.28 032 0.36 0 4 0 0 5 0 0 6 0 [NHzOH-N ] ,ag mL-I BBM
Fig. 3. Growth of axenic Chlorella culture in tubes of Bold's Basal Medium treated with different concentrations of hydroxylaminc. Incubation time was 7 days under fluorescent lighting. Average of 3 tubes.
30°/°
a. 20%
~0%
--
0%
_
la
Yz t a ~ i ~ ,
Fig. 2. Effect on growth of Chlorella in P(TA pour plates of various intermediates of nitrification as measured by chlorophyll-a concentrations. Initial concentration of all intermediates in the plates was lO,ug-N ml * sample. Incubation time was 5 days in the dark. of the zone of inhibition. These results provided initial evidence that Arthrobacter sp. QI in oxidizing the reduced organic nitrogen in the medium to hydroxylamine was thereby generating the compound responsible for Chlorella inhibition. We next examined the sensitivity of Chlorella to commercially-available hydroxylamine. Initial studies using PCA strongly indicated a low tolerance by this alga to the chemical. Later experiments carried out using BBM, since this medium more closely approximates the physical and chemical attributes of lakewater than does solid agar medium. In addition. measurement of algal tolerance levels were found to be more sensitive with this medium than in agar. As can be observed in Fig. 3, inhibition m BBM was evident at an initial hydroxylamine-N concentration of 0.24 #g ml ~. Further experiments demonstrated that hydroxylamine concentrations varied over time within the zone of inhibition (Fig. 4). The hydroxylamine-N levels within the zone increased to 5 l~g ml ~ 4 days after bacterial streaking and then dropped. No hydroxylamine was detected outside the zone of inhibition until after green algal colonies had developed. This reflected some slow diffusion of the small hydroxylamine molecule through the agar had occurred. In a related study, it was found that Arthrobacter sp. QI also produced hydroxylamine in agar plates lacking algae. Thus, Chlorella was not needed for induction.
Since filtrates of Arthrobacter sp Q1 placed in penicylinders on Chlorella-containing pour plates failed to inhibit the alga, it was reasoned that the toxic product should be expected to decompose in the agar with time. This was shown to be the case for hydroxylamine, as is shown in Fig. 5. The concentration of commercially-available hydroxylamine dropped to about one-half the initial concentration after about 1.5 days in both sterile PCA and BBM. In view of the rapid decay time, it is probable that Chlorella cells are inhibited by considerably lower concentrations of hydroxylamine-N than 0.24 Itg mlSupporting the hydroxylamine inhibition theory, we found in another study that the zone of algal inhibition failed to occur when plates were streaked with 6 --
5--
_°~
ifed zone
..~ 4B
,5, 2.z. p
--
Non-Inhibited zone r
2
3
Days afterArthroOacter
4
sp.
5 O I
Fig. 4. Hydroxylamine-N levels in the inhibited zone and in the non-inhibited zone of Chlorella-inocutated PCA streaked once with Arthrobacter sp. Ql. Average of 2 sets of 3 plates.
Bacterial suppression of Chlorella
271 DISCUSSION
100%
Chlorella, grown either in the light or dark on solid organic media, was inhibited by Arthrobacter sp. Ql. 80% Growth in agar strips from zones of inhibition transferred to Noble Agar plates, and lack of growth 70% stimulation in these zones upon the addition of various organic and inorganic nutrients indicated E 60% _o competitive inhibition was not involved. Moreover, no evidence was obtained by ultrafiltration proo 50% cedures that implicated stable, high molecular weight compounds (above 1000). "6 4 0 % The fact that nitrifying bacteria alone caused algal oo 3 0 % inhibition indicated a correlation between nitrificaO. tion and inhibition. Low molecular weight interme20% diaries from nitrification, therefore, were tested for Chlorella toxicity, and hydroxylamine was found to I0% be the most likely candidate. I I I Microbiologically-mediated hydroxylamine pro0% I 2 3 duction is not only produced by nitrification, but also Time, doys by nitrogen fixation (Jones, 1973) and denitrification Fig. 5. Decay profile of hydroxylamine in sterile Bold's as well as other processes of nitrite and nitrate reducBasal Medium and in uninoculated Plate Count Agar tion (Jensen, 1951; Tanaka, 1953). A number of plates. O represents BBM, [] represents PCA. Plates contain an initial hydroxylamine-N level of 10#gml-I and heterotrophs have already been shown to produce were incubated in the dark at 26°C. Flasks of BBM con- hydroxylamine under various cultural conditions tained an initial hydroxylamine-N level of 0.6 #g ml- ~ and (Verstraete, 1975), and there is evidence that this comwere incubated under constant fluorescent lighting at 20°C. pound can accumulate in lakes (Tanaka, 1953; Baxter et al., 1973; Jones, 1973). Verstraete & Alexander a mixed culture of Arthrobacter sp. Q1, and a second (1973) found evidence that hydroxylamine produced lake isolate, tentatively identified as another Arthro- by heterotrophic nitrifiers occurs in some lakes, river bacter species. If this second bacterium were indeed water, and sewage. capable of metabolizing the hydroxylamine produced Several publications have examined the tolerance by Arthrobacter sp. Ql, then Chlorella-inoculated of specific microorganisms to hydroxylarriine. The pour plates possessing a high initial concentration of compound is a known mutagen (Hayes, 1968). Castell commercially-available hydroxylamine and streaked & Mapplebeck (1956) cite several articles which indionce with this bacterium should show algal growth cate generally that concentrations between l and 250 #g ml- ~ of hydroxylamine are required for inhibionly in the vicinity of the streak. When the experiment was performed with plates containing tion of bacteria, depending on the species. Their own 12#gm1-1 hydroxylamine-N, this effect was clearly data indicate that a concentration of 20-30 #g mlallowed growth of most of the 119 bacterial cultures discernible (Fig. 6). 90%
i
iiii~
Fig. 6. Effect of a hydroxylamine-metabolizing bacterium on Chlorella-inoculated PCA plates containing 12/~gml-1 hydroxylamine-N. The algal colonies are observed growing only around the bacterial streak.
