Effects of temperature and pH on the growth of heterotrophic bacteria in waste stabilization ponds

~

Pergamon

0043-1354(95)00150-6

Wat. Res. Vol. 30, No. 2, pp. 447-455, 1996 Copyright © 1996 ElsevierScience Ltd Printed in Great Britain. All rights reserved 0043-1354/96 $15.00 + 0.00

EFFECTS OF TEMPERATURE A N D pH ON THE GROWTH OF HETEROTROPHIC BACTERIA IN WASTE STABILIZATION PONDS A L O I C E W. M A Y O I* and T A T S U Y A N O I K E 2 ~Civil Engineering Department, University of Dar es Salaam, P. O. Box 35131, Dares Salaam, Tanzania and 2Department of Civil Engineering, Tohoku University, Aoba Sendai 980, Japan (First received February 1994; accepted in revised form May 1995) A~traet--This paper presents the effects of temperature and pH on the growth of heterotrophic bacteria in Chlorella vulgaris-heterotrophic bacteria culture. The growth of heterotrophic bacteria was studied at 10, 15, 20 and 30°C, and pH was controlled from 3.0 to 11.5 in a series of fed-batch chemostat reactors supplied with glucose as the sole source of carbon. Samples were analyzed for heterotrophic bacteria by tryptone glucose extract agar in triplicate. The agar pH of 7.0 was the best for enumeration of heterotrophic bacteria. The bacteria grown at pH near neutral were more sensitive to the variation of agar pH than those grown at very alkaline pH. No significant difference in the number of cells capable of forming colonies was noted for incubation temperature of 20 and 35<'C, but the lag time for colony formation was longer at 20°C. Samples for enumeration of heterotrophic bacteria collected from algal-bacterial systems such as waste stabilization ponds are recommended to be incubated at 35°C for 72 h. Depending on the pH of the culture, about 86 98% of the ceils capable of forming colonies will be visible to the naked eyes after incubation at 35°C for 72 h. At the steady state conditions, heterotrophic bacteria were not sensitive to temperature in the range of 10-20°C. However, at 30°C, a notable competition for glucose between Chlorella vulgaris and heterotrophic bacteria was observed. This competition was responsible for the low bacterial density near neutral pH. No evidence was found to support the view that the discharge of bactericidal substances from Chlorella vulgaris was responsible for reduction of heterotrophic bacteria at high pH. Key words--heterotrophic bacteria, temperature, pH, algal-bacterial system, Chlorella vulgaris, waste stabilization ponds, competition for substrate.

INTRODUCTION Temperature and p H are among the important environmental parameters governing the activities and growth rates of algae and heterotrophic bacteria in waste stabilization ponds. Temperature is known to influence the biomass composition, nutrient requirement, nature of metabolism (Pirt, 1971) and the metabolic reaction rate (Novak, 1974). Microorganisms do not have the ability to regulate their internal temperature (Esener et al., 1981). This may in turn affect the microorganism growth and substrate utilization rate. On the other hand, p H of the medium in algal-bacterial system is known to influence the biomass regulation, ion transport system (Guffanti et al., 1984) and metabolic rate (Mayo and Noike, 1994b). Temperature varies widely in waste stabilization ponds depending on pond location and season of the year, In domestic wastewaters, the p H is the function of organic loading rate and may vary from 6.0 in the influent to as high as 11.5 in the maturation ponds. The p H in waste stabilization ponds located in tropical and sub-tropical climates has been re*Author to whom all correspondence should be addressed.

ported to be correlated to water temperature (Mayo, 1995a), Pour plate method is widely used for enumeration of heterotrophic bacteria in natural environments (Hattori, 1982; Brozel and Cloette, 1992), because most probable number (MPN) technique is unsuitable where precision is desired (Jen and Bell, 1982). Many researchers differ significantly on the choice of incubation temperature for enumeration of heterotrophic bacteria (Boylen and Brock, 1973; Guthrie et al., 1976; Kaminski and Ferroni, 1980 and Jen and Bell, 1982). Hattori (1982) for instance used three different temperatures, 30, 27 and 20°C. The Standard Methods ( A P H A et al., 1985) recommend incubation temperature of 35°C for 48 h for tryptone glucose extract agar using plate count technique. Jen and Bell (1982) and Brozel and Cloette (1992) recommended the temperature of 30°C for 72h. The agar pH recommended by the Standard Methods ( A P H A et al., 1985) is strictly 7.0 + 0.2, although the bacteria cytoplasm pH varies widely. The internal p H of bacteria ranges from 6.5 to 7.0 in acidophiles, 7.5 to 8.0 in neutrophiles and 8.4 to 9.0 in alkophiles (Booth, 1985; Guffanti et al., 1978; Guffanti et al., 1984; Hackstadt, 1983). This means that enumeration 447

