Effect of glucose loading on the growth behavior of Chlorella vulgaris and heterotrophic bacteria in mixed culture

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War. Res. Vol. 28, No. 5, pp. 1001-1008,1994 Copyright ~ 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0043-1354/94$6.00+ 0.00

Pergamon

E F F E C T OF GLUCOSE L O A D I N G ON THE GROWTH BEHAVIOR OF CHLORELLA VULGARIS A N D HETEROTROPHIC BACTERIA IN M I X E D C U L T U R E ALOICE W. MAYOand TATSUYANOIKE*@ Department of Civil Engineering, Tohoku University, Aoba, Sendai 980, Japan (First received March 1993; accepted in revised Jbrm October 1993)

Abstract--The effect of glucose concentration on the growth of Chlorella vulgaris and heterotrophic bacteria in mixed culture was studied. Granular glucose was fed daily as a source of organic carbon to provide an input concentration ranging from 25-700 mg/l. Growth rates of heterotrophic bacteria and algae increased with glucose loading rate but excessive loading rates were detrimental to the survival of the algae and bacteria. The maximum specific growth rates for the algae and bacteria were 1.58 and 1.70 d-t, respectively.However, the saturation constants were significantlydifferent with values of 27 and 174mg/l for bacteria and algae, respectively, Under anaerobic conditions, bacterial density and algae growth were significantlyaffected by accumulation of volatile fatty acids (VFA). Although in low glucose loaded reactors, pH reached as high as 10.6, no significant effect of pH was observed on the survival of Chlorella vulgaris and heterotrophic bacteria. Inorganic carbon production increased with increasing glucose loading rate, When volatile fatty acids accumulated in anaerobic reactors, pH dropped resulting in a shift of equilibrium between H2CO3, HCO i- and CO~- in favor of H2CO3 production, most of which was released as CO2(g) to the atmosphere. Key" words--heterotrophic bacteria, Chlorella vulgaris, glucose loading rate, growth rate, oxidation ponds

INTRODUCTION Algae play a major role as producers in waste stabilization ponds and other polluted aquatic environments. Because of this role, algae have been used extensively for domestic as well as industrial wastewater treatment. The major role of algae is providing oxygen to bacteria whose oxidative activity is more efficient (Martinez et al., 1987). Thus, the relationship of algae and bacteria is very important for pond systems. In addition, algae also have a very important ability to utilize nutrients such as nitrogen and phosphorus responsible for the eutrophication of receiving waters as well as the uptake of some heavy metals. For growth, while bacteria utilize organic matter as a sole source of carbon (Azov et al., 1982), algae utilize carbon from bicarbonate alkalinity, CO2 released by bacterial degradation of organic matter as well as CO 2 dissolved in the pond from the atmosphere. Partially degraded organic compounds may also be utilized by algae through photoassimilation (Neilson and Lewin, 1974) and through algal heterotrophism (Abeliovich and Weisman, 1978). Martinez et al. (1987) reported that acetate affects the photoautotrophic growth of Pseudoanabaena catenata, but was assimilated by Chlorella sp., Scenedesmus quadricauda and Stichococcus bacillaris. Sucrose was assimilated well by Chlorella sp. and Stichococcus *Author to whom all correspondence should be addressed.

bacillaris but Scenedesmus quadricauda showed lower rates compared to the controls under photo- and chemoheterotrophic conditions. Chlorella was reported to grow well in the presence of glucose. In waste stabilization ponds and other polluted environments, CO, for algal growth is obtained from bacterial degradation of organic matter. Concern has been focused on the organic carbon concentration which, depending on the concentration, may support or inhibit biological activities in waste stabilization ponds. Because the growth of algae and bacteria is directly or indirectly affected by organic carbon loading rate, a study was carried out to investigate the response of algae and bacteria to organic carbon concentration. The specific objectives of this study were:

- - t o investigate the effect of organic carbon concentration on the growth of algae and bacteria, and - - t o investigate the effect of organic carbon concentration on the formation of by-products and their influence on the survival of algae and bacteria. Glucose was provided as a sole source of organic material and degradation behavior was studied over a wide range of glucose loading covering both aerobic and anaerobic conditions.