272
PAt I S, BIRGIR, JINNQI I: RHo and HAIM B. (~I:xNI I~
used, and several thngi and yeasts tolerated 200 itg ml J hydroxylaminc. In our investigation up to 12/~gml ~ hydrox31amine (5 ltg ml ~ hydroxylamine-N) was detected in agar plates streaked with Arthrohacter sp. Q1. Yet Chlorella was strongly inhibited, in addition, Chlorella grown in BBM under continuous lighting was inhibited by 0.57/~g m l 1 hydroxylamine (0.24 itg m l - J hydroxylamine-N). From previous reports most bacteria, yeasts and fungi tolerate thEsE concentrations. Thus hydroxylamine is a much more potent inhibitor of Chlorella t?ulgaris than of other organisms. Besides hydroxylamine and oximes, other nitrogenous c o m p o u n d s may have contributed to Chlorella inhibition. Although the t-nitropropane failed to inhibit Chlorella (Fig. 2) other nitro c o m p o u n d s might. Nitroso c o m p o u n d s were not tested for their ability to inhibit Chlorella. Certain N-nitroso substances are reported to be carcinogenic and mutagenic (Ayanaba & Alexander, 1974; Tate & Alexander, 1975), and indeed 201~g ml ~ of a nitroso c o m p o u n d produced by Pseudomonas ./~'agi inhibited Chlorella on agar plates (Tamura et al., 1967). According to Lund (1973) and Smith (1966), aliphatic primary and secondary nitroso c o m p o u n d s are unstable and tautomerize to oximes, often quite rapidly. It is noteworthy that Verstraete & Alexander (1973) discovered that 1-nitrosoethanol, a nitrification product from an Arthrohacter strain, was quite persistent in aqueous suspensions unlike our toxic factor. In addition, Murthy et al. (1966) found that a nitroso compound produced by Streptomyces alanosinicus was stable at a neutral pH. Another type of c o m p o u n d which may arise from heterotrophic nitrification is a hydroxamic acid. Hydroxamic acids, which are very stable c o m p o u n d s (Smith, 1966), can be produced from the reaction of hydroxylamine with several other c o m p o u n d s (Siggia, 1963: Waelsch, 1952). Verstraete & Alexander (1972) detected this substance as a product of heterotrophic nitrification and found that the a m o u n t observed depended sharply on ferric iron levels in the medium. Approximately an order of magnitude difference in the yields of hydroxamic acid was noted between 0.0 and 0.1/~gml r a n d between 0.1 and 1.0~gml -~ iron added. In our study, 2.0,ugml-~ Fe 3÷ failed to decrease the zone of inhibition. Therefore hydroxamic acid is evidently not involved in Chlorella inhibition. Although hydroxylamine may not be the exclusive toxic factor operative, the essential fact is that this c o m p o u n d is produced by Arthrohacter sp. Q1 a n d does inhibit the alga. Further research should illuminate the significance of this p h e n o m e n o n in lakewater and sewage. REFERENCES
American Public Health Association (1975) Standard Methods for the Examination of Water and Wastewater. 14th Edn. American Public Health Assodation, Washington, D.C.
