Effects of temperature on biodegradation ...

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The authors have developed a new solid phase aerobic biological treatment process. (Lim et al. ..... Wastewater treatment with rotating biological contactor. Wat.
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B-R. Lim*, X. Huang**, H-Y. Hu*, N. Goto* and K. Fujie *Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, 441-8580 Japan **Department of Environmental Science and Engineering, Tsinghua University, Beijing, 100084 China Abstract The BOD removal rate and microbial community structure in a solid phase aerobic bioreactor using polyvinyl alcohol gel particles as packing material for the treatment of high strength organic wastewater were investigated at various temperatures. The BOD removal rate in the bioreactor increased when the temperature increased from 20ºC to 30ºC, 40ºC, and 50ºC, but it decreased when the temperature increased from 50ºC to 60ºC. Higher temperature enhanced the endogenous respiration of microbes in the bioreactor. The microbial community structure in the bioreactor was analyzed with quinone profile. The experimental results showed that the microbial community structure in the bioreactor was significantly affected by temperature. The dominant quinone of the microbes inhabiting the bioreactor was ubiquinone-8 at 30ºC, but that at 50ºC and 60ºC was menaquinone-7. It was estimated that the thermophilic Bacillus having menaquinone-7 dominated in the bioreactor at higher temperature. The microbial diversity in the bioreactor varied with temperature. Keywords Solid phase aerobic bioreactor; polymer gel; BOD removal rate; endogenous respiration; quinone profile; high strength organic wastewater; microbial community structure; microbial diversity

Introduction

High strength organic wastewaters discharged from food manufactures, breweries, cattle farming yards are usually treated with activated sludge processes or anaerobic digestion process. However, the activated sludge process pushes up the operating cost due to the huge oxygen demand in the aeration tank and generates a mass of excess sludge. On the other hand, the anaerobic digestion process cannot achieve an effluent with high quality. The authors have developed a new solid phase aerobic biological treatment process (Lim et al., 1997) for the treatment of high strength organic wastewater. In this treatment process, wastewater is trickled on to the top of the vessel packed with adsorbent polymer gel particles. The organic pollutants in the wastewater will be adsorbed and concentrated in the packed bed, and will be biologically degraded under aerobic conditions. The heat energy generated from the degradation of organic pollutants will elevate the temperature of the bioreactor. As a result, the biological degradation of organic pollutants, the endogenous degradation of microbial cells, and the water evaporation in the solid phase bioreactor will be greatly accelerated. This process has been successfully used for the treatment of easily biodegradable organic pollutants with higher concentrations in wastewaters (Lim et al., 1997). The purpose of this study is to clarify the effects of temperature on the degradation rate of organic pollutants, the growth rate and the endogenous respiration of microbes in the bioreactor. In addition, change in microbial community structure in the solid phase bioreactor was also investigated using quinone profile as a new approach (Fujie et al., 1994; Hiraishi, 1988; Hu et al., 1997, 1998 and 1999).

Water Science and Technology Vol 43 No 1 pp 131–137 © 2001 IWA Publishing and the authors

Effects of temperature on biodegradation characteristics of organic pollutants and microbial community in a solid phase aerobic bioreactor treating high strength organic wastewater

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Experiments Experimental apparatus and operating conditions

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The same apparatus described in our previous report (Lim et al., 1997) was used here. The main part of the apparatus was a column made of transparent polyvinyl chloride (11 cm in diameter and 30 cm in height; packing volume: 190 cm3), and placed in a water bath to maintain a constant temperature. Polyvinyl alcohol (PVA) gel particles coated with powdered activated carbon (ACP particle, hereafter) were used as the adsorbent medium for the solid phase aerobic bioreactor. The size of the ACP particles was 1.0 cm with irregular shape and the void fraction in packed condition was 60–65%. The maximum water holding capacity, i.e., the moisture contents, of ACP particles was as high as 70–75%. The artificial wastewater was composed mainly of glucose and peptone (1:1, w/w). ACP particles were soaked into the artificial wastewater in a vessel to have the particles adsorb the wastewater for one hour. Then the particles were packed into the column and air was fed to the bottom. ACP particles were removed from the column to soak into the wastewater again after 23 hours from the previous packing. This operating manner was repeated once a day. Volume, total and dissolved organic carbon (TOC and DOC, respectively) concentrations of the wastewater in the vessel before and after the soaking were observed. CO2 concentration in the exhaust gas was continuously recorded. DOC concentration of the artificial wastewater was changed from 30 to 250 g · L–1. The temperature of the column was maintained constant in the range from 20ºC to 60ºC. The feed rate of air was controlled from 0.5 to 3 L . min–1. Analytical methods