Aloice W. Mayo and Tatsuya Noike

448

of bacteria at pH 7.0 _+ 0.2 as recommended by the Standard Methods' (APHA et al., 1985) may not necessarily produce the best results in algal-bacterial system. Several studies have been carried out on the enumeration of heterotrophic bacteria in natural environments. Samples for these studies were collected from forest and paddy soils, sea, lake and river waters (Hattori, 1982), a continually flowing storm drain (Jen and Bell, 1982) and cooling waters (Brozel and Cloette, 1992). These natural environments are different from waste stabilization ponds where nutrients, pH and other physical-chemical factors affecting cell growth differ. Application of incubation conditions derived from other environmental conditions may therefore not be suitable in algal bacterial system. The specific objectives of this study were: (i) to study simultaneous effect of pH and temperature on the growth of heterotrophic bacteria and the efficiency of glucose consumption; (ii) to determine the correct enumeration procedure for bacteria growing in algal-bacterial system; (iii) to investigate the competition for substrate between algae and bacteria. Fed-batch chemostat reactors were used as model ponds for the experiments. Glucose was supplied daily as the sole source of organic carbon at the concentration of 75 mg/l. Previous study indicated that at this concentration the anaerobic conditions may not prevail (Mayo and Noike, 1994a). METHODS

AND MATERIALS

Microorganisms and inoculum preparation Bacterial seed for inoculum was obtained from final sedimentation basin of the Sendal City activated sludge wastewater treatment plant and was maintained in the laboratory on glucose fed-batch seed T a b l e 1. N u t r i e n t composition Chemical KNO 3 MgSO4 • 7 H 2 0 K2HPO 4 KH2PO 4 NaC] CaCI 2 • 2 H 2 0 EDTA-Na T a p water Fe solution T r a c e minerals

Amount 1000 mg 250 mg 110 mg 110 mg 100 mg 10 mg 16 mg 998 ml I ml 1 ml

Fe solution FeSO4 • 7 H 2 0 Distilled water

= 500 mg = 250 ml

T r a c e mineral composition H2BO 3 M n S O 4 "4 H 2 0 ZnSO4 • 7 H 2 0 CuSO4" 5 H 2 0 NazMoO a Distilled water

= = = = = =

286 mg 130 mg 320 mg 183 mg 2.l nag 100 ml

Gas release

T

=

Reactor 5 liters

[

10-30 ° C

I

I Magnetic I I

mixer

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±H I

~/}.~ o

Sampling port

II

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Fig. I. Experimental apparatus. culture. A nutrient composition shown in Table l was used to maintain bacterial seed culture. About 400 rag/1 of glucose was fed daily and oxygen was provided by an air pump. The seed was transferred once every two weeks to maintain good viability and stability of heterotrophic bacterial population. Alga, Chlorella vulgaris, was used for the experiments and was grown at 6000 lx in a 51 conical flask containing about 21 of liquid medium with the composition shown in Table 1. Carbon was supplied by sparging in air at the bottom of the flask. Medium and chemostat reactor operation The nutrient composition shown in Table 1 was used for the experiments. Tap water was used in the preparation of the medium and trace metals were supplemented. Chemostat reactors made of acrylic plastic were used as the model stabilization ponds, and lights, temperature and time were controlled by model FLI-301N incubator (Eyela, Japan). Each reactor had 61 gross volume with 51 of useful capacity and was provided with sampling port near the bottom end and a cover with gas release ports (Fig. 1). A light intensity of 6000 lx was provided by cool white fluorescent lamps, fixed to the three inner sides of the incubator. Lights were operated at 12 h alternating photoperiods of light and darkness controlled by a timer. This light intensity may not be a limiting factor for growth of algae because saturation light intensity is less than 5400 Ix (Goldman, 1979). Actively growing bacterial cells from a newly prepared seed culture was acclimatized at a temperature similar to that of the culture, in a 500 ml conical flask containing 200 ml liquid medium. After acclimatization for about 24-36 h, seeds of algae and bacteria were mixed and used to inoculate cultures for the experiments. The cultures were grown at the temperatures of 10, 15, 20 and 30~C and were completely mixed by magnetic stirrers. For each temperature, several runs were made at pH 3.0-11.5. The pH of the medium was controlled by adjusting with IN HCI or 1N NaOH solutions. Glucose was daily added in granular form to provide an input concentration of 75 mg/l of the remaining volume of culture. With this