MATERIALSANDMETHODS Nutrient composition and reactor operation Table l shows the nutrient composition used in the experiments. These nutrients were provided at the beginning

1001

1002

ALOICE W. MAYO and TATSUYANOIKE Table 1. Nutrient composition and glucose loading rate Glucose loading

Chemical

Amount

Fe solution

KNO 3 K2HPO4 MgSO4.7H20 NaCI CaCIz • 2H20 EDTA-Na Distilled water Fe solution Trace minerals

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

FeSO4 - 7H2O = 500 mg Distilled water = 250 ml Trace mineral composition H3BO3 MnSO4'4H 20 ZnSO4 • 7H20 CuSO4 . 5H20 Na2MoO4 Distilled water

of the experiments and were supplemented as the experiments progressed to maintain the C : N ratio at about 4-5:1. Ferrous iron solution and micro-nutrients were added in liquid form and macro-nutrients were added in solid form. At the beginning of the experiments, the culture pH was adjusted to about 7.0 by 20% hydrochloric acid, but when the nutrients were supplemented the pH was maintained at the original culture pH by addition of either HC1 or NaOH. The reactors, each of 5 1. (Fig. I), were made from acrylic plastic and were provided with a sampling port near the bottom end and a cover with gas release ports. A light intensity of 6000 lux 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 algal growth because the saturation light intensity for growth of algae is less than 5400 lux (Goldman, 1979). The cultures were incubated at 30°C and were mixed continuously by magnetic stirrers. The alga Chlorella vulgaris was used for the experiments and bacterial inoculum was collected from the final sedimentation tank of the Sendai city activated sludge wastewater treatment plant. Glucose was added daily to provide input concentrations of a series of reactors of 25, 50, 75, I00, 150, 300 and 700 mg/l of the remaining volume of water in the reactor. The reactors were named according to the amount of glucose they received (see Table I). Prior to sampling, biomass attached on the reactor walls was carefully resuspended by swirling the culture contents. Sampling was done at 10.00a.m., 3 h after the lights were switched on. Daily additions of glucose were made immediately after sampling, or at I0:00 a.m. on days when sampling was not done.

Sample analyses Algal biomass was measured by chlorophyll a as detailed in Standard Methods (APHA et al., 1985). Chlorophyll a

= 286 mg =130mg = 320 mg = 183 mg = 2.1 mg = 100ml

Reactor

(mg/I/d)

R25 R50 R75 RI00 R150 R300 R700

25 50 75 100 150 300 700

was extracted by 90% acetone and was measured by spectrophotometric method (Hitachi, model U-ll00). Heterotrophic bacteria were determined by the pour-plate count method on serial dilution of samples mixed with tryptone glucose extract agar medium (APHA et al., 1985). The plates were incubated at 35°C for 48 h. Particulate organic carbon (POC), inorganic carbon (IC) and dissolved organic carbon (DOC) were analyzed by a TOC analyzer (Shimadzu, model TOC-5000). Prior to measurement of particulate organic carbon, the biomass was first disrupted by a sonifier (Branson Sonic Power, model 185) for 5 min in order to break the particles into small pieces and extend the settling time during the measurement. Dissolved organic matter (DOC) was analyzed after removing particulate matter by centrifuging at 3500 rpm for 15min. No effect of cell disruption by the sonifier on inorganic carbon was observed. Glucose concentration was determined by the phenolsulfuric acid method (Dubois et al., 1956). Glucose concentration was measured spectrophotometrically at 490nm (Hitachi, model 100-20). Glucose concentration in the samples was then determined by comparing the optical density reading with the standard glucose solution calibration curves. Volatile fatty acids were measured by gas chromatography (Shimadzu, GC-8A) equipped with an integrator (Shimadzu, C-R6A). The operational temperatures for the injection block and column were, respectively, 180 and 140°C. Dissolved oxygen was measured by a DO meter (TOA Electronics, DO-IB) and pH was measured by a pH meter (TOA Electronics, HM-18E). Alkalinity was measured titrimetrically using 0.02 N H,.SO 4 standardized by 0.05 N N%CO3 solution. RESULTS

