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The importance of the surface charge on support media for microbial adhesion
JotrgNaL OF FERMENTATIONAND BIOENGINEERING Vol. 73, No. 4, 323-325. 1992
The Importance of the Surface Charge on Support Media for Microbial Adhesion KENJI KIDA, 1. SHIGERU MORIMURA, I YORIKAZU SONODA, l AND TOSHINOBU YANOH 2
Department of Applied Chemistry, Faculty of Engineering, Kumamoto University, 2-39-1 Kurokami, Kumamoto-City, Kumamoto 860, ~ and Nittetsu Mining Co. Ltd., New Material Department of Research and Development Center, 8-10-16, Shimorenjaku, Mitaka-City, Tokyo 181, 2 Japan Received 6 December 1991/Accepted28 January 1992 Using cristobalites treated at various temperatures, methane fermentation tests were carried out in an anaerobic fluidized-bed reactor (AFBR). Cristobalites treated below 1,000°C had almost the same qualities in terms of components, physical properties, and surface structures. However, points of zero charge were different between cristobalites treated below 600°C and those treated above 800°C. Points of zero charge on the former numbered about 5 and their surfaces were positively charged in the fermentor controlled pH at 7, while points of zero charge of the latter numbered above 8 and surfaces were negatively charged. In the methane fermentation tests, a TOC removal efficiency of 78% was obtained using positively charged cristobalite at a TOC loading rate of 8 g/l. h, but with negatively charged cristobalite, the efficiency was only 63% under the same conditions. This result confirms the importance of the surface charge on the support medium for the anaerobic digestion in an AFBR.
In order to reduce the hydraulic retention time (HRT) in anaerobic digestion, it is very important to keep the microorganisms concentration high in the reactor. There are many studies aimed at keeping microorganisms at high concentrations in reactors using an upflow anaerobic sludge blanket (UASB) (1), an anaerobic fluidized-bed reactor (AFBR) (2), an upflow anaerobic filter process (UAFP) (3), and so on. We also have studied the anaerobic treatment of various kinds of wastewater and have tried to generalize a methane fermentation process for treatment of wastewater. In the AFBR process, it is very important to select a support medium with high fluidity to reduce the energy required for fluidization. And the support media must be hard and must allow microbial adhesion. We evaluated eight kinds of support medium, namely, cristobalite, zeolite, vermiculite, granular active carbon, granulated clay, pottery stone, volcanic ash, and slag, by methane fermentation tests, as reported previously (4). From our results, cristobalite was found to be the best support medium and zeolite was the second best. Both media had rough surfaces under the scanning electron microscope (SEM: JSM-T200, Jeol, Tokyo), but their surface charges were different. Cristobalite was positively charged and zeolite was negatively charged under conditions of methane fermentation. The cristobalite used in that test was treated at 400°C, and it was found that the surface charge of cristobalite changed with different temperatures of treatment. In this report, we describe the importance of surface charge as well as the roughness of the surface, as assessed by measurements of physical properties and a methane fermentation test. First, we measured the components of cristobaiites treated at various temperatures. As shown in Table 1, metal contents were almost the same in every sample of cristobalite, but the SiO2 content increased with increases in
temperature. The increase of SiO2 content was caused by a decrease in ignition loss with increased temperatures, that is, by the decrease in organic matter with treatment at high temperatures. With this exception, the components of the cristobalites were almost identical. Table 2 shows the bulk density (Pd, g/cm3), the wet density (Pw, g/cma), the specific surface area (m2/g), the average pore radius (mm), and the total pore volume (p, cm3/g) of cristobalites. Bulk and wet densities were measured by using a pycnometer, specific surface area by use of Monosorb (Yuasa-Quantachrome, Osaka), average pore radius and total pore volume by use of a Porosimeter (MOD 220, Carlo-Erba Co., Milano, Italy) as reported previously (4). The ratio of total pore volume to total volume of the medium was calculated from the following equation: Ratio of pores to medium (%) ---- { P d ' p / ( 1 --Pw+P~+Pd'P)} x 100
(1)
Bulk and wet densities were almost the same for every temperature of treatment, but specific surface area rapidly decreased above 1,000°C. Since average pore radii increased with decreases in specific surface area, small-sized pores might be destroyed by treatment at high temperature. With treatment at 1,200°C, the ratio of total pore volume to total volume of a medium was reduced to 50% of the ratio after treatment below 1,000°C. Surface structures of media were examined by SEM. Figure 1 shows scanning electron micrographs of surface structures of cristobalites treated at 400, 600, 800, and 1,000°C. Surface structures were rough and almost the same for every temperature. Points of zero charge were also measured by the method reported previously (4). As shown in Fig.2, points of zero charge of cristobalites treated below 600°C numbered about 5 and their surfaces were positively charged in a reactor in which the pH was controlled at 7. In the case of treatment above 800°C, points of zero charge numbered above 8 and surfaces were negatively charged. This distinction might be caused by differences in the numbers of
* Corresponding author. 323
324
KIDA ET AL.