Temperature and pH on oxidation pond bacteria glucose concentration, anaerobic conditions may not occur in algal-bacterial system (Mayo and Noike, 1993; Mayo and Noike, 1994a). Before sampling, biomass attached on the reactor walls was carefully resuspended by swirling the culture content. Sampling was done at 10.00 a.m., 3 h after the lights were switched on. Daily additions of glucose were made immediately after sampling, or at 10.00 a.m. on the days when sampling was not done. Experiments on the effects on agar p H and incubation temperature for enumeration o f bacteria

To determine the correct enumeration procedure for the number of heterotrophic bacteria grown in algal-bacterial system, it was necessary to study the effects of agar pH and incubation temperature on the cells capable of forming colonies. The heterotrophic bacteria used for the experiments were grown in mixed culture of Chlorella vulgaris--heterotrophic bacteria according to the procedure described above. The cultures were maintained at 2ff'C and at pH 3.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 in a series of chemostat reactors, until the steady state densities were reached. The steady state density of heterotrophic bacteria was assessed by enumerating bacteria by pour plate method as outlined in the Standard Methods (1985). Depending on the pH of the culture the beterotrophic bacteria attained the steady state density in about 8-11 days. Samples for the examination of the effects of agar pH and incubation temperature on the cells capable of forming colonies were drawn on the 20th day after steady state conditions were confirmed for a sufficiently long period of time. The effect of agar pH was investigated at pH 6.0, 7.0, 8.0, 10.0, and 11.0 because this range is typical in waste stabilization ponds. Agar was adjusted to the desired pH by addition of either NaOH or HC1, before sterilization by autoclaving. The pH of agar did not change after autoclaving. Colony formation was determined by serial dilution of the samples, and each dilution was spread onto 15 plates. Three plates of each dilution were spread with agar at a specified pH. For the study of incubation temperature a similar procedure was followed, but agar pH was adjusted to pH 7.0 before autoclaving. Each dilution was spread onto 6 plates. Three plates of each dilution were then incubated at 35°C as recommended in the Standard Methods (APHA et al., 1985) and at 2OC, the temperature similar to that at which bacteria were grown. The temperature of the environment at which the bacteria were grown was chosen because according to Jen and Bell (1982) higher densities of bacteria are recovered at the incubation temperature similar to the ambient temperature. Sample analyses

Heterotrophic bacteria population was determined by pour-plate count on serial dilution of samples

449

mixed with tryptone glucose extract agar (Difco, U.S.A.). Incubation was done at 35°C and colonies were counted after 72 h of incubation for reasons that will be discussed later in the text. Sterilization was done in accordance to the Standard Methods (APHA et aL, 1985). Algal biomass was determined by chlorophyll a method in the Standard Methods (APHA et aL, 1985). Chlorophyll a was extracted by 90% acetone and was measured by spectrophotometric method (Hitachi, model U-1100). Glucose concentration was determined by the procedure proposed by Dubois et al. (1956). Glucose concentration in the samples was prepared in appropriate dilution and was measured spectrophotometrically at 490 nm (Hitachi, model 100-20). The glucose concentration in the samples was then determined by comparing the optical density reading with the standard glucose solutions calibration curves. The pH was checked by a pH meter (TOA Electronics Limited, model HM-10P). RESULTS The choice o f incubation conditions Jot the enumeration of bacteria

Heterotrophic bacteria were first grown in mixed culture of algae and bacteria at 20°C for 20 days and at various pH values. Bacteria were then enumerated at different agar pH, incubation temperature and incubation times. Jen and Bell (1982) reported that at 95% confidence limit the accuracy of enumeration of heterotrophic bacteria using pour plate method is within +20%. This means that the minimum incubation time can be determined as the duration after which heterotrophic bacterial count exceeds 80% of the number of cells capable of forming a colony. This minimum incubation time was mathematically determined using equation (1) (Hattori, 1982) and (2) as described below In(No - N) = In No - 2t,

(1)

where No is the number of cells capable of forming colonies, N is the number of cells at time t,, tc is the time since the colonies started to form, 2 is the probabilistic parameter and was calculated from the slope of [ln(N0 - N ) / l n No] on re. A linear regression was obtained when In(N0 - N) was plotted against tc(tx> tr). The lag time, tf, is defined as the time when the colonies started to form since the plates were poured. The lag time was determined as shown in Fig. 2. Equation (2) was used to estimate the total time, tv, required for N cells to form since the plates were poured 1 ti- = tr + t~ = tr + ~ ln[No/(No - N)].