Ayanaha A. & Alexander M. (1974) Transtbrmalions ol methylamines and formation of u hazardous product, dimeth31nitrosaminc, m samples of treated se,.~age and lake water..I, c;lrir. Quality 3, $3 89. Baxter R. M., Wood R. B. & Prosser M. V !1973)I'he probab b occurrence of hydrox~lamine in the water of art Ethiopian lake. Limnol. Oceam~gr. 18, 470 472. Berland B. R_ Bonin D..I. & Maestrini S. Y. (1972) Are some bacteria toxic for marine algae? Mar. Biol. 12, I89 193. Buchanan R. E. & Gibbons N. E. (Editors)(1974} Bergey's ),tanual q/'Detcrmimttirc Bacteriolo#y. 8th Edn. Williams and Wilkins. Baltimore. Castell C H. & Mapplebeck t. G. (1956) A nolc on the production of nitrite fi'om hydroxylamine by some heterotrophic bacteria. Fish. Res. Bd Canad. 13, 20t 206. Coughlan S. (1977) Comparative studies of glycollic acid uptake by four marine algae. Be. phycol. J. IL 55 62. Granhall U. & Berg B. (I972) Antimicrobial effects of Cellrihrio on bhle green algae, Arch. Mikrohiol. 84, 234 242. Gromov B. V,. h'anov O. G,, Mamkaeva K A. & 4vilov 1. A. (1972) A flexibacter that lyses blue-green algae. Microhiolo#y 41, 952 956. Hayes W. (1968} The (;c,etics o/Bacteria aml their V/ruses. Wiley. New York Jensen H. L, (1951) Nitritication of oxime compounds by heterotrophic bacteria. J. Gen. Mierohiol. $, 360 368. Jones K. {1973) Hydroxylamine. Comprehen,sive Inorganic Chemistry (Edited by Bailar J. C. et al.), Vol. 2, pp. 265 276. Pergamon Press, Oxford. Land H. (1973) Cathodic reduction of nitro compounds. Orgatlic Electrochemistry (Edited by Baizer M. M.), pp. 315 345. Marcel Dekker, New York. Magee W. E. & Burris R. H. (1954) Fixation of Nz and utilization of combined nitrogen by Nostoc muscorum. Am. J. Bot. 41, 777 782. Murthy Y. K. S.. Thiemann J. E.. Coronelli C. & Sensi P. (1966) Alanosine, a new antiviral and antitumour agent isolated from a Streptomyees. Nature {Lond.) 211, 1198 1199. Nichols H. W. & Bold H. C. (1965) Frichosarcina polymorpha Gen. ct sp. nov..I. Phycol. I, 34~38. Reim R. L.. Shane M. S. & Cannon R. E. (1974)I'hc characterization of a Bacillus capable of blue-green bactericidal activity. Can. J. ?vlicrobiol. 20, 986986. Rhee G.-Y. (1972) Competition between an alga and an aquatic bacterium for phosphate. Limnol. Oceanogr. 17, 505 514. Rho J., Berger P. & Gunner H. B. (1978) Characterization of heterotrophic nitrifying Arthrohaeter spp. of lacustrine origin. Abstr. 78th Amuial Meetin# Amer. Soc..li~r Microbiol. p. 171. Shiaris M. P. & Morrison S. M. (1976) The inhibition of blue green algae by Pseudomonas.fluorescens. Ahstr. 76th Annual Meetin# Amer. So(!. Jor Microhiol. p. 180. Shilo M. (1970) Lysis of blue green algae by myxobacter. J. Bact. 104, 453 461. Siggia S. (1963) Oxime formation. Quantitative Organic Analysis via Functional Groups (Edited by Siggia S.), 3rd Edn.. pp. 73 79. Wiley, New York. Smith P. A. S. (1966) The Chemistry of Open Chain Oryanic Nitro#en Compounds. Vol. 2. W. A. Benjamin, New York. Tamura S., Murayama A. & Hata K. (1967) Isolation and structural elucidation of Fragin, a new plant growth inhibitor produced by a Pseudomonas. Agric. Biol. Chem. 31, 758 759. Tanaka M. (1953) Occurrence of hydroxylamine in lake waters as an intermediate in bacterial reduction of nitrate. Nature (Lond.) 171, 1160 1161. Tate R. L. III & Alexander M. (19751 Stability of nitrosamines in samples of lake water, soil and sewage. J. natn. Cancer Inst. 54, 327 330. Verstraete W. (19751 Heterotrophic nitrification in soils
Bacterial suppression of Chhn'ella and aqueous media (a review), l:restiya Akademii Nauk SSSR, Seriya Biolooicheskaya. No. 4. pp. 541-558. Translated from Russian by Plenum, New York. Verstraete W. & Alexander M. 0972) Heterotrophic nitrification by Arthrohacter sp. J. Bact. II0, 955-961. Verstraete W. & Alexander M. (1973) Heterotrophic nitrification in samples of natural ecosystems. Enrir. Sci. Technol. 7, 39-42.
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Waelsch H. (1952) Certain aspects of intermediary metabolism of glutamine, asparagine, and glutathionc. Adr. En:ymol. 13, 237-319. Zavarzina N. B. (1964) Lysis of Chlorelta cultures in the absence of bacteria. Microhiolo.qy 33, 595- 509.
BACTERIAL SUPPRESSION OF CHLORELLA BY HYDROXYLAMINE PRODUCTION* PAUL S. BERGER, JINNQUE RHO and HAIM B. GUNNERt Department of Environmental Sciences, University of Massachusetts, Amherst, MA 01003, U.S.A.