Effect o f glucose concentration on p H G a s release

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Figure 2 shows the variation o f p H with o p e r a t i o n time measured 3 h after the lights were switched on. In the first 5 - 2 0 days o f operation, p H increase was faster in reactors loaded with a high glucose concentration. As glucose additions were continued, low glucose loaded reactors m a i n t a i n e d higher p H values than high glucose loaded reactors. In R25, p H remained near neutral for the first 20 days, then rose rapidly to as high as 10.6 on day 37, after which it suddenly d r o p p e d to 8.2 in 12 days. It seems that at a p H o f 10.6 the enzymatic activities o f algae might have been temporarily affected, resulting in the loss o f algal p h o t o s y n t h e t i c utilization o f CO,. The variations o f pH in RI50, R300 and R700 where a n a e r o b i c conditions appeared, were different

Growth of algae and heterotrophic bacteria from the other reactors. In RI50 and R300, pH increased rapidly at the beginning but decreased when volatile acids accumulated in the reactors, declining to as low as 4.6 in R300. Carberry and Brunner (1991), in their experiments with the algal bacterial clay system, observed a similar behavior, but concluded that the lack of photosynthetic activity and concomitant oxygen production prevented the pH from recovering. The decline of pH in the anaerobic reactors was caused by accumulation of volatile fatty acids (VFA). VFA contained in anaerobic reactors were mostly acetic acid and propionic acid with iso-butyric acid contributing, at most, 300 mg/l. Whereas R300 and R150 contained acetic acid as a major contributor of VFA, R700 had roughly equal shares of acetic acid and propionic acid. These results are consistent with other researchers' findings in the literature (Brockett, 1976).

10 s

Effect of glucose concentration on bacterial growth

lO s

The growth of heterotrophic bacteria shows a similar trend in all reactors for the first 20 days (Fig. 3). Bacterial growth exhibited no lag period and the time bacteria took to grow to maximum density was shorter with increased glucose loading rate. The maximum bacterial density in aerobic reactors was a p p r o x . 10 7 cfu/ml, whereas in reactors loaded with a high glucose concentration as high as 3 x 108 cfu/ml were observed. Bacterial growth rates were between 0.54 and 3.07d -~ and were influenced by glucose concentration. In R25, pH rose to above 9.0 from day 22 for about 25 days, but there was no indication that heterotrophic bacteria were affected by such high pH values. In contrast, when pH started to rise gradually from 9.3 on day 27 to a maximum of about 10.6 on day 37, heterotrophic bacterial density rose from about 106 to 2 × 10 7 cfu/ml. Because of this peculiar behavior, we studied the effect of pH on the heterotrophic bacteria in separate batch experiments.

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Fig. 3. Variation of heterotrophic bacterial density with glucose concentration.

The results showed that at least up to pH 10.5, the survival of heterotrophic bacteria was similar to the control sample at pH <9.0 (unpublished data). In R150, R300 and R700, bacterial densities were higher than reactors fed with a low glucose concentration in the first 20 days. As addition of glucose was continued, VFA accumulated in the high glucose concentration fed reactors, resulting in the decline of bacterial density. Interestingly, although pH fell to an acidic range in R700, bacterial density declined temporarily by only about 1 log~o scale from day 16 to day 21, but thereafter continued to grow gradually, particularly when pH started to increase. It seems that acetic acid is probably more toxic than propionic acid because reactors with a higher proportion of acetic acid (R300 and R150) showed a remarkable decline in bacterial number than reactor R700 with a lower proportion. The effect of pH on bacteria at the pH range observed is probably minimum because heterotrophic bacteria survive well at pH values as low as 4.5 (unpublished data).

Effect of glucose concentration on growth of Chlorella vulgaris

8

6 5 4

1003

100

20 ~ 30 40 50 Operation time (d)

60

Fig. 2. Variation of pH with glucose concentration and operation time.