Concentrations of dissolved organic carbon (DOC) and total organic carbon (TOC) were determined using a TOC analyzer (Model TOC-500, Shimadzu Co., Japan) with and without microfiltration using a membrane filter with a pore size of 0.45 µm, respectively. And thus the difference between TOC and DOC was the particulate organic carbon concentration (POC, hereafter). BOD concentration was calculated from DOC. Our preliminary experimental results showed that the ratio of BOD to DOC for the artificial wastewater used in this study was 1.9. CO2 concentration in the exhaust gas was measured using a gas meter (PG-230, Horiba Co., Japan). Respiratory quinones in the solid phase bioreactor were analyzed using a method previously described (Hu et al., 1999). HPLC (ZorbaxODS, 4.6 (I.D.) mm×250 mm, Shimadzu-Dupont, Japan) and a photodiode array detector (SPD,M10A, Shimadzu Co., Japan) were used to determine the quinone species and their concentrations. Results and discussion Organic removal characteristics

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The rate of organic removal in the bioreactor was observed. Figure 1 shows the experimental results at 30ºC. The volume of wastewater absorbed into the particles was almost constant at around 50 mL. The top figure in Figure 1 shows the change of DOC concentration in the artificial wastewater before and after the particles soaking. The DOC concentration of wastewater was increased from 30 g · L–1 to 250 g · L–1 in these experimental runs. The fact that DOC in the wastewater after soaking was not beyond the concentration before the soaking shows that the organic pollutants adsorbed on and in the particles were biologically degraded through the runs. The organic removal rate was equal to the organic loading. From the volumes of supplied and residual wastewater per one day, (Vi and Vr), DOC concentrations before and after the particles soaking, (Ci and Cr), the rate of total organic removal per unit volume of packing bed, (–Rsv), can be determined as: –Rsv=(Vi Ci–VrCr)/V, where V is the effective volume of the bioreactor. It was clarified that

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Figure 1 Changes of DOC concentration in wastewater before and after the particles soaking and the organic removal rate with time at 30ºC

Figure 2 Effect of temperature on the BOD removal rate per one cell of bacteria (–υs), and bacterial number per unit volume of packed ACP particles (Bs), and volumetric BOD removal rate (–Rsv)

the DOC removal rate was increased up to about 8.5 kg–C · m–3 · d–1 with increasing organic concentration. The same experiments were carried out at 20, 40, 50 and 60ºC. Effect of temperature on BOD removal rate

BOD removal rate per unit volume of packed bed was determined from the observed DOC removal rate. The ratio of BOD to DOC for the artificial wastewater used in this study was 1.9 as above-mentioned. The results are shown in Figure 2 as functions of the temperature. The maximum organic removal rate under the present experimental conditions, i.e., the organic concentration of 30–250 g–C · L–1 and one-time supplement of wastewater to the particles in a day, was as high as 20 kg-BOD · m–3 · d–1 as shown in Figure 2. Note that the higher temperatures gave higher organic removal rate during the range of temperature from 20ºC to 50ºC, while the temperature beyond 50ºC abruptly pulled down the organic

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removal rate. The effects of temperature and organic removal rate on the bacterial number per unit volume of packed materials (Bs) are shown in Figure 2. The number of bacterial cells per unit volume of packing materials, Bs, was calculated from the quinone content, based on the fact that the quinone content of activated sludge sample averages about 1 µmol · g-dry cell–1 and the cell weight was about 2.5×10–13 g-dry cell (Nozawa, 1998). The value of Bs ranged from 0.9×1014 cell · L–1 · Bs was decreased with increasing temperature. Large values of Bs at lower temperatures could compensate the decreased activity of individual bacteria for BOD removal. BOD was mainly removed by the bacteria attached to the surface of packed materials in the bioreactor. The specific BOD removal rate, defined as the rate of BOD removal per one bacterial cell, –υs (g-BOD · cell–1 · d–1), was determined from –Rsv divided by Bs. The –υs had a maximum value at 50ºC, and decreased at 60ºC, showing a similar tendency to the BOD removal rate per unit volume of packed bed as above-mentioned. From this result, the organic degradation activity of microbes in the present treatment process may be maximum at around 50ºC. A similar phenomenon was also observed in the composting processing of sewage sludge (Vicky et al., 1984). The treatment characteristics of the wastewater used in this study in the solid phase bioreactor were compared with that in a submerged bioreactor reported previously (Hu et al., 1994). The effect of temperature on –υs of the two bioreactors is presented in Figure 3. The value of –υs of the solid phase bioreactor was 0.95×10–13–1.58×10–13 g-BOD . g–cell–1 · d–1 at 20–60ºC. The value of –υs of submerged bioreactor was 0.2×10–12–5.0×10–12 g-BOD · g–cell–1 · d–1 at 5–35ºC. Compared to the submerged bioreactor, –υs of the solid phase aerobic biological treatment process was low for the wastewater used in this study.