J. FERMENT. BIOENG., TABLE 1. Components of cristobalite treated at various temperatures
Conditions of heat treatment
SiO2 (%)
Na20 (%)
K,O (~)
TiO2 (%)
CaO (%)
MgO (~)
S (%)
AI,O3 (/a~)
Fe.,O3 (o/0)
Ig. loss (%)
Raw material 400°C, 2 h 600°C, 2 h 800°C, 2 h 1000°C, 2 h 1200°C, 2 h
76.5 78.9 80.6 80.8 81.9 82.9
0.39 0.41 0.40 0.40 0.42 0.41
0.94 0.94 0.97 0.98 0.98 0.99
0.24 0.26 0.26 0.26 0.27 0.28
1.80 2.21 1.73 1.88 1.95 1.69
0.89 0.90 0.91 0.92 0.94 0.94
0.21 0.27 0.24 0.22 0.15 0.04
6.75 7.23 7.17 7.24 7.42 7.45
3.91 4.81 3.93 4.16 4.32 4.30
8.30 4.02 3.43 2.62 1.23 0.22
TABLE 2.
Physical properties of cristobalite treated at various temperatures
Conditions of heat treatment
Bulk density (g/cm ~)
Wet density (g/cm 3)
Specific surface area (m2/g)
Average pore radius x ( 10- s mm)
Total pore volume (cm3/g)
Ratio of pores a (%)
Raw material 400°C, 2 h 600°C, 2 h 800°C, 2 h 1000°C, 2 h 1200°C, 2 h
0.82 0.82 0.77 0.78 0.82 0.89
1.33 1.46 1.47 1.44 1.46 1.50
49.2 51.9 49.4 52.4 28.3 1.8
8.8 19.2 7.5 12.9 26.9 130.1
0.299 0.281 0.265 0.276 0.312 0.131
33 39 40 39 42 23
'
This value was calculated from Eq. I.
silanol groups, so the n u m b e r s o f silanol groups were measured by the lithium h y d r i d e - a l u m i n i u m m e t h o d . The re-
(a)
(b)
suits are s h o w n in Fig.2. The n u m b e r s o f silanol groups decreased with increases in the t r e a t m e n t t e m p e r a t u r e . Thus, the difference in the n u m b e r s o f points o f zero charge on cristobalites treated at various t e m p e r a t u r e s d e p e n d e d on the change o f silanol groups f r o m [Si-OH] to [Si-O-Si]. While the surface o f cristobalite which was not heated was positively charged, the a b r a s i o n loss was as high as 7 . 5 % (see Fig. 2). T h e r e f o r e , m e t h a n e f e r m e n t a t i o n tests were carried out using cristobalites treated at 400 and 1,000°C. T h e particle sizes, the m i n i m u m fluidization velocities, and the linear velocities to allow twice the exp a n d e d v o l u m e , which were the s a m e a m o n g b o t h cristobalits, were 0.1-0.3 r o m e , 0.02 c m / s , and 0.28 c m / s , respectively. A total o f 2 0 % ( w / v ) cristobalite was put in a digestor, m a d e o f acrylic resin with a w o r k i n g v o l u m e o f 0.45 l, and the test was started at 53°C, at p H 7.0, with a circulation rate o f culture m e d i u m o f 100 m l / m i n (4). Synthetic wastewater c o n t a i n i n g (g//): P o l y p e p t o n , 2.6; m e a t extract, 2.0; urea, 0.5; N a C l , 0.15; KC1, 0.07; MgSO4. 7 H 2 0 , 0.05; Na2HPO4, 0.5; CaCI2, 0.07, was used for the test. T o t a l organic c a r b o n ( T O C ) in the wastewater was 1,900 mg/l. As s h o w n in Fig.3, the T O C r e m o v a l efficiencies were a l m o s t the s a m e with b o t h cristobalites at T O C A
10
"7"
g
,6- o "6
(c)
(d)
FIG. 1. Scanning electron micrographs of surface structures of cristobalite treated at various temperatures. (a) Treated at 400oc; (b) at 600°C; (c) at 800°C; (d) at t,000°C.
gg
,¢
]F, I, ~ 0
200
400
,' /, 600
800
n~ 1000
1200
Temperatures of heat treatment (~C)
FIG. 2. Effects of heat treatment on points of zero charge (z~), abrasion losses ( o ), and numbers of silanol groups ( • ).
