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Comparative studies on utilization of fatty acids and hydrocarbons in Nocardia amarae and Rhodococcus spp.
JOURNAL OF FERMENTATION AND BIOENC~NEERING Vol. 79, No. 2, 186-189. 1995
Comparative
Studies on Utilization of Fatty Acids and Hydrocarbons in Nocardia amarae and Rhodococcus spp.
KEISUKE Department
IWAHORI,
of Environmental
MIN WANG,
HIRONORI
TAKI,
AND
Engineering, Faculty of Engineering, Suita, Osaka 565, Japan Received
8 June
1994jAccepted
MASANORI
FUJITA”
Osaka University, 2-1 Yarnadaoka,
1 November
1994
Comparative investigations on the substrate utilization of Nocardia amarae and Rhodococcus spp. (R. rhodochrous and R. erythropolis) were carried out using various fatty acids and paraffinic hydrocarbons. Upon calculation and comparison of their specific growth rates (,u) during the logarithmic phase, it was shown that the lower fatty acids (C2-C5) were utilized preferably and the highest p value was obtained in octadecane (C18) for all three strains. The initial total organic carbon (TOC) concentration affecting the growth of the 3 strains was investigated, using octadecane as the carbon source. From a comparison of the kinetic parameters of the 3 strains with those obtained in the past studies, it was found that Rhodococcus spp. had higher growth rate and lower affinity for octadecane than N. amarae.
[Key words: fatty
acid,
Nocardia amarae, Rhodococcus rhodochrous, Rhodococcus erythropolis, substrate
Abnormal foaming and scum production have been reported as unusual operation problems in the activated sludge process (l-4). The microorganisms responsible for these problems are actinomycetes such as Nocardia amarae (2) or Rhodococcus spp. (5). Various studies on N. amarae, e.g., its morphological and physiological characteristics (6), generation of higher fatty acids (micoric acids) in its cell walls (7) and survey of its profiles in the sewage treatment plant (8) have been performed. Judging from the results of physiological tests by Lechevalier and Lechevalier (2) and Goodfellow et a/. (7), monosaccharides and lower fatty acids were utilized favorably by N. amarae. It was also reported that hydrophobic substrates such as n-hexadecane and aromatic substrates were decomposed by N. amarae (9). Thus, it is well known that N. amarae has extensive substrate utilization characteristics, as mentioned above. However, the substrate utilization of Rhodococcus spp. has been investigated scarcely. In this paper, comparative investigations on the substrate utilization of N. amarae and Rhodococcus spp. were carried out, using various fatty acids and paraffinic hydrocarbons. N. amarae (10) isolated from the foaming activated sludge and Rhodococcus spp. (R. rhodochrous ATCC 13808 and R. erythropolis ATCC4277) were subcultured in MS medium (peptone, 5.0 g; yeast extract, 2.5 g; glucose, 1.O g; sodium propionate, 4.5 g; agar, 17.5 g; deionized water, 1 r) and used throughout the experiments. Rhodococcus spp. were confirmed by preliminary examination to cause viscid and abnormal foaming like N. amarae. Various fatty acids and paraffinic hydrocarbons listed in Table 1, which were added to nutrient salts, were used in the experiments. Fatty acid media were abbreviated as FA[I] to FA[16] to indicate the number of carbons, and paraffinic hydrocarbon media were similarly abbreviated as PHC[6] to PHC[20]. Each strain pre-cultured in MS medium was adjusted to an initial concentration of 5 to lOmg//, and was ad* Corresponding
utilization.
hydrocarbon]
ded into 100 ml of each of the above media in a 300 ml Erlenmeyer flask, and then cultured at 28°C on a rotary shaker at 120 rpm. For each medium, 8 to 10 flasks were prepared. The ODem of the cell suspension after ultrasonic dispersion for 2 min (275 /‘A) or the dry weight obTABLE I.