(Received 2 October 1978) Abstract--An actively nitrifying Arthrobacter sp. isolated from a lake inhibited the growth of Chlorella vulgaris. This was found to be due to hydroxylamine released by the bacterium during the oxidation of ammonium or other reduced nitrogen compounds. Plates containing Plate Count Agar streaked with Arthrobacter accumulated 5/~gml - t hydroxylamine-N. Chlorella was sensitive to less than 0.24/~g ml - 1 hydroxylamine-N. Non-nitrifying bacterial isolates of lacustrine origin did not demonstrate inhibiting activity. Our results indicate that nitrifying microorganisms may thus affect the population dynamics of algae in eutrophic lakes.
INTRODUCTION Algal growth has been shown to be stimulated or repressed by bacteria, depending upon species and environmental factors. F o r example, bacteria-mediated algal inhibition may be due to cell-wall lysis upon direct contact with several types of myxobacteria (Shilo, 1970; G r o m o v et al., 1972), unsuccessful competition for limiting nutrients (Rhee, 1972; Coughlan, 1977), phage transfer (Zavarzina, 1964) and toxic substances released by bacteria into the surrounding medium (Berland et al., 1972; Reim et al., 1974). Several investigators have found that low molecular weight c o m p o u n d s liberated by bacteria suppress algal growth (Granhall & Berg, 1972; Shiaris & Morrison, 1976), but seldom have been identified. This investigation was undertaken to identify the mechanism and causative agent by which a lake bacterium strongly inhibited the growth of Chlorella vulgaris. MATERIALS AND METHODS
Culture and media The bacterium in this study was originally isolated by standard dilution plate techniques from water in the littoral zone of Quabbin Reservoir in central Massachusetts. Its biochemical and morphological characteristics were most consistent with those for the genus Arthrobacter (Bergey's Manual, 1974), and henceforth is referred to as Arthrobacter sp. Q I. Other lacustrine bacteria were also isolated. A strain of Chlorella vulgaris (IUCC no. 398) was obtained from the Carolina Biological Supply Co. Confirmation of cellular purity was achieved by successive streaking on Plate Count Agar (PCA) (Difco). * This work was supported in part by the Massachusetts Agricultural Experiment Station Project NE 39 and Department of the Interior, Water Resources Research Grant WRB 058. t Please forward all correspondence to Dr. Haim B. Gunner, Principal Investigator, Department of Environmental Sciences, University of Massachusetts, Amherst, MA 01003, U.S.A.
A stock culture of Chlorella was maintained on a rotary shaker at room temperature under two 40-W cool-white fluorescent lights in Bold's Basal Medium (BBM) (Nichols and Bold, 1965). Fresh cultures were prepared as required. Most interactions between the Arthrobacter and Chlorella were studied in Petri dishes containing PCA. Pour plates of Chlorella were prepared, incubated at 26°C in the dark for 1 day, then streaked once with the bacterial culture and reincubated in the dark. In one experiment, plates were incubated at 20°C under 40-W cool-white fluorescent light. Algal colonies appeared within 3-4 days. Other media were used as indicated.
Chemical tests Chemical tests were performed on slices of agar medium to determine the presence of nitrogenous compounds. To prepare agar for these tests, a total of 4 g agar medium taken in equal aliquots from three different plates was used in all tests. Agar was sliced from the plates and transferred to a tissue grinder (glass tube and glass pestle). Deionized, distilled water was then added, the agar macerated, and the resultant slurry transferred quantitatively to a test tube. A total of 10 ml water was used. The.test tube containing the agar slurry was mixed thoroughly on a Vortex mixer and the sample subjected to immediate chemical testing. Hydroxylamine was measured by the 8-hydroxyquinoline method (Magee & Burris, 1954). Nitrite was determined by the sulfanilamide procedure (Standard Methods, 1975). After color development, the reaction mixtures were centrifuged and the optical density of the clarified supernatant was determined spectrophotometrically. No significant amounts of hydroxylamine or nitrite adhered to the centrifuge-formed pellet. Chlorophyll-a determinations Chlorophyll-a determinations were performed by a modification of the trichromatic method (Standard Methods, 1975) with the following modifications. Four grams of Chlorella-inoculated agar from three plates were transferred to a tissue grinder and 6--8 ml 90% acetone added. The agar was macerated and quantitatively transferred to a screw-cap test tube. The total volume of the mixture was brought to 10ml with 90% acetone. The mixture stood overnight at 4°C in the dark and was then centrifuged. The volume of the clarified extract was measured and the optical density determined at wavelengths of 663, 645 and 630 nm. Chlorophyll-a concentrations were calculated by the equation presented in Standard Methods. 267
268
PA~L ~. Bi]~(ilR, JINNQI I RHO and HAIM B. (}t NNIR !