The effect of glucose concentration on the growth of Chlorella vulgaris is presented in Fig. 4. The reactors with a low glucose loading rate possessed a lag growth, particularly in R25. Reactors with a high glucose loading showed high initial algal growth rate, but as anaerobic conditions developed, growth of algae ceased and then declined. Our experience shows that Chlorella vulgaris is not significantly affected by a pH as low as 4.5 (unpublished data). From the literature (King, 1970; Shapiro, 1973; Ip et al., 1982),

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ALOICEW. MAYo and TATSUYANOIKE

20

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15

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30, , 40, , 50, , 60 Operation time (d)

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20 30 40 50 Operation time (d)

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Fig. 4. Variation of chlorophyll a with glucose concentration.

Fig. 5. Effect of glucose concentration on glucose utilization.

it appears that green algae are growing more efficiently in abundant CO2 or a lowering of pH. Shapiro (1973) concluded that lowering of pH or addition of CO 2 stimulated growth of green algae. Ip et al. (1982) have reported that unicellular green algae such as Chlorella, Scenedesmus, etc. were predominant in high CO2 samples. This information suggests that at low pH and high glucose concentration, Chlorella vulgaris should have survived better than at high pH and low glucose concentration. This was, however, not the case in anaerobic reactors. The inhibition of Chlorella vulgaris growth at high glucose concentration, may therefore be explained by VFA production.

The remaining glucose concentrations in the reactors are shown in Fig. 5. Since glucose concentration in R75 was generally low ( < 8 mg/l), nearly all daily additions were consumed by algae and bacteria. In R300, a rapid increase of undegraded glucose was observed from days 16 to 23 which corresponds to a sharp fall in viable bacterial density (Fig. 3), and specific glucose utilization rate (Table 2). Although bacterial density did not increase after day 23, glucose was once again readily degraded. It could be possible that the bacteria adapted to the new culture conditions or that a VFA tolerant species grew in the reactors. The rate of increase of undegraded glucose was high in R700 from day 43 following a decrease in specific glucose degradation rate. The reasons for the occurrence of this behavior were not quite clear because bacterial density was reasonably high (about 108cfu/ml) in this reactor.

Effect of glucose concentration on glucose degradation Six runs were carried out for each of the reactors R150, R300 and R700, for determination of specific glucose utilization rates. On the sampling days, as soon as glucose additions were made, glucose utilization was measured at 1 h intervals. The graphs of glucose degradation were plotted against time, from which the tangent slopes were determined as the glucose utilization rates. The specific glucose utilization rates were determined as the glucose utilization rates per unit biomass (as particulate organic carbon), and are shown in Table 2. Over the period of batch experiments (less than 10 h), the biomass change was assumed negligible compared to the initial biomass. It appears from Table 2 that high glucose loaded reactors had higher specific glucose utilization rates than low glucose loaded reactors. Table 2. The specific glucose utilization rate in mg glucose/mg

POC/h Time since operation started (d)

RI50

R300

R700

15 25 33 38 47 56

0.117 0.050 0.043 0.039 0.035 0.042

0.185 0.068 0.047 0.044 0.052 0.058

0.206 0.207 0.285 0.147 O. 142 0.104

Effect of glucose concentration on inorganic carbon production Inorganic carbon production from organic carbon by the algal-bacterial system was found to be influenced by the amount of glucose added to the system (Fig. 6). As addition of glucose was continued, inorganic carbon in R300 and RI50 accumulated to values as high as 185 mg/l, then declined to values below 15 mg/l. The decline in inorganic carbon concentration in RI50 and R300 may be explained by either consumption of free carbon dioxide by algae or by production of organic acids. The first possibility is unlikely because consumption of CO2 by algae will normally be accompanied with an increase in pH rather than a decrease in pH as observed. To determine whether pH influenced the reduction of inorganic carbon, a standard solution containing 500mg/l as inorganic carbon was made from NaHCO3 and Na2CO3 each contributing about 50%. The pH of this solution was about 10.2. The effect of pH on inorganic carbon was studied by varying pH in I 1 samples between 4.7 and 12.9 by the addition

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4000 8000 12000 16000 CumulativeglucG6eadded (mg/I)

Fig. 6. Effect of glucose concentration on inorganic carbon accumulation.