Compositions of media for various carbon sources
Carbon source *Fatty acid Sodium formate Sodium acetate Sodium propionate Butyric acid Valerie acid Sodium hexanoate Sodium caprylate Sodium caprate Sodium lauriate Sodium palmitate *Paraffinic hydrocarbon Hexane Octane Decane Dodecane Tetradecane Hexadecane Octadecane Eicosane Nutrient salts” Yeast extract NaCl KC1 CaCl, MgSO, NH,Cl Deionized water TOC pHb
Abbreviation 5.67 3.42 2.67 1.91 1.81 1.92 1.73 1.62 1.54 1.45
g g g ml ml g g g g g
FA[IO] FA[12] FA[l6]
1.81 1.70 1.62 1.58 1.55 1.53 1.18 1.18
ml ml ml ml ml ml g g
PHC[6] PHC[8] PHC[lO] PHC[lZ] PHC[14] PHC[16] PHC[lS] PHC[ZO]
0.1 0.303 0.14 0.18 0.2 0.35 1.0
g g g g g g I
1000
FALlI FA[2]
FA[31 FAL41
FA[51 FA[61 FAl81
mg/l
7.5
a Nutrient salts were added to various carbon sources. h Adjustment of pH was carried out using phosphate buffer, Na2HP0, (M/15) : KZHPO, (M/15)=4: 1. Numbers in parentheses are expressed as number of carbons.
author. 186
i.e.,
VOL. 79, 1995
NOTES
Number
10°0W
Time(d) FIG. 1. Time course of biomass concentrations of N. atnarae in FA media. Symbols: Cl, FA[I]; 0, FA[2]; a, FA[3]; A, FA[4); 0, FA[5]; l , FA[6]; D, FA[8]; n , FA[IO]; and V, FA[16].
tained by the paper filter method (1 Irrn Millipore filter) per flask was measured within a certain period of time. The ODsoo or the dry weight of two flasks without strains was similarly measured at the start and end of each experiment. These control values were used to correct the biomass concentration. The filter paper method was applied to the case of FA[lO]-FA[16] and PHC media containing an insoluble source. Time courses of biomass concentrations of N. amarae in FA media and R. rhodochrous in PHC media are shown in Figs. 1 and 2, respectively. The same tendencies were observed in the cases using other media and strains. The specific growth rates (,f) during logarithmic phase were calculated and the relationship between number of carbons and /f values of the 3 strains in both FA and PHC media is shown in Fig. 3. Lower fatty acids (C2-C5) were utilized preferably by the 3 strains and FA media with number of carbons higher than 8 were not utilized at all. It is well known that sodium acetate and sodium propionate were utilized by N. amarae (2, 7). Therefore, additional substrates such as butyric and valeric acids (C4 and CS) could be utilized by N. amarae. In contrast, 11 values of N. amarae and Rhodococcus spp. increased with increase in number of carbons in PHC media. The highest /I value was obtained in octadecane (Cl@. Such peculiar substrate utilization is also rec-
187
of carbons
FIG. 3. Relationship between number of carbons and ,t logarithmic phase. Symbols: ci, N. amarae in FA media; rhodochrous in FA media; 0, R. erythropolis in FA media; amarae in PHC media; A, R. rhodochrous in PHC media; and erythropok in PHC media.
during n, R. 0, N. l , R.
ognized in LAS (linear alkylbenzene sulfonates), composed of the same straight chain as paraffinic hydrocarbons (11). Octadecane and eicosane (C20) have melting points of 28.O”C and 36.6”C, respectively. The former was a liquid while the latter was a solid during our experiments. In general, liquid is more likely to be utilized than solid by microorganisms; however, the /t value in the case of eicosane was shown to be nearly the same as that of dodecane (C12) or tetradecane (C14). Therefore, the affinity of the 3 strains to eicosane was estimated to be higher in spite of it being in the solid state. Although hydrocarbons with more than 21 carbons were not examined, it was suggested that the /I values would be considerably lower since they would be present as solid under our experimental conditions. From these results, it was found that N. amarae and Rhodococcus spp. exhibit the peculiar substrate utilization of fatty acids and hydrocarbons. As shown in Fig. 3, fatty acids and hydrocarbons were utilized in the same way by N. amarae and Rhodococcus spp. The /t values of Rhodococcus spp. were 3.6 and 2.0 times those of N. amarae in FA[3] and PHC[18] media, respectively. The /l values of N. amarae in FA[2]-FA[5] media were almost equal, but sodium propionate utilization was exceptionally high by Rhodococcus spp. The 1’
--o
0
500 lnltlal TOC concentration
Time (h) FIG. 2. Time course of biomass concentrations dochrous in PHC media. Symbols: (3, PHC[lO]; 0, PHC[14]; A, PHC[16]; 0, PHC[lI]; and +, PHC[20].