(2)

Figure 3 shows that the colony formation is considerably slower at 20°C than at 35°C. Similar results were observed at pH 7.0 and 9.0 but at pH 3.0 colony

Aloice W. Mayo and Tatsuya Noike

450 1.1

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,-e---a~r.. _6.__.e----- u

DO., " f

•~ 15 "-- 0 . 9 Z

-- 0 . 8

,,

f.:/

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50 100 150 Inc~abalion time (h)

0

200

formation rate was the same regardless of incubation temperature. The incubation temperature of 35°C was adopted because the number of cells capable of forming colonies at incubation temperature of 20°C did not differ significantly to those at 35°C and the incubation time at 35°C was shorter. The incubation time of 72 h, a multiple of 24 h period was adopted because it gave 86-98% of the heterotrophic bacteria cells. The adopted incubation time differs to those of Jen and Bell (1982) and Standard Methods ( A P H A et al., 1985) who suggested 48 h incubation at 30 and 35°C, respectively. If 48 h had been adopted, only about 73% of the colonies able to grow at culture pH of 5.0 may be recorded, Figure 4 shows a good fit of equation (2) for the culture grown at pH of 3.0, 5.0, 7.0 and 9.0 whose plates were enumerated at the incubation temperature of 35°C. When the bacteria were incubated at

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Fig. 4. Effects of incubation time on the heterotrophic bacteria colony formation at 35°C for cultures grown at various pH values at 20°C for 20 days. The dotted curves are data fit according to equation (2) for tv > t,.

20°C, lag time of at least 24 h was observed, except for heterotrophic bacteria grown at pH 3.0 (Table 2). As seen from Table 2, the number of cells capable of forming colonies (No) for a given culture pH was independent of incubation temperature. No values at 20°C differed by only about 11, 6, 1 and 3% of those at 35°C for culture pH of 3.0, 5.0, 7.0 and 9.0, respectively. Except for culture pH of 3.0, No were higher at 35°C than at 20°C, although bacteria were grown at 2ff'C. Figure 5 shows that more colonies were formed at pH 7.0, regardless of pH at which the bacteria were initially grown• This suggests that the optimum p H for bacterial growth is probably near neutral pH. Bacteria cultured at pH 10.0 were less sensitive to agar pH than those cultured at pH 6.0. It is possible that the growth of heterotophic bacteria adapted to high p H is better when medium pH is neutral. When agar pH was increased to 11.0, only 1.67% of the cells capable of forming colonies was observed for bacteria originally grown at pH 6.0 (Table 3). For the same conditions 21.2% of cells formed colonies for bacteria originally grown at p H 10.0. This suggests that cells grown at pH 10.0 were more tolerant to increased pH because of adaptation of high pH. F r o m the results observed here, the most suitable set of conditions for enumeration of heterotrophic bacteria was incubation at 35°C for 72h with agar pH of 7.0.

Effect of temperature on growth o f heterotrophie bacteria in fed batch cultures

i •

o

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Fig. 2. Effect of incubation temperature for enumeration of bacteria on lag time. Bacteria were initially grown at 20°C, pH 5.0 for 20 days before sampling. The arrows indicate the lag time for the incubation temperature of 20°C and 35°C.

"f -

f ..~£

5

0.7

,~../~

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Fig. 3. Effect of incubation temperature on the heterotrophic bacteria colony formation for culture grown at pH 5.0. The dotted curves shows the fit according to equation 2 for tv > t r.