Fig. 8. Effect of glucose concentration on carbon recovery.

of carbon-free HC1 or NaOH. Samples were then tested for inorganic carbon, total carbon and total alkalinity, and pH was accurately measured by a pH meter. Figure 7 shows that pH did not affect the inorganic carbon concentration at pH values above 8.3 but alkalinity continued to increase because of OH ions. Below pH 8.3, which corresponds to pHHco3 at 25°C, inorganic carbon decreased linearly with decreasing pH. The interpretation of this behavior is that HCO3 and CO~- ions in water were converted to H2CO 3 by the following reactions:

concentration of HCO 3 and CO~- will start to decrease at pH 8.3, which suggests that part of the HCO~- and CO~- that converted to HzCO3 at pH < 8.3 was actually released to the atmosphere as CO2(g), thus reducing total inorganic carbon in the solution. Because the culture temperature was higher (30°C) and the solubility of CO2(aq) decreases with increasing temperature (Loewenthal and Marais, 1976), the inorganic carbon in the reactors will decrease significantly at high temperature and low pH values. The decline of inorganic carbon in the

CO~ + 2 H + = H C O 3 + H

+

= H2CO3 = H20 + CO2(aq) The equilibrium constant at 25°C using Henry's law suggests that H:CO3 dominates at pH < 6.3, HCO3 at 6.3 < pH < 10.3 and CO~ at pH > 10.3. H:CO3 is usually in the form of CO2(aq) because the equilibrium constant suggests that carbonic acid has the properties of a strong acid (K = 10-35). The total

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Fig. 9. Effect of aerobic and anaerobic conditions on carbon distribution.

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Fig. 10. Lineweaver-Burk plot of the dependence of glucose uptake by algae and bacteria on glucose concentration. anaerobic reactors when the pH declined could therefore be explained by the release of CO2 to the atmosphere rather than uptake by algae. This experiment also explains why, in spite of the drop in pH in R25 from 10.6 on day 37 to 8.2 on day 49 (Fig. 2), the inorganic carbon concentration did not decline, but increased from 25 to 70 mg/1.

Effect of glucose concentration on carbon recovery The total carbon recovery was calculated as the sum of inorganic carbon, particulate organic carbon (biomass) and dissolved organic carbon (unutilized glucose, VFA and other intermediate soluble carbon). Figure 8 shows the effect of glucose addition on total carbon recovery in 236 samples collected from 7 reactors operated at glucose loading rates ranging from 25 to 700 mg/ld for 58 days. In general, at a cumulative glucose concentration of less than 4000 mg/l as carbon, an average of about 50% of carbon remained in the reactors. Above 4000 mg/1, the net recovery decreased and beyond the cumulative glucose concentration of 8000 mg C/! added to the reactors, over 60% of the input glucose added was lost as gases. For example, at a cumulative loading of 16,000 mg C/l, about 80% of the input carbon was lost as gases. This is in agreement with the findings of Gang et aL (1992) who observed CO 2 release ranging from 82.8 to 100% of input carbon in a thermophilic oxic process treating high strength food processing wastewater.

Effect of glucose concentration on carbon distribution Figure 9 shows the carbon mass balance in R50 and R300 representing aerobic and anaerobic reactors, respectively. It is interesting to note that glucose fed to the reactors was more efficiently converted to biomass in aerobic reactors (R50) than in anaerobic reactors (R300). Undegraded glucose and inorganic carbon concentrations were low in both cases, representing less than 5 and 20% of the total carbon, respectively. Inorganic carbon accumulation was uniform in R50, but decreased in R300 as

TATSUYA NOIKE

VFA increased. This information is consistent with the results depicted in Figs 6 and 7, and further supports the influence of VFA on inorganic carbon. Figure 9 also shows that gas release was inversely related to biomass concentration in both aerobic (R50) and anaerobic (R300) reactors. Gas release in R300 was high for the entire operation period, and increased as biomass decreased. In contrast, gas release decreased in R50 as biomass concentration increased. It appears from these results that the high proportion of gas release in the anaerobic reactors was caused by a low concentration of algae (Fig. 4), which could not readily utilize the CO 2 produced by bacterial respiration of glucose. DISCUSSION