of R. rhoPHC[IZ]; A,
1000 of medium (mgii)
FIG. 4. Relationship between initial TOC concentration of medium and ,l during logarithmic phase in PHC[18] medium. Symbols: 0, N. amarae; A, R. rhodochrous; c\, R. erythropolis. Each curved line was calculated and plotted using kinetic parameters.
188
IWAHORl
J. FERMENT.BIOENG.,
ET AL
TABLE Microorganism
2.
Comparison
of kinetic
parameters Kinetic
Medium /l,,,
(I/d)
parameter
K (w/O
11.8
29
9.1
11
Ref.
Glucose
8.6
41
7.0
47
9.1
6
8.9
17
8.2
8
Acetic acid Activated
sludge microorganisms
Propionic
acid
Glucose Sphaerotilus sp. Sphaerotilus natat@
Glucose
2.9
-
6.5
10
2.6
2
2.8
0.5
3.0
2.5
5.8
98.2b
15.9
212.3h
15.2
557.7h
14 14 15
Filamentous
bacterium
Type
1701”
N. amarae ASF3” N. anzarae ASAC 1a
Acetic acid
13
N. amarae R. rhodochrous
Octadecane
R. erythropolis a Kinetic parameters were calculated under steady-state chemostat h These values are K,’ values and are higher than k; values
values of both the activated sludge and the dominant bacteria in activated sludge, which were obtained during the logarithmic phase of batch culture using artificial sewage, were reported to be 13.8 I/d and 4.0-19.7 l/d, respectively (12). These values are similar to those obtained from substrates which could be utilized by Rhodococcus spp. Therefore, it was shown that R. rhodochrous and R. erythropolis are capable of overcoming competition with the activated sludge microorganisms depending on the components of the substrate or sewage. The initial TOC concentration affecting the growth of N. amarae and Rhodococcus spp. was investigated in the as the carbon source. same way, using octadecane PHC[ 181 medium listed in Table 1 was diluted by deionized water and its TOC concentrations were adjusted to 100, 300 or 500mg//. The lag phase was observed in case of N. amarae as the TOC concentration decreased, but it was not observed in the case of Rhodococcus spp. The 11 values during logarithmic phase were calculated and their relationship with the initial TOC concentrations is shown in Fig. 4. The ~t values of the 3 strains obviously decreased with decrease of the initial TOC concentration. The relationship between the specific growth rate (/f) and TOC concentration (S) can be generally expressed by the following equation: ,‘I
I?;‘,
S
where P,,, is the maximum specific growth rate and K, is the saturation constant. Assuming that S, and ,j are the initial TOC concentration and the removal efficiency, respectively, Eq. 1 can be rewritten as follows:
culture.
This study
The others were carried out in batch culture.
.s
d?_--,iSO If ,i is constant, K,/(l-,?) becomes constant (K,‘) and the relationship between /1 and S, is represented by a Monod-type equation. It was found that ,j values in the case of the 3 strains were constant regardless of the initial TOC concentration, because good correlations were obtained from the Hofstee plot. The /L,,, and KS’ values are summarized in Table 2 along with those obtained from the past studies, using activated sludge microorganisms, filamentous bacteria and actinomycetes. Since the culture method and the carbon source are different, it is difficult to compare the kinetic parameters accurately. The /I,,, values of Rhodococcus spp. are higher than those of N. amarae, filamentous bacteria and activated sludge microorganisms. On the other hand, the KS’ value is twice or 10 times the KS value if ,i is 50% or 90%, respectively. Judging from this conversion, the KS value of N. amarae is of the same order as those of activated sludge microorganisms, while K, values of Rhodococcus spp. are estimated to be higher than those of activated sludge microorganisms, filamentous bacteria and N. amarae. Therefore, Rhodococcus spp. are found to have higher growth rate and lower affinity for octadecane than N. amarae. In other words, R. rhodochrous and R. erythropofis are capable of rapid growth in the activated sludge process if higher-strength octadecane is fed and contacted with them, whereas N. amarae grows rapidly in lower-strength octadecane. These trends for N. amarae and Rhodococcus spp. seem to be recognized in other substrates such as lower fatty acids and hexadecane. Consequently, it is thought that their growth char-
VOL. 79, 1995
acteristics
NOTES
in the activated
sludge process
are dissimilar.
