Impact of main factors on growth and ability of aerobic thermophilic bacteria for wastewater

treatment

Cao Jingguo School of Environ. Sci. & Eng.

Tianjin Univ. Tianjin, China cjg@tju.edu.cn

Zhao Shuxin Tianjin Key Lab. of Industrial

Microbiology Tianjin University of Science &

Technology, Tianjin, China

Mojmir Rychtera Institute of Chemical Technology

Prague Prague, Czech Republic

Abstract—In order to study technological properties of aerobic thermophilic bacteria for degrading organic material of selected food wastewater thermophilic bacterium Thermus ruber(CCM4212) was adopted. The specific growth conditions and the ability for wastewater treatment of the thermophilic bacteria were studied and results are listed as follows: The optimal growth temperature: is among 50 - 70 , and pH: is 7 - 9. As waste- water was exploited processing maize dipping water. Tests were carried out on rotary shaker and laboratory bioreactor (effective volume – 4.4 L) according to suggested objectives. Original waste-water was diluted before cultivation started to reach COD about 2000 mg/L - 3000 mg/L; rotation speed of the bioreactor impeller was 300 rpm and the air flow rate was 60 L/h. After 12 hours treatment, the COD removal efficiency was about 82.5 %.

Keywords-thermophilic bacteria; thermophilic aerobic waste -water treatment; cultivation in laboratory bioreactor

I. INTRODUCTION

Thermophilic aerobic biological wastewater treatment systems have many advantages compared to conventional techniques for high-strength wastewaters including higher biodegradation rates, greater overall process efficiencies and low rates of residual biomass (sludge) production [1-6]. However, due to the water physico-chemical characteristics changed by the high temperature, it makes the treating process more complex. For example, dissolved oxygen declines with increasing temperature, oxygen transfer rate demands and organic material solubility increase [7]. This arguments lead to changes of operation conditions. The key factor of the thermophilic aerobic biological wastewater treatment is the activity of thermophilic bacteria.

One collection strain of thermophilic bacteria, their growth and ability of wastewater treatment affected by the environmental factors were studied in this paper.

II. MATERIALS AND METHODS

A. Inoculum Thermophilic bacterium Thermus ruber (CCM 4212)

(abbreviated as T. ruber) was purchased from the Czech

Collection of Microorganisms (Masaryk University Brno, Czech Republic). Its ability to degrade organic substances in wastewater was enhanced by a series of adaptation steps starting from a recommended medium up to a standard wastewater. Seed culture (10 mL) was used to inoculate 100 mL of a complex medium in a 250 mL flask. The flask was placed on a rotary shaker (60 , 200 rpm). Such inoculum was used to seed both flask in rotatory shaker and in fermentor vessel.

B. Medium • Semi-synthetic medium preparation with supplement

of: Citric Acid (500 mg), calcium sulphate dihydrate (60 mg), calcium sulphate dihydrate (60 mg), magnesium sulphate heptahydrate (100 mg), sodium chloride (8 mg), potassium nitrate (103 mg), sodium nitrate (689 mg), potassium hydrogen phosphate dehydrate (140 mg), ferric trichloride hexahydrate (0.47 mg), manganese sulphate monohydrate (2.2 mg), zinc sulphate heptahydrate (0.5 mg), boric acid (0.5 mg), cupric sulphfate pentahydrate (0.025 mg), sodium molybdate dehydrate (0.025 mg), cobalt (II) chloride hexahydrate (0.046 mg), yeast extract (1 g), peptone (1 g). All components were mixed with 1 L tap water, adjusted to pH 8.0 with 20 % solution of NaOH.

• Maize dipping water was prepared as follows: maize (350 g) and sulphuric acid (50 mL) were mixed with water (1L) and dipped for 48 hours at 50 . For the experiments only supernatant was used. pH 8.0 adjusted with 20 % solution of NaOH

C. Experiment set-up The experiment was designed as single factor to study the

bacterial growth conditions and effectiveness of wastewater treatment both in shaken flasks and in aerated bioreactor.