In these experiments, algae survived and even grew in the absence of oxygen. It may be noticed that algae grew in R300 and R700 when dissolved oxygen was completely depleted from the first day of operation, although micro levels may be available through liquid-air interfacial transfer or the algae's own photosynthetic activity in the presence of light. The growth of algae in anaerobic conditions has been repeatedly reported, in spite of the belief that algae are strictly aerobic organisms. Stewart and Pearson (1970) showed that the blue-green algae Anabaena flos-aquae and Nostoc muscorum reduce acetylene to ethylene most actively at pO2 levels below 0.2 atm and grow well in the presence of Na2 S. Baas-Becking and Wood (1955a, b) also observed survival of blue-green algae in estuarine muds at Eh values of - 1 0 0 m V , whereas Knobloch (1966) observed growth of Chlorella, Ankistrodesmus, Scenedesmus, Coelastrum, Chlamydomonas and Porhyridium in the presence of H2S. Martinez et al. (1987) also reported that Chlorella sp. can assimilate acetate in photoautotrophic conditions. In R700, algae were still growing even in the presence of VFA, the concentration of which was stable at 1250 mg/l of acetate, and increasing for propionic and iso-butyric acids. If acetate was consumed by algae then the rates of assimilation and production were equal, but no clear evidence of acetate consumption was observed. This may however, be one possibility because from days 37 to 58 the dissolved organic carbon in R700 declined and was accompanied by an increase in pH (from 5.3 to 6.8) and chlorophyll a (from 7.5 to 12 mg/l). It is possible that the mechanisms of VFA production in reactors loaded with high organic concentration were different from those loaded with low organic concentration. Slight growth of algae in RT00 towards the end of operation may be explained by the assimilation of glucose or CO: which is available even in anaerobic conditions. In waste stabilization ponds, assimilation of acetate by algae may still be a contributing source of carbon because glucose, which is easily assimilated by algae (Martinez et al., 1987),

Growth of algae and heterotrophic bacteria is not usually available (Abeolivich and Weisman, 1978). Even though algae grow under anaerobic conditions, the growth rate seems to be affected by the absence of dissolved oxygen. The growth rates of algae and bacteria were calculated as the slope of the natural logarithm of chlorophyll a concentration and bacterial density versus time, respectively. The data presented in Fig. 10 indicate that the growth rate of algae decreased at a glucose loading rate exceeding 150 mg/1/d. R300 and R700, however, were the only reactors which were devoid of dissolved oxygen during the algal exponential growth phase. Reactor RI50 had dissolved oxygen for as long as 15 days, sufficient to support algal growth during the entire algal exponential growth phase. While in R700, VFA might have contributed to slow growth, no VFA limitations were observed in R300 during the exponential growth phase. VFAs were produced from day 7 in R300, the period outside the exponential phase considered for analysis of growth rate. In addition, in the first 2 weeks, the pH in all reactors was between 5.5 and 8.0, the range which is favorable for growth of Chlorella vulgaris. It seems therefore that dissolved oxygen might have been a growth limiting factor during the exponential growth phase. Beyond the exponential growth phase, VFA accumulation was observed to be the most important parameter affecting algal growth. At elevated pH conditions (low glucose loading), there was no obvious evidence of pH effects on the survival of algae and bacteria within the range of pH observed (as high as 10.6) in the reactors (see Fig. 2), although values exceeding this level were detrimental. Batch culture of Chlorella vulgaris and heterotrophic bacteria at a glucose concentration of 25 mg/l and at various pH values also shows that, at pH above 10.5, the chlorophyll a decreased compared to the control at pH < 9.0. In a culture dominated by Chlorella, self pH adjustment to a steady maximum value of about 8.5-9.2 was repeatedly observed in R50, R75 and RI00. Under field conditions, where various species coexist, Chlorella vulgaris dominance in maturation ponds may be affected by high pH values which may rise to as high as 11.5. King (1970) has suggested that under alkaline conditions, green algae should not predominate. Wringley and Toerin (1990) in their limnological study of mixed species of algae in small scale sewage ponds in series, observed that at least 14 genera were dominant at various times in their 21 month study. Their data indicated that Chlorella occasionally dominated in the first two ponds but never in the third and fourth ponds. Interestingly, it seems that algal consumption of CO2 ceases at inhibiting pH values, therefore resulting in self pH control. This self pH control is important to bacteria because elevated pH values above 9.2 are detrimental to their survival (Parhad and Rao, 1974). Batch culture studies showed that the survival of Chlorella vulgaris and heterotrophic