Fig. 1. Pour plates of Chlorella in Plate Count Agar streaked once with a bacterial isolate. Zone of inhibition is evident around Arthrohacter streak (center plate) but not around Flavobacterium (upper plate) or Nocardia (lower platc). RESULTS
In order to investigate in depth our preliminary observation that Arthrobacter sp. Q1 colonies were hindering the growth of Chlorella t~ulgaris on PCA plates, pour plates of PCA containing this alga were streaked once with selected bacterial isolates of lacus-
trine origine. Our results are indicated in Fig. 1. The wide zone of inhibition of Chlorella by Arthrobacter sp. Q1 on PCA failed to appear in the presence of two other bacterial species--Flavobacterium sp. and Nocardia sp. The zone of inhibition always occurred with Arthrobacter regardless of whether the plates
Bacterial suppression of Chlorella
269
robacter cultures never rose above 7.4 in m-Plate Count Broth. Earlier experiments by our group had revealed that Arthrobacter sp. Q1 and several other lake isolates were heterotrophic nitrifiers (Rho et al., 1978). To ascertain whether any correlation existed between nitrification and algal inhibition on agar plates, both nitrifiers and non-nitrifiers were streaked once onto two types of Chlorella-inoculated agar plates. The results are shown in Table 1. Ability to nitrify in a particular medium was indicated by the production of hydroxylamine and nitrite. It can readily be observed that for both PCA and Nutrient Agar (with 0.5% glucose added to permit algal growth) a strong correlation existed between algal inhibition and nitrification. Interestingly, Arthrobacter sp. Q1 failed to produce a zone of inhibition in Nutrient Agar. The lack of hydroxylamine or nitrite in this medium was a further indication of a link between nitrification and Chlorella inhibition and indicated that a nitrogenous compound might be the cause of the inhibition. Besides hydroxylamine and nitrite, other nitrogenous intermediates may also be produced and released by Arthrobacter sp. QI. An examination was made of a number of possible intermediates to determine whether any were toxic to Chlorella. All tests were conducted by using PCA pour plates containing Chlorella and 10pgm1-1 sample-N. As can be observed in Fig. 2, ammonium, nitrite, nitrate, and the nitro compound proved nontoxic at this concentration. Only free hydroxylamine and the oxime (a type of bound hydroxylamine) proved toxic, with no algal growth at all noted even after 5 days incubation with the former. One other group of nitrogenous compounds was tested indirectly for inhibitory effect. Fe (as FeC13) was added in the preparation of Chlorella-inoculated agar pour plates such that its final concentration was 2 pg ml-l. The magnitude of the zones of inhibition around the Arthrobacter streak did not depart from that previously observed. Thus the possible class of nitrification intermediates known as hydroxamic acids, which is produced in an irondeficient medium, was unlikely as the causative agent
were incubated in the light or dark, and persisted over time. In peripheral zones of the plates, Chlorella grew well. Undoubtedly the alga was using the glucose in the medium as a carbon source for heterotrophic growth. Strips of agar transferred from the zone of inhibition (1, 2, 3 and 6 day old plates) to fresh Chlorellaincubated pour plates showed no inhibitory activity. Indeed colonies of the alga appeared within the transferred strips. Thus, the growth-restraining factor did not kill the algal cells, but merely inhibited their growth. Later evidence suggested that this phenomenon was not due to a dilution of an inhibitory substance by diffusion. To investigate the mechanism of inhibition more closely, Arthrobacter sp. QI was subjected to various treatments and then placed in penicylinders on freshly prepared pour plates containing Chlorella. Bacteria cells which had been treated with chloroform or boiled for 5 min, and cell filtrates from actively growing or disrupted cells failed to inhibit the alga. Only actively growing cells inhibited Chlorella. Nutrient enrichment studies which involved the addition of different organic and inorganic compounds to the PCA failed to permit algal growth in the zone of inhibition. In addition, Chlorella in agar strips from zones of inhibition (6 day old plates) grew on plates containing an uninoculated, highly purified preparation of agar (Noble Agar). No growth resulted when washed Chlorella cells were streaked across this agar, precluding the possibility that the Noble Agar was providing the necessary ingredients for growth. The results of these experiments indicated that competition for nutrients was probably not a factor in the algal inhibition observed. Studies using ultrafiltration procedures indicated that stable high molecular weight (> 1000) bacterial products such as enzymes were not directly responsible for Chlorella inhibition, even when concentrated up to 20 times. The pH of the agar media was also apparently not a factor in algal inhibition. Chlorella grew well with agar pH values between 3.0 and 9.2. Moreover, the pH of exponential of stationary Arth-
Table 1. Inhibition of Chlorella by intermediates of heterotrophic nitrification Plate count agar Organism* Nocardia sp. Flavobacterium sp. Q1 Q2 Q3 P1 P2 P3 P4
Zone of inhibition + . . + + + .
NH2OH-N . . + . .
NO~-N . .
. .
. .
+ . .
. .
+ + + .
Nutrient agart Zone of inhibition NHzOH NO~-N
. .
+ + + .
.
. .
+ + + .
-
-
+ + +
+ + +
. .
.
* "Q" strains isolated from Quabbin Reservoir, "P" strains isolated from Pontoosuc Lake. t 0.5% glucose added to permit Chlorella growth. :~Chemical tests performed three days after bacterial streak.