REFERENCES 1. Milwaukee Mystery: Unusual operating problem develops. Water & Sew., 116, 213 (1969). 2. Lechevalier, M. P. and Lechevalier, H. A.: Nocardia amarae sp. nav., an actinomycete in foaming activated sludge. Int. J. Syst. Bacterial., 24, 278-288 (1974). W. 0.: Actinomycete scum production in activated 3. Pipes, sludge processes. J. WPCF, 50, 628-634 (1978). 4. Dhaliwal, B. S.: Nocardia amarae and activated sludge foaming. J. WPCF, 51, 344-350 (1979). 5. Mori, T., Sakai, Y., Honda, K., Yano, I., and Hashimoto, S.: Stable abnormal foam in activated sludge process produced by Rhodococcus sp. with strong hydrophobic property. Environ. Technol. Lett., 9, 1041-1048 (1988). 6. Lemmer, H. and Kroppenstedt, R. M.: Chemotaxonomy and physiology of some actinomycetes from scumming activated sludge. Sys. Appl. Microbial., 5, 124-135 (1984). 1. Goodfellow, M., Minnikin, D. E., Todd, C., Alderson, G., and Minnikin, S. M.: Numerical and chemical classification of Nocurdia amarae. J. Gen. Microbial., 128, 1283-1297 (1982). 8. Hiraoka, H. and Tsumura, K.: Suppression of actinomycete scum production-A case study at Senboku wastewater treat-
189
ment plant. Wat. Sci. Tech., 16, 83-90 (1984). 9. Baumann, M., Lemmer, H., and Ries, H.: Scum actinomycetes in sewage treatment plants. I. Growth kinetics of Nocurdia amarae in chemostat culture. Water Res., 22, 755-759 (1988). IO. Sakai, Y., Mori, T., Honda, K., and Matsumoto, T.: Scum formation by Actinomycetes (Nocardia sp.) in final clarifier of activated sludge process. J. Jpn Sew. Works Assoc., 19(214), 56-65 (1982). (in Japanese) Il. Swisher, R. D.: Surfactant biodegradation, p. 126-133. Surfactant science series. Marcel Dekker Inc., New York (1970). 12. Hashimoto, S., Iwahori, K., Kamiya, T., and Kato, S.: Fundamental studies on microbial interaction in activated sludge. Res. J. Jpn Sew. Works Assoc., 28(334), 33-43 (1991). (in Japanese) cul13. Blackall, L. L., Tandoi, V., and Jenkins, D.: Continuous ture studies with Nocardia amarae from activated sludge and their implications for Nocardia foaming control. Res. J. WPCF, 63(l), 44-50 (1991). 14. Yasuda, M.: Quantitative photographic measurement of Sphaerotilus sp. and mechanism of filamentous bulking of activated sludge. J. Jpn Sew. Works Assoc., 15(168), 2-8 (1978). (in Japanese) 15. Richard, M. G., Hao, O., and Jenkins, D.: Growth kinetics of Sphaerotilus species and their significance in activated bulking. J. WPCF, 57. 68-81 (1985).
Comparative
Studies on Utilization of Fatty Acids and Hydrocarbons in Nocardia amarae and Rhodococcus spp.