The effect of temperature on the growth of heterotrophic bacteria is shown in Fig. 6. The growth of bacteria showed no lag phase except at 10°C when pH was above 10.0 or below 4.5. The time bacteria took to grow to the maximum density was shorter with increasing temperature• It was interesting to observe

Temperature and pH on oxidation pond bacteria

451

Table 2. Minimum incubation time as function of culture pH and incubation temperature pH o f medium for growth o f bacteria in mixed culture

Incubation temperature for enumeration o f bacteria CC)

N0 ( x 106)

(d J)

3,0 3.0 5.0 5.0 7.0 7.0 9.0 9.0

20 35 20 35 20 35 20 35

0.86 0.78 14.9 15.8 20.7 20.9 3.25 3.35

0.0467 0.0353 0.0230 0.0272 0.0299 0,0296 0,0259 0,0313

lower bacterial densities at 30°C regardless of pH of the culture. The mean steady state bacterial populations at culture pH of 10.0 were 2.2-+ 1.3 × 10 6 7 2 . 0 + 2 7 . 5 x 106, 73.5+ 53.1x 106 and 71.9__+ 19.4 x 106 cfu/ml at 30, 20, 15 and 10°C, respectively. At pH 7.0, the heterotrophic bacterial densities were 1.3 _+ 0.6 x 10 6, 14.7 + 3.2 x 10 6 and 20.1 + 5.5 × 10 6 cfu/ml at 30, 20 and IO°C, respectively. Statistical analyses using student t-distribution at 95% confidence interval shows that mean values for cultures grown at 10, 15 and 2ff~C were statistically the same but those at 30°C were statistically different. The reason for the same number of heterotrophic bacteria at 10-20°C was not clearly evident. It is possible that the density attained is the maximum possible in algal-bacterial system for the experimental conditions given in this work. It was also noticed that as the temperature of culture decreased, it took longer for bacteria to reach the maximum density. Low bacterial populations at 30°C might have been

s.o-[ 6.0-

2

R for data fitting o f equation (1) ------

0,994 0.988 0.986 0.970 0.975 0.960 0.976 0.984

Lag time (h)

Minimum incubation time (h)

2.5 2.1 38.6 8.8 24.4 2.3 43.5 19.4

37 48 109 68 78 54 106 71

influenced by high activities of algae and bacteria at this temperature, that has caused glucose to become a growth limiting factor. At 30°C, 6000 Ix and pH 7,0, for instance, the efficiency of glucose consumption was nearly 100% and biomass yield was 0.385mg particulate organic carbon (POC)/mg glucose (Mayo and Noike, 1994b). This yield is equivalent to 96.3% because theoretically, 1 mg glucose has 0.4 mg C. The biomass yield observed here is considerably higher than those reported in other microbial systems (Pipyn and Verstraete, 1978). Evidently, with the biomass yield and the efficiency of glucose consumption observed at 30°C, glucose was a limiting factor for bacterial growth. In contrast, biomass yield at 20 and 15°C were considerably lower at 0.245 and 0.218 mg POC/mg glucose, respectively. These values are equivalent to 61.3 and 54.5% of the biomass yield, respectively. It is also worth mentioning that in algal-bacterial system fed with glucose, apart from symbiotic relationship (Oswald et al., 1953), algae and bacteria compete for glucose, a substance assimilated well by Chlorella (Kommor and Tanner, 1974; Martinez et al., 1987; Mayo and Noike, 1994a). Mayo and Noike (1994a, b) also reported evidence of heterotrophic bacteria-Chlorella vulgaris competition for glucose, with Chlorella having higher ability for uptake than bacteria.

~4.0Effect o f p H on bacterial population in fed batch cultures

lture pH 6.0 2.0-

"~,

~

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0.0.

~£6 . 0 ~ 4.0

F÷Fr~ 6.o

N

1 ÷ pH 7.0 I-~ p n 8.0 I ÷ P H t0-0

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24

48 72 96 120 Time of incubation (h)

144

Fig. 5. Effect of agar pH on the number of heterotrophic bacteria. Heterotrophic bacteria were initially grown in mixed algal-bacterial culture at 20°C for 20 days. WR 30/2--N

Figure 7 shows the effect of pH of culture on the heterotrophic bacterial density at 10°C. The bacterial densities were 2 0 . 1 + 5 . 5 x 106cfu/ml at pH 7.0, but decreased slightly to 12.5+0.9 x 106 and 4.9 __+1.25 x 106cfu/ml at the culture pH of 6.0 and 4.5, respectively. In alkaline pH, bacterial densities decreased only slightly to 10.9__+ 1.7 × 106cfu/ml at pH 9.0. At pH 10.0, bacterial populations were higher than at pH 7.0. Similar results were repeated at 30 and 20°C (Fig. 6). Statistical analysis at 95% confidence interval, shows that the differences in bacterial population at pH 7.0 and 10.0 were insignificant at 30°C but significant at 10, 15 and 20°C. It is possible that high activities of algae and bacteria near neutral pH were responsible for this behavior by making glucose a growth limiting factor. At pH 10.0, only about 64% of glucose was assimilated. This means algae and bacteria did not have to compete for