270
PAt I. S. BIl~{iJ~, .lIxN{)t I RH{}and [tAIM 13. (h NXI ~,' 120%
,GI2 r
--
I ©Jr
1!0%
g o~oj N
009
o} _o
~-
"6.
008~
a 90%
cE 0 0 7
100%
§
OD6 80%
c
g
,,o
--
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70%
0.05-
E
o~ 6 0 %
I i 001
50%
£8 5
0
0
4O*/o
016 0.20 024 0.28 032 0.36 0 4 0 0 5 0 0 6 0 [NHzOH-N ] ,ag mL-I BBM
Fig. 3. Growth of axenic Chlorella culture in tubes of Bold's Basal Medium treated with different concentrations of hydroxylaminc. Incubation time was 7 days under fluorescent lighting. Average of 3 tubes.
30°/°
a. 20%
~0%
--
0%
_
la
Yz t a ~ i ~ ,
Fig. 2. Effect on growth of Chlorella in P(TA pour plates of various intermediates of nitrification as measured by chlorophyll-a concentrations. Initial concentration of all intermediates in the plates was lO,ug-N ml * sample. Incubation time was 5 days in the dark. of the zone of inhibition. These results provided initial evidence that Arthrobacter sp. QI in oxidizing the reduced organic nitrogen in the medium to hydroxylamine was thereby generating the compound responsible for Chlorella inhibition. We next examined the sensitivity of Chlorella to commercially-available hydroxylamine. Initial studies using PCA strongly indicated a low tolerance by this alga to the chemical. Later experiments carried out using BBM, since this medium more closely approximates the physical and chemical attributes of lakewater than does solid agar medium. In addition. measurement of algal tolerance levels were found to be more sensitive with this medium than in agar. As can be observed in Fig. 3, inhibition m BBM was evident at an initial hydroxylamine-N concentration of 0.24 #g ml ~. Further experiments demonstrated that hydroxylamine concentrations varied over time within the zone of inhibition (Fig. 4). The hydroxylamine-N levels within the zone increased to 5 l~g ml ~ 4 days after bacterial streaking and then dropped. No hydroxylamine was detected outside the zone of inhibition until after green algal colonies had developed. This reflected some slow diffusion of the small hydroxylamine molecule through the agar had occurred. In a related study, it was found that Arthrobacter sp. QI also produced hydroxylamine in agar plates lacking algae. Thus, Chlorella was not needed for induction.
Since filtrates of Arthrobacter sp Q1 placed in penicylinders on Chlorella-containing pour plates failed to inhibit the alga, it was reasoned that the toxic product should be expected to decompose in the agar with time. This was shown to be the case for hydroxylamine, as is shown in Fig. 5. The concentration of commercially-available hydroxylamine dropped to about one-half the initial concentration after about 1.5 days in both sterile PCA and BBM. In view of the rapid decay time, it is probable that Chlorella cells are inhibited by considerably lower concentrations of hydroxylamine-N than 0.24 Itg mlSupporting the hydroxylamine inhibition theory, we found in another study that the zone of algal inhibition failed to occur when plates were streaked with 6 --
5--
_°~
ifed zone
..~ 4B
,5, 2.z. p
--
Non-Inhibited zone r
2
3
Days afterArthroOacter
4
sp.
5 O I
Fig. 4. Hydroxylamine-N levels in the inhibited zone and in the non-inhibited zone of Chlorella-inocutated PCA streaked once with Arthrobacter sp. Ql. Average of 2 sets of 3 plates.
Bacterial suppression of Chlorella
271 DISCUSSION
100%
Chlorella, grown either in the light or dark on solid organic media, was inhibited by Arthrobacter sp. Ql. 80% Growth in agar strips from zones of inhibition transferred to Noble Agar plates, and lack of growth 70% stimulation in these zones upon the addition of various organic and inorganic nutrients indicated E 60% _o competitive inhibition was not involved. Moreover, no evidence was obtained by ultrafiltration proo 50% cedures that implicated stable, high molecular weight compounds (above 1000). "6 4 0 % The fact that nitrifying bacteria alone caused algal oo 3 0 % inhibition indicated a correlation between nitrificaO. tion and inhibition. Low molecular weight interme20% diaries from nitrification, therefore, were tested for Chlorella toxicity, and hydroxylamine was found to I0% be the most likely candidate. I I I Microbiologically-mediated hydroxylamine pro0% I 2 3 duction is not only produced by nitrification, but also Time, doys by nitrogen fixation (Jones, 1973) and denitrification Fig. 5. Decay profile of hydroxylamine in sterile Bold's as well as other processes of nitrite and nitrate reducBasal Medium and in uninoculated Plate Count Agar tion (Jensen, 1951; Tanaka, 1953). A number of plates. O represents BBM, [] represents PCA. Plates contain an initial hydroxylamine-N level of 10#gml-I and heterotrophs have already been shown to