KEISUKE Department
IWAHORI,
of Environmental
MIN WANG,
HIRONORI
TAKI,
AND
Engineering, Faculty of Engineering, Suita, Osaka 565, Japan Received
8 June
1994jAccepted
MASANORI
FUJITA”
Osaka University, 2-1 Yarnadaoka,
1 November
1994
Comparative investigations on the substrate utilization of Nocardia amarae and Rhodococcus spp. (R. rhodochrous and R. erythropolis) were carried out using various fatty acids and paraffinic hydrocarbons. Upon calculation and comparison of their specific growth rates (,u) during the logarithmic phase, it was shown that the lower fatty acids (C2-C5) were utilized preferably and the highest p value was obtained in octadecane (C18) for all three strains. The initial total organic carbon (TOC) concentration affecting the growth of the 3 strains was investigated, using octadecane as the carbon source. From a comparison of the kinetic parameters of the 3 strains with those obtained in the past studies, it was found that Rhodococcus spp. had higher growth rate and lower affinity for octadecane than N. amarae.
[Key words: fatty
acid,
Nocardia amarae, Rhodococcus rhodochrous, Rhodococcus erythropolis, substrate
Abnormal foaming and scum production have been reported as unusual operation problems in the activated sludge process (l-4). The microorganisms responsible for these problems are actinomycetes such as Nocardia amarae (2) or Rhodococcus spp. (5). Various studies on N. amarae, e.g., its morphological and physiological characteristics (6), generation of higher fatty acids (micoric acids) in its cell walls (7) and survey of its profiles in the sewage treatment plant (8) have been performed. Judging from the results of physiological tests by Lechevalier and Lechevalier (2) and Goodfellow et a/. (7), monosaccharides and lower fatty acids were utilized favorably by N. amarae. It was also reported that hydrophobic substrates such as n-hexadecane and aromatic substrates were decomposed by N. amarae (9). Thus, it is well known that N. amarae has extensive substrate utilization characteristics, as mentioned above. However, the substrate utilization of Rhodococcus spp. has been investigated scarcely. In this paper, comparative investigations on the substrate utilization of N. amarae and Rhodococcus spp. were carried out, using various fatty acids and paraffinic hydrocarbons. N. amarae (10) isolated from the foaming activated sludge and Rhodococcus spp. (R. rhodochrous ATCC 13808 and R. erythropolis ATCC4277) were subcultured in MS medium (peptone, 5.0 g; yeast extract, 2.5 g; glucose, 1.O g; sodium propionate, 4.5 g; agar, 17.5 g; deionized water, 1 r) and used throughout the experiments. Rhodococcus spp. were confirmed by preliminary examination to cause viscid and abnormal foaming like N. amarae. Various fatty acids and paraffinic hydrocarbons listed in Table 1, which were added to nutrient salts, were used in the experiments. Fatty acid media were abbreviated as FA[I] to FA[16] to indicate the number of carbons, and paraffinic hydrocarbon media were similarly abbreviated as PHC[6] to PHC[20]. Each strain pre-cultured in MS medium was adjusted to an initial concentration of 5 to lOmg//, and was ad* Corresponding
utilization.
hydrocarbon]
ded into 100 ml of each of the above media in a 300 ml Erlenmeyer flask, and then cultured at 28°C on a rotary shaker at 120 rpm. For each medium, 8 to 10 flasks were prepared. The ODem of the cell suspension after ultrasonic dispersion for 2 min (275 /‘A) or the dry weight obTABLE I.
Compositions of media for various carbon sources
Carbon source *Fatty acid Sodium formate Sodium acetate Sodium propionate Butyric acid Valerie acid Sodium hexanoate Sodium caprylate Sodium caprate Sodium lauriate Sodium palmitate *Paraffinic hydrocarbon Hexane Octane Decane Dodecane Tetradecane Hexadecane Octadecane Eicosane Nutrient salts” Yeast extract NaCl KC1 CaCl, MgSO, NH,Cl Deionized water TOC pHb
Abbreviation 5.67 3.42 2.67 1.91 1.81 1.92 1.73 1.62 1.54 1.45
g g g ml ml g g g g g
FA[IO] FA[12] FA[l6]
1.81 1.70 1.62 1.58 1.55 1.53 1.18 1.18
ml ml ml ml ml ml g g
PHC[6] PHC[8] PHC[lO] PHC[lZ] PHC[14] PHC[16] PHC[lS] PHC[ZO]
0.1 0.303 0.14 0.18 0.2 0.35 1.0
g g g g g g I
1000
FALlI FA[2]
FA[31 FAL41
FA[51 FA[61 FAl81
mg/l
7.5
a Nutrient salts were added to various carbon sources. h Adjustment of pH was carried out using phosphate buffer, Na2HP0, (M/15) : KZHPO, (M/15)=4: 1. Numbers in parentheses are expressed as number of carbons.