452

Aloice W. Mayo and Tatsuya Noike Table 3. Effect of agar pH on colony forming units pH of medium Percentage of cells for growth Agar pH for R for data Lag capableof forming of bacteria in enumeration N~ 2 fitting of time colonies at 35 C mixed culture of bacteria ( × 10") (d ~) equation(1) (fi) for 72 ha 6.0 6.0 1.72 0.0666 0.996 18.3 34. I (35. I ) 6.0 7.0 4.90 0.0389 0.934 6.3 92.2 (100) 6.0 8.0 1.47 0.0527 - 0.979 11.9 28.6 (29.9) 6.0 10.0 0.74 0.1326 0.950 17.3 15.0 (15.1) 6.0 I 1.0 0.083 0.0665 0.997 3.2 1.67 (1.69) 10.0 6.0 14.9 0.0886 - 0.997 19.0 54.5 (55.0) 10.0 7.0 27.0 0.0520 0.958 3~8 97.1 (100) 10.0 8.0 24.5 0.0737 0.959 1.6 90.2 (90.8) 10.0 10.0 16.1 0.0587 -0.956 12.4 57.8 (59.61 10.0 I1.0 5.75 0.0980 -0.993 13.1 21.2 (21.3) Values in parenthesesshow the cells capable of formingcolony at the respectiveagar pH based on the maximum value at pH 7.0.

glucose. The bacterial population at pH creased to 1.1 +_ 0.8 x 104cfu/ml.

1 1.0 de-

Growth of heterotrophic bacteria in the presence and absence of algae To determine whether the low n u m b e r o f heterotrophic bacteria at 3 0 C was a result o f temperature or o f starvation, further studies were carried out in the absence o f algae. H e t e r o t r o p h i c bacteria were first isolated from the colonies that have formed on the plates. Actively growing bacterial cells were prepared in a 1000 ml flask containing 2000 mg o f tryptone glucose extract and were cultured at 30°C. After 2 days, the heterotrophic bacterial cells were transferred to reactors adjusted to pH 7.0 and 10.0. Glucose was added daily in granular form to provide

10 9 .

A: pH7.0 10 8 10 7

~

10 6

an input concentration of 75 mg/l o f the remaining volume of culture and nutrient composition listed in Table 1 was provided. F r o m Fig. 8, it can be seen that in the presence o f algae, the n u m b e r of heterotrophic bacteria was lower c o m p a r e d to a similar system in the absence of algae. This evidence suggests that some factor inhibits growth o f heterotrophic bacteria in the presence o f Chlorella vulgaris. Two possible causes for this behavior were considered to be production o f bactericidal agents by algae or competition for substrate.

Effects of pH and temperature on the degradation of glucose Glucose uptake by algal-bacterial system was calculated as a slope o f cumulative glucose c o n s u m e d against glucose added and the results obtained are presented in Fig. 9. Temperature did not significantly influence the efficiency o f glucose c o n s u m p t i o n at low p H values, but a significant influence was observed at p H above 9.0. The uptake o f glucose is p H dependent with a b r o a d o p t i m u m from p H 6.0 to 8.0, a steep decline above pH 9.0 and relatively mild decline below p H 6.0. In a mixed Chlorella vulgaris-heterotrophic bacterial culture, the efficiency of glucose c o n s u m p t i o n in alkaline pH range was adversely

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Fig. 6. Effect of temperature on the growth of heterotrophic bacteria at pH 7.0 and 10.0.

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Fig. 7. Effect of pH on the growth of heterotrophic bacteria at 10~C.