Cultivation conditions: pH value was changed between 4 -10, temperature was maintained between 30 and 80 with intervals of 10 . Rotation frequency of the shaker was set from 100 rpm to 250 rpm with an interval of 50 rpm. 10 mL of

The work was financially supported by the Ministry of Education, Youth and Sport of the Czech Republic (Project KONTAKT No. 1P05ME 806) and Chinese government. The authors highly appreciate this financial support.

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inoculum was used for seeding 100 mL of a semi-synthetic medium in a 250 mL flask. The experiments were always performed in duplicates. Samples (10 mL) were taken for analyses.

Simulation of wastewater was represented by a maize dipping water prepared in laboratory. Bioreactor is BIAOF-2005 7-litre fermentor (B.Braun, Germany) with a working volume of 4.4 litres. Temperature of the liquid in bioreactor was maintained at 60 . 400 mL of adapted inoculum was used for seeding 4000 mL of wastewater. Stirrer rotation frequency was adjusted from 200 rpm to 500 rpm with an interval of 100 rpm. Air flow rates were changed from 60 L/h to 360 L/h with a 100 L/h interval. Concentration of dissolved oxygen (dissolved oxygen concentration) means percentage of dissolved oxygen from its saturation value at corresponding temperature. The experiments were done in duplicates. Samples (50 mL) were taken for analyses.

D. Analytical methods • Bacterial growth was determined by measuring optical

density at 660 nm.

• Biochemical oxygen demand (BOD5), chemical oxygen demand (COD) and biomass were determined by standard methods [8].

• pH were determined by PHS-25 type pH meter.

• Temperature in the bioreactor was continuously monitored.

III. RESULTS AND DISCUSSION

A. Growth character in shaken culture 1) Effect of initial pH value Microorganisms grow well

and metabolize nutrients within a wider pH range. If ambient pH value is out of this range, the growth and metabolism will certainly be limited or even restrained. Besides direct influence of pH, there are several indirect factors affecting activity of cells. For example, pH value can interfere a sorption and transport of nutriments; it can change toxicity of some components of the medium for microbial cells or only for some particular enzyme in metabolic reactions. The impact of initial pH on T. ruber growth is shown in Figure 1.

Figure 2. Effect of temperature on bacterial growth in batch culture.(Seed (10 mL) was used to inoculate 100 mL of a complex semi-synthetic medium in a

250 mL flask. The flask was placed on a rotary shaker at rotation frequency of 200 rpm. pH 8.0 adjusted with 20 % solution of NaOH.)

As shown in Figure 1, the initial value of pH for good bacterial growth was found between 6 and 9. The optimal value of pH for the growth was 8.0 and the optical density at this pH value was 1.05.

2) Temperature effect Vitality and viability of microorganisms is composed by a series of biochemistry reactions, and the temperature effect on them is significant, thus the temperature is one of the most important factors for the growth of microorganisms. The effect at pH 8.0 during 8 hours cultivation was studied. The bacterial growth expressed as optical density is shown in Figure 2. The bacterium T. ruber grew best at 60 and attained optical density 1.05.

3) Effect of rotation speed of the shaker As generally known, dissolved oxygen concentration (DOC) in medium can affect the growth of aerobic bacteria very significantly. It is necessary to consider a decrease of DOC with increasing temperature (solubility of oxygen in water at 60 is 5.44 mg/L). Therefore, in order to avoid a serious problem of oxygen limitation and maintain the oxygen transfer rate at a appropriate value, Impact of rotation speed of the shaker was studied (see Table I). With an increased rotation speed of the shaker oxygen transfer rate to liquid phase increases and thus enhances bacterial growth. At the rotation speed of 200 rpmthe optical density value reached the highest value equal to 1.05. However, it appeares a decrease of growth at 250 rpm. One of reasons of lower growth in this case could be a very strong foaming probably connected with changes of physico-chemical properties of medium which also affects oxygen and nutrients transfer.

4) Growth curve of bacterium T. ruber Bacterial growth is usually demonstrated by a growth curve consisting of lag, exponential, stationary and eventually decay phases.