Figure 3 The Ahrrenius plot of BOD removal rate per one bacterial cell (–υs) in the solid phase and submerged bioreactors Table 1 Microbial cell yield (a) and endogenous respiration rate coefficient (b) Microbes

Temp. (ºC)

a (g-cell · g-BOD–1)

b (d–1)

Wastewater

References

Solid phase

This work

30

0.47

0.10

Artificial wastewater

bioreactor

40

0.79

0.11

(Glucose and peptone)

50

0.79

0.25

Thermophilic

52

0.48

0.32

Slaughterhouse

bacteria

58

0.51

0.78

wastewater

20

0.4–0.8

0.04–0.07

Domestic wastewater

Activated sludge 134

Couillard et al., (1989) Tahara (1980)

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Figure 4 Relationship between specific growth rate (µ) and specific BOD removal rate per unit amount of microbes (–υs) at 30ºC

With the assumption that the Arrhenius equation is applicable to the determination of the activation energy for BOD removal in this study, the activation energy, Ea, was determined from Figure 3. The average value of Ea for the solid phase bioreactor and that for the submerged bioreactor were about 14.6 kJ · mol–1 at 20–50ºC and 60 kJ · mol–1 at 5–35ºC, respectively. The activation energy obtained in this study for the solid phase bioreactor was lower than that for the submerged bioreactor. It was reported that Ea may decrease with increased temperature (Hoshino et al., 1977). In addition, the difference in bacterial community between the two bioreactors may lead to the different values of Ea. Microbial growth and endogenous respiration

A material-balance equation for the bacterial cell mass in the bioreactor can be written as: dX/dt=a · (–Rsv)–b · X .–rev

(1)

where X is the bacterial cell mass in the bioreactor (g–cell · m–3), –Rsv the organic removal rate per unit volume of packed bed in the bioreactor (g–BOD · m–3 · d–1), –rev the rate of the bacterial cell mass sloughed from the bioreactor (g–cell · m–3 · d–1), a the cell yield (g–cell · g–BOD–1), and b the endogenous respiration rate coefficient (d–1). –rev was determined from the observed data of POC in the wastewater after the particles soaking. The specific growth rate of bacteria, µ, in the bioreactor can be written as follows: µ=(dX/dt+rev)/X=a · (–υs)–b

(2)

where µ is the specific growth rate of bacteria in the bioreactor (d–1) and –υs the specific organic removal rate (g-BOD · g–cell–1 · d–1). In this study, from the results of the microbial growth rate and organic removal rate measured at 30–50ºC, values for a and b have been calculated using Eq. (2). The results at 30ºC are illustrated in Figure 4. There was a linear relationship between the specific microbial growth rate and the specific organic removal rate. From the slope and intercept of the straight line, a and b were determined to be 0.47 g–cell · g–BOD–1 and 0.10 d–1, respectively. The results obtained at 40ºC and 50ºC compared with the results previously reported were summarized in Table 1. The values of a obtained in the present research ranged from 0.47 to 0.79. The endogenous respiration rate coefficient of the bacteria increased with increasing temperature. The value of a was 0.25 at 50ºC, this value is 3–6 times that of the activated sludge process. The thermophilic bacteria with high endogenous respiration rate

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might dominantly multiply under the high temperature. Couillard et al. (1989), who investigated the thermophilic aerobic process when applied to a pig slaughterhouse effluent, reported that the value of b was 0.32 at 52ºC. Change in microbial community

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Microbial respiratory quinones are components of the bacterial respiratory chain and play an important role in electron transfer during microbial respiration. Quinones exist in almost all bacteria, and in general, one species or genus of bacteria has only one dominant type of quinone (Collins et al., 1981). So the quinone profile, which is usually represented as the mole fraction of each quinone type, should be specific for a microbial community structure. Changes in microbial community structure in a mixed culture of microbes could effectively be quantified using the quinone profiles. Note that some change in the bacterial phase of mixed culture may not bring about a change in the quinone profile because some different bacteria have the same dominant quinone. Any change, however, in the quinone profile certainly shows a bacterial phase change. A change in quinone profile is a sufficient condition for bacterial phase change in the mixed culture. The quinone profiles of the solid phase bioreactor at varying temperatures are shown in Figure 5. The dominant quinones were ubiquinone (UQ)-8 at 20ºC and 30ºC, UQ-8, menaquinone (MK)-6 and MK-7 at 40ºC, and UQ-8 and MK-7 at 50ºC and 60ºC. The total mole fraction of menaquinones increased gradually with increasing temperature and reached 70–80% at the temperatures of 50 and 60ºC. The mole fraction of MK-7 in the quinones sharply increased with increasing temperature. Mori et al. (1993) previously reported that MK-7, which is well known as the dominant quinone of thermophilic Bacillus, took a large part of the quinones observed in the thermophilic aerobic digestion process of the high strength organic wastewater. In addition, the microbial diversity using quinone as an index (DQ) is defined with the following equation (Hu et al., 1999):  DQ =   

n

∑( k =1

 fk   

)

2

(3)

where fk is the mole fraction of quinone species k and n is the number of quinone species with the mole fractions higher than or equal to 0.001.