Temperature and pH on oxidation pond bacteria 1010

453

Table 4. Effect o f pH on K,, and Vm,~ at 3 0 ' C

,10 7

pH

Km (mg glucose/I)

vm~x (nag glucose/mg POC/h)

5 7 8 9 10 12

45 43 47 112 248 668

0.501 0.674 0.610 0.435 0.224 0.051

lo 6

lytical approach developed by Halwach (1978) shown by equation (4)

.~ 105"

~

v-

104 0

Operation time (d)

U

Vm,xXo

Ymax S

In

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Fig. 8. Growth of heterotrophic bacteria in cultures in the presence and in the absence of algae. affected than in acidic pH range. The decrease in influx velocity with increasing pH might have been caused by the decline of affinity of glucose of the algal uptake system. The kinetics of the effect of pH on the degradation of glucose was studied in batch culture using one experiment for each pH investigated. The input biomass used for the batch culture was about 550 mg POC/1 (range 546-558 rag/l) and was collected at the end of exponential growth phase from the seed culture. The batch experiments were carried out at 30°C in a series of completely mixed batch cultures maintained at pH 5.0, 7.0, 8.0, 9.0, 10.0 and 12.0. A light intensity provided was 6000 lx operated at 12 h alternating photoperiods of light and darkness, Glucose concentration of 2000 mg/l was added and a fraction of nutrient composition listed in Table 1 was provided, By using the data obtained from batch culture, the maximum specific glucose degradation rate, v~,~, and the half rate saturation coefficients, Kin, were determined according to a generic Micfiaelis-Menten rate law [equation (3)] using aria100 OOO

13°

so

ID

gk []

60 40

20

I:1 m ,

0 2

i

i

i

i

4

6

8

10

pH

12

Fig. 9, Effects ofpH and temperature on the glucose uptake efficiency.

(3)

Km+S 4 S°+K~ Vr~.xXO

(4)

where U = [(S0 - S)/So], So is the initial substrate concentration, S is the substrate concentration at the time, t, and X0 is the biomass concentration (rag/l). By plotting (t/U) against [(l/U) • In[{I/ (1 - U ) } - 1]], Km and vm,x were calculated from the slope, [Km/(Vm,~Xo)] and vertical intercept, [(So + K,O/(Vm,xXo)]. Table 4 shows the effect of pH on the maximum specific glucose degradation rate, v. . . . and the half rate saturation coefficients, Km. From the data shown in Table 4, the high affinity uptake occurs at pH near neutral. Kommor and Tanner (1975) have observed that protonation changes the conformation of the carrier and thus increases Chlorella vulgaris affinity for sugar. DISCUSSION

The results presented in Table 2 show that the number of heterotrophic bacteria grown at the incubation temperature of 35~'C did not differ significantly to those grown at 20°C, the original temperature of the culture. These results differ from the work by Jen and Bell (1982) who concluded that higher recovery of heterotrophic bacteria at the incubation temperature of 30~C, which was similar to the temperature of the environment from which the bacteria were obtained (28-30°C). Interestingly, at the incubation temperatures of 20, 25 and 35=C, Jen and Bell (1982) observed no significant difference in bacterial number at the incubation time exceeding 96 h. These results are similar to those shown in Table 2. If ambient temperature was the governing factor for the selection of incubation temperature, then the higher number of bacteria should have been obtained at 20°C than at 35°C. This means that ambient temperature of the environment was not the factor governing the higher recovery of the heterotrophic bacteria. This work has shown that glucose metabolism decreases significantly at high pH values particularly above pH 9.0. Interestingly, although heterotrophic bacterial densities were higher at pH 10.0 than at the neutral pH of 7.0 for all temperatures investigated, glucose metabolism sharply decreased. The sharp decline in glucose utilization efficiency at alkaline pH