produce were incubated in the dark at 26°C. Flasks of BBM con- hydroxylamine under various cultural conditions tained an initial hydroxylamine-N level of 0.6 #g ml- ~ and (Verstraete, 1975), and there is evidence that this comwere incubated under constant fluorescent lighting at 20°C. pound can accumulate in lakes (Tanaka, 1953; Baxter et al., 1973; Jones, 1973). Verstraete & Alexander a mixed culture of Arthrobacter sp. Q1, and a second (1973) found evidence that hydroxylamine produced lake isolate, tentatively identified as another Arthro- by heterotrophic nitrifiers occurs in some lakes, river bacter species. If this second bacterium were indeed water, and sewage. capable of metabolizing the hydroxylamine produced Several publications have examined the tolerance by Arthrobacter sp. Ql, then Chlorella-inoculated of specific microorganisms to hydroxylarriine. The pour plates possessing a high initial concentration of compound is a known mutagen (Hayes, 1968). Castell commercially-available hydroxylamine and streaked & Mapplebeck (1956) cite several articles which indionce with this bacterium should show algal growth cate generally that concentrations between l and 250 #g ml- ~ of hydroxylamine are required for inhibionly in the vicinity of the streak. When the experiment was performed with plates containing tion of bacteria, depending on the species. Their own 12#gm1-1 hydroxylamine-N, this effect was clearly data indicate that a concentration of 20-30 #g mlallowed growth of most of the 119 bacterial cultures discernible (Fig. 6). 90%
i
iiii~
Fig. 6. Effect of a hydroxylamine-metabolizing bacterium on Chlorella-inoculated PCA plates containing 12/~gml-1 hydroxylamine-N. The algal colonies are observed growing only around the bacterial streak.
272
PAt I S, BIRGIR, JINNQI I: RHo and HAIM B. (~I:xNI I~
used, and several thngi and yeasts tolerated 200 itg ml J hydroxylaminc. In our investigation up to 12/~gml ~ hydrox31amine (5 ltg ml ~ hydroxylamine-N) was detected in agar plates streaked with Arthrohacter sp. Q1. Yet Chlorella was strongly inhibited, in addition, Chlorella grown in BBM under continuous lighting was inhibited by 0.57/~g m l 1 hydroxylamine (0.24 itg m l - J hydroxylamine-N). From previous reports most bacteria, yeasts and fungi tolerate thEsE concentrations. Thus hydroxylamine is a much more potent inhibitor of Chlorella t?ulgaris than of other organisms. Besides hydroxylamine and oximes, other nitrogenous c o m p o u n d s may have contributed to Chlorella inhibition. Although the t-nitropropane failed to inhibit Chlorella (Fig. 2) other nitro c o m p o u n d s might. Nitroso c o m p o u n d s were not tested for their ability to inhibit Chlorella. Certain N-nitroso substances are reported to be carcinogenic and mutagenic (Ayanaba & Alexander, 1974; Tate & Alexander, 1975), and indeed 201~g ml ~ of a nitroso c o m p o u n d produced by Pseudomonas ./~'agi inhibited Chlorella on agar plates (Tamura et al., 1967). According to Lund (1973) and Smith (1966), aliphatic primary and secondary nitroso c o m p o u n d s are unstable and tautomerize to oximes, often quite rapidly. It is noteworthy that Verstraete & Alexander (1973) discovered that 1-nitrosoethanol, a nitrification product from an Arthrohacter strain, was quite persistent in aqueous suspensions unlike our toxic factor. In addition, Murthy et al. (1966) found that a nitroso compound produced by Streptomyces alanosinicus was stable at a neutral pH. Another type of c o m p o u n d which may arise from heterotrophic nitrification is a hydroxamic acid. Hydroxamic acids, which are very stable c o m p o u n d s (Smith, 1966), can be produced from the reaction of hydroxylamine with several other c o m p o u n d s (Siggia, 1963: Waelsch, 1952). Verstraete & Alexander (1972) detected this substance as a product of heterotrophic nitrification and found that the a m o u n t observed depended sharply on ferric iron levels in the medium. Approximately an order of magnitude difference in the yields of hydroxamic acid was noted between 0.0 and 0.1/~gml r a n d between 0.1 and 1.0~gml -~ iron added. In our study, 2.0,ugml-~ Fe 3÷ failed to decrease the zone of inhibition. Therefore hydroxamic acid is evidently not involved in Chlorella inhibition. Although hydroxylamine may not be the exclusive toxic factor operative, the essential fact is that this c o m p o u n d is produced by Arthrohacter sp. Q1 a n d does inhibit the alga. Further research should illuminate the significance of this p h e n o m e n o n in lakewater and sewage. REFERENCES
American Public Health Association (1975) Standard Methods for the Examination of Water and Wastewater. 14th Edn. American Public Health Assodation, Washington, D.C.