TABLE I. EFFECT OF ROTATION SPEED ON THE BACTERIAL GROWTH

Shaker rotation speed (rpm) 100 150 200 250

Optical density 0.57 0.05 0.78 0.05 1.05 0.05 0.59 0.05

(Liquid inoculum (10mL) was used for seeding 100 mL of a complex medium in a 250 mL flask. The flask was placed on a rotary shaker at 60 �. Initial pH value of this medium was 8.0.)

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Figure 1. Effect of the initial pH value on bacterial growth in batch culture (pH was not controlled during the process. Liquid seed (10 mL) was used to

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Figure 3. Growth curve of T. ruber .(Cultivation conditions: pH 8, temperature: 60 , rotation speed of the shaker: 200 rpm. 10 mL of the seed volume was used for inoculation of 100 mL of a semi-synthetic medium in a 250 mL flask. The experiments were done in duplicates. Samples were taken

every 2 hours for analyses.)

Temperature, however, affects bacterial growth significantly in dependence on the type of bacteria and the time period of temperature effect. Growth curve is shown in Figure 3. At temperature of 60 micro-organisms demonstrated 2 hours lag phase, exponential phase took 5.5 hours and then biomass growth rate decreased (8. hr) and a decay phase started. After totally 20 hours of cultivation the medium turned to red color caused by formation of pigment accompanied with a slight odor. This feature is quite typical for this species of bacterium Thermus.

B. Maize dipping water treatment in 7 L fermentor Some factors such as control parameters, physico-chemical

properties of gas-liquid phase and bioreactor configuration can affect the level of dissolved oxygen concentration [9]. Among the control parameters, the most important ones are temperature, pressure in fermentor, air flow rate and stirrer rotation speed. To characteristics affecting the microbial growth belong above all the reaction (growth) kinetics, surface tension, oxygen solubility etc. The bioreactor configure is determined by its geometry, impeller, air sparger and baffles constructions and some other factors.

The maize dipping water was selected as a model wastewater to study the processing ability of T. ruber affected by operation parameters.

1) Effect of stirrer rotation speed: Effect of different stirrer rotation speeds on COD values and COD removal rates are demonstrated in Figure 4. COD value decreased with time till 8th hour then more or less kept constant. Even though the higher stirrer speeds can slightly improve the oxygen transfer rate, it does not mean that COD removal rate increase also. In case stirrer rotation speeds vary between 200 rpm- 500 rpm, the COD removal rates change from 55 % to 75 %. In preliminary studies the authors found out that the COD removal was lower below the rotation speed of 200 rpm. It seems that the COD removals are not significantly different between rotation speeds of 300 rpm and 500 rpm . The speed of 300 rpm, however, is a little more efficient than others. The dissolved oxygen tension is affected by many factors such as composition of media, total pressure in the bioreactor, oxygen

uptake rate caused by microorganisms, air hold-up in the liquid, stirring and aeration functions given by the bioreactor construction and operation. Volumetric coefficient of oxygen transport (KLa) can be influenced by diameter of air bubbles [10]. The increased stirrer rotation speed can also lessen the diameter of bubbles and as such can increase the retention time of air bubbles. Positive effect of bubble size is however limited till its optimum below which is the oxygen transfer rate lower. S. Aiba [10] showed that diameter of air bubbles appearing in dilute fermentation fluid ranges from 1.5 mm to 10 mm and the ascending velocity of bubbles is then 20 cm/s - 30 cm/s. However, the values of ascending velocity may be retarded in dependence on characteristic values such as flow behavior index, consistency index etc. Increase of COD at the end of batch cultivation is mostly caused by endogenous processes taking place in bacterial cells due to depletion of some nutrients, cell intracellular material released into medium is mostly organic and therefore contributes to an increase of COD values.

2) Effect of aeration: Different values of air flow rates on COD values and COD removal rates are shown in Figure 5. Itcan be seen that when the aeration rates are fixed to 60 L/h, 160 L/h, 260 L/h and 360 L/h respectively and kept constant during 12 hours of cultivation then the COD removal rates are 82.5 %, 75 %, 79.9 % and 77.9 % respectively. The highest COD removal was attained at the air flow of 60 L/h. Nevertheless, it can be concluded that flow rate has no significant effect on COD reduction in the range between 60 and 360 L/h.