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Figure 5 Change in quinone profiles with temperature at BOD loading 8.4 kg · m–3 · d–1

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The microbial diversities calculated using Eq. (3) at 20ºC, 30ºC, 40ºC, 50ºC and 60ºC were 8.0, 4.2, 5.0, 6.0 and 6.1, respectively. The microbial diversities varied with the temperature. Our previous report showed that higher temperature resulted in a diverse microbial community structure in the submerged bioreactor (Hu et al., 1994). Conclusion B-R. Kim et al.

The effect of temperature on the organic degradation rate, the growth rate and endogenous respiration of bacteria in the solid phase bioreactor were investigated. The conclusions are summarized as follows. 1. BOD removal rate in the solid phase bioreactor increased with increasing temperature from 20ºC to 50ºC, but decreased at 60ºC. 2. The endogenous respiration rate coefficient of microbes in the solid phase aerobic bioreactor increased with increasing temperature. The generation of sludge in the bioreactor at high temperature may be reduced. 3. It was estimated that the thermophilic Bacillus having menaquinone-7 dominated in the bioreactor at higher temperature. References Collins, M.D. and Jones, D. (1981). Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implications. Microbiol. Rev., 45(2), 316–354. Couillard, D., Gariepy, S. and Tran, F.T. (1989). Slaughterhouse effluent treatment by thermophilic aerobic process. Wat. Res., 23(5), 573–579. Fujie, K., Hu, H-Y., Tanaka and Urano, K. (1994). Ecological studies of aerobic submerged biofilter on the basis of respiratory quinone profiles. Wat. Sci. Tech., 29(7), 373–376. Hiraishi, A. (1988). Respiratory quinone profiles as tools for identifying different bacterial populations in activated sludge. J. Gen. Appl. Microbiol., 34, 39–56. Hoshino, S. and Kubota, H. (1977). Wastewater treatment with rotating biological contactor. Wat. Purific. Liq. Waste Treatment, 18(1), 29–35. Hu, H-Y., Fujie, K. and Urano, K. (1994). Effect of temperature on the reaction rate of bacteria inhabiting the aerobic microbial film for wastewater treatment. J. Ferment. Bioeng., 78(1), 100–104. Hu, H-Y., Fujie, K., Tanaka, H., Makabe, T. and Urano, K. (1997). Respiratory quinone profile as a tool for refractory chemical biodegradation study. Wat. Sci. Tech., 35(8), 103–110. Hu, H-Y., Fujie, K., Nozawa, M., Makabe, T. and Urano, K. (1998). Effects of biodegradable substrates and microbial concentration the acclimation of microbes to acrylonitrile in aerobic submerged biofilter. Wat. Sci. Tech., 38(7), 81–89. Hu, H-Y., Fujie, K., Nakagome, H., Urano, K. and Katayama, A. (1999). Quantitative analyses of the change in microbial diversity in a bioreactor for wastewater treatment based on respiratory quinones. Wat. Res., 33(15), 3263–3270. Hu, H-Y., Fujie, K. and Urano, K. (1999). Development of a novel solid phase extraction method for the analysis of bacterial quinones in activated sludge with a higher reliability. J. Biosci. and Bioeng., 87(3), 378–382. Lim, B-R., Huang, X., Hu, H-Y. and Fujie, K. (1997). Solid phase aerobic digestion of high strength organic wastewater using adsorbent polymer gel. Wat. Sci. Tech., 35(7), 13–20. Mori, T., Liu, B.G. and Cho, K.S. (1993). High strength wastewater treatment by thermophilic aerobic process: complete decomposition of organic matter and water evaporation (in Japanese). Kaguku Kougyou, 11, 52–58. Nozawa, M. (1998). Microbial population dynamics and materials balance in open system environment (in Japanese). Doctoral thesis, Department of Environment and Life Engineering, Toyohashi University of Technology, Japan. Tahara, Y. (1980). Haisuino seibutsusyori, Chikyusya, Tokyo, pp. 363. Vicky, L.M. and Vestal, J.R. (1984). Biokinetic analyses of adaptation and succession: microbial activity in composting municipal sewage sludge. Appl. Environ. Microbiol., 47(5), 933–941.

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