Ayanaha A. & Alexander M. (1974) Transtbrmalions ol methylamines and formation of u hazardous product, dimeth31nitrosaminc, m samples of treated se,.~age and lake water..I, c;lrir. Quality 3, $3 89. Baxter R. M., Wood R. B. & Prosser M. V !1973)I'he probab b occurrence of hydrox~lamine in the water of art Ethiopian lake. Limnol. Oceam~gr. 18, 470 472. Berland B. R_ Bonin D..I. & Maestrini S. Y. (1972) Are some bacteria toxic for marine algae? Mar. Biol. 12, I89 193. Buchanan R. E. & Gibbons N. E. (Editors)(1974} Bergey's ),tanual q/'Detcrmimttirc Bacteriolo#y. 8th Edn. Williams and Wilkins. Baltimore. Castell C H. & Mapplebeck t. G. (1956) A nolc on the production of nitrite fi'om hydroxylamine by some heterotrophic bacteria. Fish. Res. Bd Canad. 13, 20t 206. Coughlan S. (1977) Comparative studies of glycollic acid uptake by four marine algae. Be. phycol. J. IL 55 62. Granhall U. & Berg B. (I972) Antimicrobial effects of Cellrihrio on bhle green algae, Arch. Mikrohiol. 84, 234 242. Gromov B. V,. h'anov O. G,, Mamkaeva K A. & 4vilov 1. A. (1972) A flexibacter that lyses blue-green algae. Microhiolo#y 41, 952 956. Hayes W. (1968} The (;c,etics o/Bacteria aml their V/ruses. Wiley. New York Jensen H. L, (1951) Nitritication of oxime compounds by heterotrophic bacteria. J. Gen. Mierohiol. $, 360 368. Jones K. {1973) Hydroxylamine. Comprehen,sive Inorganic Chemistry (Edited by Bailar J. C. et al.), Vol. 2, pp. 265 276. Pergamon Press, Oxford. Land H. (1973) Cathodic reduction of nitro compounds. Orgatlic Electrochemistry (Edited by Baizer M. M.), pp. 315 345. Marcel Dekker, New York. Magee W. E. & Burris R. H. (1954) Fixation of Nz and utilization of combined nitrogen by Nostoc muscorum. Am. J. Bot. 41, 777 782. Murthy Y. K. S.. Thiemann J. E.. Coronelli C. & Sensi P. (1966) Alanosine, a new antiviral and antitumour agent isolated from a Streptomyees. Nature {Lond.) 211, 1198 1199. Nichols H. W. & Bold H. C. (1965) Frichosarcina polymorpha Gen. ct sp. nov..I. Phycol. I, 34~38. Reim R. L.. Shane M. S. & Cannon R. E. (1974)I'hc characterization of a Bacillus capable of blue-green bactericidal activity. Can. J. ?vlicrobiol. 20, 986986. Rhee G.-Y. (1972) Competition between an alga and an aquatic bacterium for phosphate. Limnol. Oceanogr. 17, 505 514. Rho J., Berger P. & Gunner H. B. (1978) Characterization of heterotrophic nitrifying Arthrohaeter spp. of lacustrine origin. Abstr. 78th Amuial Meetin# Amer. Soc..li~r Microbiol. p. 171. Shiaris M. P. & Morrison S. M. (1976) The inhibition of blue green algae by Pseudomonas.fluorescens. Ahstr. 76th Annual Meetin# Amer. So(!. Jor Microhiol. p. 180. Shilo M. (1970) Lysis of blue green algae by myxobacter. J. Bact. 104, 453 461. Siggia S. (1963) Oxime formation. Quantitative Organic Analysis via Functional Groups (Edited by Siggia S.), 3rd Edn.. pp. 73 79. Wiley, New York. Smith P. A. S. (1966) The Chemistry of Open Chain Oryanic Nitro#en Compounds. Vol. 2. W. A. Benjamin, New York. Tamura S., Murayama A. & Hata K. (1967) Isolation and structural elucidation of Fragin, a new plant growth inhibitor produced by a Pseudomonas. Agric. Biol. Chem. 31, 758 759. Tanaka M. (1953) Occurrence of hydroxylamine in lake waters as an intermediate in bacterial reduction of nitrate. Nature (Lond.) 171, 1160 1161. Tate R. L. III & Alexander M. (19751 Stability of nitrosamines in samples of lake water, soil and sewage. J. natn. Cancer Inst. 54, 327 330. Verstraete W. (19751 Heterotrophic nitrification in soils
Bacterial suppression of Chhn'ella and aqueous media (a review), l:restiya Akademii Nauk SSSR, Seriya Biolooicheskaya. No. 4. pp. 541-558. Translated from Russian by Plenum, New York. Verstraete W. & Alexander M. 0972) Heterotrophic nitrification by Arthrohacter sp. J. Bact. II0, 955-961. Verstraete W. & Alexander M. (1973) Heterotrophic nitrification in samples of natural ecosystems. Enrir. Sci. Technol. 7, 39-42.
w.R.
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Waelsch H. (1952) Certain aspects of intermediary metabolism of glutamine, asparagine, and glutathionc. Adr. En:ymol. 13, 237-319. Zavarzina N. B. (1964) Lysis of Chlorelta cultures in the absence of bacteria. Microhiolo.qy 33, 595- 509.