3) Effect of dissolved oxygen level The metabolic efficiency of bacteria is affected by oxygen level. Values of dissolved oxygen concentrations may influence the process efficiency [11]. In order to find out impact of various levels of dissolved oxygen concentration, the experiments arranged at various values of dissolved oxygen concentration (DO) by changing both the air flow and the rotation speed of the impeller .

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Figure 4. Stirrer rotation speed effect on COD values (Air flow rate was fixed to 160 L/h, and initial COD of wastewater was adjusted to about 3000mg/L, stirrer rotation speed from frequency of 200 rpm to 500 rpm on COD

removal.)

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A combination of air flow rate and stirrer rotation speed was adjusted manually. Three dissolved oxygen states were designed to study the effect of DOC levels on the waste COD values and COD removal rates (Figure 6). Dissolved oxygen levels were maintained manually at following ranges:

Regime 1: (0-12 h) dissolved oxygen level - between 20 % and 40 %

Regime 2: (0-12 h) dissolved oxygen level – between 50 % and 70 %.

Regime 3: (0-12 h) dissolved oxygen level - between 75 % and 95 %.

Bacterium T. ruber could be adapted to different DO levels. The COD removal rates were 73.64 %(regime 1), 58.5 % (regime 2) and 60.7 % (regime 3) respectively under three DO levels, the lower DO level can get a higher COD removal rate. It demonstrates that higher DO level is obviously necessary to keep exponential growth and lower DO level benefits the treatment in the stationary and in declination phases of the bacterial growth. Opposite case shown in Figure 6 gives apparently worse results.

IV. CONCLUSION

Characteristics and the growth of thermophilic bacterium T.ruber (CCM 4212) under various conditions were studied and from the investigation it can be concluded that:

For maximum degradation of organic matters (COD) in maize dipping water and in the laboratory 7 L bioreactor following optimal conditions were found:

a) rotation speed of the bioreactor impeller 300 rpm and

b) the air flow rate 60 L/h At the initial COD (2000 mg/L – 3000 mg/L.) the COD can

be easily reduced by about 67 %, i.e. to the residual COD of 1 g/L in 12 Hours of the batch process. Beside these parameters the very important role in degradation of organic compounds plays dissolved oxygen in the range from 20 % to 40 %.

Process using thermophilic microorganisms can be used also as an autothermic process if process kinetics is optimal and the scale of the reactor is large. Another advantages is that material after the process can be hygienized (removal of pathogenic microflora) if kept for a certain time at the temperature 60 or above.

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[4] G. Sürücü, “Growth requirements of thermophilic aerobic microorganisms in mixed cultures for the treatment of strong wastes,” Water Science and Technology, vol. 40 (1), pp. 53-60. 1999.

[5] Timothy M. LaPara and James E. Alleman, “Thermophilic aerobic biological wastewater treatment,” Wat. Res., Vol. 33 (4), pp. 895-908. 1999.

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[7] J. C. T. Vogelaar, A. Klapwijk, and J. B. van Lier et al. “Temperature effects on the oxygen transfer rate between 20 and 55 ,” Wat. Res, Vol. 34 (3), pp. 1037-1041. 2000.

[8] “Analytical method of water quality,” Standards press of China, 2001. [9] S. Jia, “Theory of biology reaction engineering,”. Science press. Beijing,

China, pp. 118-122. 2003. [10] S. Aiba, A. E. Humphrey and N. F. Millis, “ Biochemical Engineering,”

2nd Edition, University of Tokyo Press 1973 [11] N. Gao, X. Zhang and D. Wu, “Dissolved oxygen effect on pyruvic acid

fermentiation,” Journal of Tianjin University of science and technology, China, Vol. 3 (3), pp. 5-7. 2004

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Figure 5. Ventilation effect on COD value and COD removal rates (Stirrer rotation speed was 300 rpm and initial COD was about 2000

mg/L - 3000 mg/L with air flow rate 60 L/h, 160 L/h, 260 L/h and 360 L/h.)

Figure 6. Effect of DO levels at regimes 1-3 on COD values and COD removal rates (Each regime was characterized by level of

DOC which was manually controlled at “constant” value.)

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