author. 186
i.e.,
VOL. 79, 1995
NOTES
Number
10°0W
Time(d) FIG. 1. Time course of biomass concentrations of N. atnarae in FA media. Symbols: Cl, FA[I]; 0, FA[2]; a, FA[3]; A, FA[4); 0, FA[5]; l , FA[6]; D, FA[8]; n , FA[IO]; and V, FA[16].
tained by the paper filter method (1 Irrn Millipore filter) per flask was measured within a certain period of time. The ODsoo or the dry weight of two flasks without strains was similarly measured at the start and end of each experiment. These control values were used to correct the biomass concentration. The filter paper method was applied to the case of FA[lO]-FA[16] and PHC media containing an insoluble source. Time courses of biomass concentrations of N. amarae in FA media and R. rhodochrous in PHC media are shown in Figs. 1 and 2, respectively. The same tendencies were observed in the cases using other media and strains. The specific growth rates (,f) during logarithmic phase were calculated and the relationship between number of carbons and /f values of the 3 strains in both FA and PHC media is shown in Fig. 3. Lower fatty acids (C2-C5) were utilized preferably by the 3 strains and FA media with number of carbons higher than 8 were not utilized at all. It is well known that sodium acetate and sodium propionate were utilized by N. amarae (2, 7). Therefore, additional substrates such as butyric and valeric acids (C4 and CS) could be utilized by N. amarae. In contrast, 11 values of N. amarae and Rhodococcus spp. increased with increase in number of carbons in PHC media. The highest /I value was obtained in octadecane (Cl@. Such peculiar substrate utilization is also rec-
187
of carbons
FIG. 3. Relationship between number of carbons and ,t logarithmic phase. Symbols: ci, N. amarae in FA media; rhodochrous in FA media; 0, R. erythropolis in FA media; amarae in PHC media; A, R. rhodochrous in PHC media; and erythropok in PHC media.
during n, R. 0, N. l , R.
ognized in LAS (linear alkylbenzene sulfonates), composed of the same straight chain as paraffinic hydrocarbons (11). Octadecane and eicosane (C20) have melting points of 28.O”C and 36.6”C, respectively. The former was a liquid while the latter was a solid during our experiments. In general, liquid is more likely to be utilized than solid by microorganisms; however, the /t value in the case of eicosane was shown to be nearly the same as that of dodecane (C12) or tetradecane (C14). Therefore, the affinity of the 3 strains to eicosane was estimated to be higher in spite of it being in the solid state. Although hydrocarbons with more than 21 carbons were not examined, it was suggested that the /I values would be considerably lower since they would be present as solid under our experimental conditions. From these results, it was found that N. amarae and Rhodococcus spp. exhibit the peculiar substrate utilization of fatty acids and hydrocarbons. As shown in Fig. 3, fatty acids and hydrocarbons were utilized in the same way by N. amarae and Rhodococcus spp. The /t values of Rhodococcus spp. were 3.6 and 2.0 times those of N. amarae in FA[3] and PHC[18] media, respectively. The /l values of N. amarae in FA[2]-FA[5] media were almost equal, but sodium propionate utilization was exceptionally high by Rhodococcus spp. The 1’
--o
0
500 lnltlal TOC concentration
Time (h) FIG. 2. Time course of biomass concentrations dochrous in PHC media. Symbols: (3, PHC[lO]; 0, PHC[14]; A, PHC[16]; 0, PHC[lI]; and +, PHC[20].
of R. rhoPHC[IZ]; A,
1000 of medium (mgii)
FIG. 4. Relationship between initial TOC concentration of medium and ,l during logarithmic phase in PHC[18] medium. Symbols: 0, N. amarae; A, R. rhodochrous; c\, R. erythropolis. Each curved line was calculated and plotted using kinetic parameters.