454

Aloice W. Mayo and Tatsuya Noike

(Fig. 9) also corresponds to sharp reduction in Chlorella vulgaris growth rates (Mayo and Noike, 1994b). These results suggest that perhaps Chlorella was more responsible for glucose metabolism than heterotrophic bacteria, and agrees with other findings in literature (Abeliovich and Weisman, 1978; Mayo, 1995b; Mayo and Noike, 1994b). Abeliovich and Weisman (1978) reported negligible growth and activity of heterotrophic bacteria in the pond and concluded that bacteria play but a minor role in the degradation of organic matter in high-rate oxidation ponds. Mayo and Noike (1994a) found that half-rate saturation constants, K~ for the growth of Chlorella ~ulgaris and heterotrophic bacteria were 181 and 27 ms/l, respectively. Wright and Hobbie (1965) observed the apparent K~ value of 7/~g/l for natural populations of glucose fed bacterial culture in oligotrophic environment. These low saturation constants for bacteria suggest their minor role in glucose assimilation. There was an evidence that heterotrophic bacteria grew less vigorously in the presence of Chlorella vulgaris than in the absence of Chlorella cells. Similar findings have previously been made in oxidation pond sewage (Oswald et al., 1953; Caldwell, 1946). Oswald et al. (1953) found the steady state bacteria density of 1 x 108 cfu/ml in the absence of algae but only 5 x 10 7 and 1 x 106cfu/ml in the presence of Euglena graeilis and Chlorella pyrenoidosa, respectively. These densites are significantly lower than those in Figs 6 and 7 probably because glucose used as a source of carbon in this study is readily degradable than raw sewage. Two possible causes of low number of heterotrophic bacteria in the presence of algae are competition for substrate or discharge of toxic substances by algae. Alga Chlorella vulgaris has been reported to discharge toxic long chain fatty acids when under stress, such as at high pH (Pratt and Fong, 1940). The discharged substances such as chlorellin are said to have marked antibacterial activities. Parhad and Rao (1974) observed that Escheriehia coli survived well in the presence of Chlorella in tropical waste stabilization ponds. Parker (1962) found no evidence to support the view that the release of bactericidal substances from algae was responsible for reduction of coliform counts. Vela and Guerra (1965) reported that Salmonella ty'phi and Salmonella paratyphi grew well in the absence of Chlorella pyrenoidosa and Shigella, Proteus and Streptococci were affected when exposed to Chlorella. However, the release of toxic extracellular products by algae is an unlikely cause for this behavior because these products are discharged only under stress (Pratt and Fong, 1940). At the temperature of 30~C and pH 7.0, which are near the best conditions for this alga (Mayo and Noike, 1994b), little or no stress is expected. In spite of the absence of stress, Fig. 8 shows higher number of heterotrophic bacteria in the absence of algae than in the presence of algae at pH 7.0. If pH is one of the factors that may cause stress

to Chlorella, then at pH 10.0 low number of heterotrophic bacteria should be expected compared to bacteria grown at pH 7.0. Figures 6 and 7 did not show any evidence that this hypothesis is valid. Moreover, the analysis of mass balance for the soluble organic carbon did not show evidence of release of organic carbon from algae. It is possible that competition for glucose was the main reason for low number of bacteria in the presence of algae in this study. As previously discussed algae have higher ability of glucose assimilation than bacteria. Glucose assimilation, however, can not be used as a reason in oxidation ponds fed with raw sewage. This is because no free glucose is available to the algae in the raw wastewater before its entry into the ponds (Abeliovich and Weisman, 1978). Interestingly, Oswald et al. (1953) observed that Chlorella pyrenoidosa grew more vigorously and had higher yields in sterile sewage than that it did in the presence of bacteria. Evidently, this indicates that algae and bacteria compete for substrate even in raw sewage. The possible sources of organic matter in oxidation ponds that algae can directly incorporate without first being oxidized include small organic molecules, such as organic acids, monosaccharides, amino acids etc. In some cases, depending on the conditions, citrate or acetate may stimulate the growth rate of algae more than sugars, although in most cases sugar is the best organic substrate (Martinez et al., 1987). For instance, the growth rate of Scenedesmus quadricauda under mixotrophic conditions can be best stimulated with citrate or acetate than with fructose or sucrose. The main source of these small organic molecules, including glucose and other sugars, is probably polysaccharides degraded by bacteria. Abeliovich and Weisman (1978) estimated that in high rate ponds, 25-50% of the algal carbon could be directly incorporated and then photoassimilated. This is a significant proportion particularly because algal biomass yields observed in algal-bacterial system is much higher than the bacterial biomass yields.

CONCLUSIONS

Based on the experimental results obtained the following conclusions are made: 1, The pH exceeding 10.0 were significantly detrimental to the number of heterotrophic bacteria, but the affinity for glucose decreased at pH above 8.0. 2. Temperatures between 10 and 20°C did not influence the number of heterotrophic bacteria. At 3 0 C however, the number of heterotrophic bacteria decreased because of increased competition for glucose by Chlorella vulgaris. 3. No evidence was found to support the view that the discharge of bactericidal substances from Chlorella vulgaris was responsible for reduction of the number of heterotrophic bacteria.

Temperature and pH on oxidation pond bacteria 4. The incubation temperature o f 35°C for 72 h is r e c o m m e n d e d for e n u m e r a t i o n o f heterotrophic bacteria in algal-bacteria system, such as in waste stabilization ponds. REFERENCES

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