1007

bacteria was simultaneously affected at pH above 10.5, but pH seems to affect the enzymatic activities of algae and bacteria at values above 8.2. Batch experiments carried out at initial glucose concentrations of 500-2000 mg/l and pH values from 5 to 12 indicate that the optimum pH for glucose degradation was between 6.6 and 8.2 (unpublished data). This range is a typical pH range in facultative ponds treating domestic wastewater. Experience in field ponds in Tanzania, shows that when influent BOD5 is in the range of 25-500 rag/l, pH will vary according to the relationship: pH=14.39-3.2191og10(BODs)

R2=0.84

This means that pH will rise above 8.5 only when influent BOD 5 falls below 67 mg/l. This concentration is too low for the design of facultative ponds, and therefore at such a concentration ponds are designed as maturation ponds, where elevated pH values are desirable for removal of coliform and pathogenic organisms. High pH values are however, not uncommon in facultative ponds, normally occurring only in the top 10-30 cm during the day. Under such conditions, the overall impact of pH on bacterial degradation of organic matter is probably negligible. if the ponds are not excessively overloaded or underloaded. The maximum specific growth rates,/~m,~ for bacteria and Chlorella vulgaris were 1.70 and 1.58 d -~, respectively, and were reasonably similar to the 2.0 and 1.0d -~ estimated by Carberry and Greene (1992). The maximum growth rate of Chlorella vulgaris was unexpectedly close to that of bacteria. although bacteria appear to have a higher specific growth rate. The observed bacterial specific growth rates for individual reactors (0.54-3.07d ~) were much higher than the observed specific growth rates for algae (0.20-0.91 d --~) at the same glucose loading rate (see Fig. 10). The observed specific growth rates for Chlorella vulgaris seem to be higher than those of other species of algae. A recent study by Lee et al. (1992) showed that Phormidium tenue and Anabaena macrospora grew at a rate of 0.13 and 0.22d ~, respectively. The observed saturation constants, K~, for bacteria and algal growth were 27 and 174 rag/l, respectively, indicating that bacteria reach a saturation value much earlier than algae. This may suggest that during the exponential growth phase, either algal activity was more responsible for glucose uptake than bacterial activity or inorganic carbon produced from glucose degradation was insufficient for algal growth. CONCLUSIONS

Based on the experimental results obtained, the following conclusions are made: (1) The growth of algae was significantly influenced by the concentration of glucose added. The growth rate of Chlorella vMgaris

1008

ALOICEW. MAYOand TATSUYANOIKE during the exponential phase was higher with the increase of glucose loading rate, but above 150mg/l/d of glucose, insufficient dissolved oxygen caused their inhibition. Further addition of glucose only favored growth of algae in reactors fed with a low glucose concentration but affected algal growth at a high glucose loading rate because of inhibition caused by VFA accumulation. (2) Bacterial population increased with the glucose loading rate. Excessive loading however, resulted in accumulation of VFA which were detrimental to their survival. (3) Elevated pH values affect the growths of Chlorella rulgaris and heterotrophic bacteria at levels above 10.5. Because the effect occurs simultaneously, algal activities may not affect the survival of heterotrophic bacteria, although enzymatic activities may be reduced. (4) Loss of carbon, possibly through emission of gases, increased with increased glucose loading. In contrast, biomass yield decreased with increased glucose concentration. (5) The amount of organic carbon converted to inorganic carbon was higher in reactors with a high glucose loading rate, but as VFAs were produced in anaerobic reactors, inorganic carbon accumulation seized and declined as CO~- and HCO3 were converted to CO2(g) and were quickly lost through interfacial transfer.

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