188
IWAHORl
J. FERMENT.BIOENG.,
ET AL
TABLE Microorganism
2.
Comparison
of kinetic
parameters Kinetic
Medium /l,,,
(I/d)
parameter
K (w/O
11.8
29
9.1
11
Ref.
Glucose
8.6
41
7.0
47
9.1
6
8.9
17
8.2
8
Acetic acid Activated
sludge microorganisms
Propionic
acid
Glucose Sphaerotilus sp. Sphaerotilus natat@
Glucose
2.9
-
6.5
10
2.6
2
2.8
0.5
3.0
2.5
5.8
98.2b
15.9
212.3h
15.2
557.7h
14 14 15
Filamentous
bacterium
Type
1701”
N. amarae ASF3” N. anzarae ASAC 1a
Acetic acid
13
N. amarae R. rhodochrous
Octadecane
R. erythropolis a Kinetic parameters were calculated under steady-state chemostat h These values are K,’ values and are higher than k; values
values of both the activated sludge and the dominant bacteria in activated sludge, which were obtained during the logarithmic phase of batch culture using artificial sewage, were reported to be 13.8 I/d and 4.0-19.7 l/d, respectively (12). These values are similar to those obtained from substrates which could be utilized by Rhodococcus spp. Therefore, it was shown that R. rhodochrous and R. erythropolis are capable of overcoming competition with the activated sludge microorganisms depending on the components of the substrate or sewage. The initial TOC concentration affecting the growth of N. amarae and Rhodococcus spp. was investigated in the as the carbon source. same way, using octadecane PHC[ 181 medium listed in Table 1 was diluted by deionized water and its TOC concentrations were adjusted to 100, 300 or 500mg//. The lag phase was observed in case of N. amarae as the TOC concentration decreased, but it was not observed in the case of Rhodococcus spp. The 11 values during logarithmic phase were calculated and their relationship with the initial TOC concentrations is shown in Fig. 4. The ~t values of the 3 strains obviously decreased with decrease of the initial TOC concentration. The relationship between the specific growth rate (/f) and TOC concentration (S) can be generally expressed by the following equation: ,‘I
I?;‘,
S
where P,,, is the maximum specific growth rate and K, is the saturation constant. Assuming that S, and ,j are the initial TOC concentration and the removal efficiency, respectively, Eq. 1 can be rewritten as follows:
culture.
This study
The others were carried out in batch culture.
.s
d?_--,iSO If ,i is constant, K,/(l-,?) becomes constant (K,‘) and the relationship between /1 and S, is represented by a Monod-type equation. It was found that ,j values in the case of the 3 strains were constant regardless of the initial TOC concentration, because good correlations were obtained from the Hofstee plot. The /L,,, and KS’ values are summarized in Table 2 along with those obtained from the past studies, using activated sludge microorganisms, filamentous bacteria and actinomycetes. Since the culture method and the carbon source are different, it is difficult to compare the kinetic parameters accurately. The /I,,, values of Rhodococcus spp. are higher than those of N. amarae, filamentous bacteria and activated sludge microorganisms. On the other hand, the KS’ value is twice or 10 times the KS value if ,i is 50% or 90%, respectively. Judging from this conversion, the KS value of N. amarae is of the same order as those of activated sludge microorganisms, while K, values of Rhodococcus spp. are estimated to be higher than those of activated sludge microorganisms, filamentous bacteria and N. amarae. Therefore, Rhodococcus spp. are found to have higher growth rate and lower affinity for octadecane than N. amarae. In other words, R. rhodochrous and R. erythropofis are capable of rapid growth in the activated sludge process if higher-strength octadecane is fed and contacted with them, whereas N. amarae grows rapidly in lower-strength octadecane. These trends for N. amarae and Rhodococcus spp. seem to be recognized in other substrates such as lower fatty acids and hexadecane. Consequently, it is thought that their growth char-
VOL. 79, 1995
acteristics
NOTES
in the activated
sludge process
are dissimilar.
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