VOL. 73, 1992
NOTES
100 m m
8o
"6 60 r-
o
m
~ 20 --
I
o
0
I 2
I
I 4
I
I 6
I
I 8
I
[
I
10
Volumetric loadlng rate of TOC ( g l l.d)
FIG. 3. Effects of surface charges on cristobalite on removal efficiency of TOC. Symbols: O, positive charge; o, negative charge. loading rates below 4 g/l.d, but above 4 g / l - d , the TOC removal efficiency with cristobalite treated at 400°C was higher than in the case o f that treated at 1,000°C. A t the T O C loading rate o f 8 g/l. d, the T O C removal efficiencies with cristobalites treated at 400°C and 1,000°C were 78% and 63%, respectively. The increase in TOC removal efficiency with increased T O C loading rate probably depends on the increase in numbers of attached microorganisms on the support medium. A n d the difference in the increased numbers o f microorganisms between cristobalites treated at 400°C and 1,000°C seemed to be caused by the difference o f charge on the surface. After completion o f the methane fermentation test, the volume o f attached microorganisms was measured as follows: all the culture medium was poured into a 500-ml cylinder and the support medium was separated from the liquid medium by decantation. The liquid medium containing floating microorganisms and granules was centrifuged at 1 0 , 0 0 0 x g for 10rain. Volatile suspended solids (VSS) in the precipitate were measured. The VSS o f attached microorganisms on the support medium were also measured after washing. The ratios of floated VSS to attached VSS for cristobalites treated at 4000C and 1,000°C were 13.8% and 10.5%, respectively. F r o m this data, the re-
325
moval o f T O C in methane fermentation seems to be dependent on the numbers o f attached microorganisms. Since attached VSS per weight o f cristobalites were 100.6mg/g for treatments at 400°C and 8 4 . 9 m g / g at 1,000°C, respectively, it seems that the support medium o f which the surface was positively charged allowed microorganisms to attach more easily than the negatively charged support medium. F r o m the results mentioned above, it was confirmed that differences in surface charge affect the efficiency o f anaerobic treatment even if the medium and its surface structure are almost identical, and that surface charge is the most important property of the support medium. There are many factors that effect the attachment o f microorganisms to support medium, such as electrostatic forces, Van der Waals forces, and polymers produced by microorganisms. It is likely that electrostatic forces are necessary for the initial adsorption to the surface of the support medium and to prevent the detachment o f microorganisms from the support medium. Silanol groups are very effective for the adsorption if inorganic materials are used for the support medium. The authors wish to thank K. Fujita and Y. Nakano for their excellent technical assistance. We also thank Prof. Dr. T. Nonaka of the Faculty of Engineering, Kumamoto University for measurements of specific surface area and distribution of pore radii, and Y. Hayashida of Department of Applied Microbiology, Kumamoto Industrial Research Institute for operating the scanning electron microscope. REFERENCES I. Lettinga, G., van Velsen, A. F. M., Homba, S.W. de Zeeum, W., and Klapwijk, A.: Use of the upflow anaerobic sludge
blanket CLIASB)reactor concept for biological wastewater treatment especially for anaerobic treatment. Biotechnol. Bioang., 22, 699-734 (1980). 2. Kida, K. and Nakata, T.: Treatment of distillery wastewater by a series of two anaerobic fluidization methods (in Japanese). Bioscience and Industry, 45, 107-116 (1987). 3. Carrondo, M. J. T., Silva, J. M. C., Figueira, M. I. I., Ganho, R. M.B., and Oliveira, J. F. S.: Anaerobic filter treatment of
molasses fermentation wastewater. Wat. Sci. Technol., 15, 117126 (1983). 4. Kida, K., Morimura, S., Sonoda, Y., Obe, M., and Kondo, T.:
Support media for microbial adhesion in an anaerobic fluidizedbed reactor. J. Ferment. Bioeng., 69, 354-359 (1990).
The Importance of the Surface Charge on Support Media for Microbial Adhesion KENJI KIDA, 1. SHIGERU MORIMURA, I YORIKAZU SONODA, l AND TOSHINOBU YANOH 2
Department of Applied Chemistry, Faculty of Engineering, Kumamoto University, 2-39-1 Kurokami, Kumamoto-City, Kumamoto 860, ~ and Nittetsu Mining Co. Ltd., New Material Department of Research and Development Center, 8-10-16, Shimorenjaku, Mitaka-City, Tokyo 181, 2 Japan Received 6 December 1991/Accepted28 January 1992 Using cristobalites treated at various temperatures, methane fermentation tests were carried out in an anaerobic fluidized-bed reactor (AFBR). Cristobalites treated below 1,000°C had almost the same qualities in terms of components, physical properties, and surface structures. However, points of zero charge were different between cristobalites treated below 600°C and those treated above 800°C. Points of zero charge on the former numbered about 5 and their surfaces were positively charged in the fermentor controlled pH at 7, while points of zero charge of the latter numbered above 8 and surfaces were negatively charged. In the methane fermentation tests, a TOC removal efficiency of 78% was obtained using positively charged cristobalite at a TOC loading rate of 8 g/l. h, but with negatively charged cristobalite, the efficiency was only 63% under the same conditions. This result confirms the importance of the surface charge on the support medium for the anaerobic digestion in an AFBR.
In order to reduce the hydraulic retention time (HRT) in anaerobic digestion, it is very important to keep the microorganisms concentration high in the reactor. There are many studies aimed at keeping microorganisms at high concentrations in reactors using an upflow anaerobic sludge blanket (UASB) (1), an anaerobic fluidized-bed reactor (AFBR) (2), an upflow anaerobic filter process (UAFP) (3), and so on. We also have studied the anaerobic treatment of various kinds of wastewater and have tried to generalize a methane fermentation process for treatment of wastewater. In the AFBR process, it is very important to select a support medium with high fluidity to reduce the energy required for fluidization. And the support media must be hard and must allow microbial adhesion. We evaluated eight kinds of support medium, namely, cristobalite, zeolite, vermiculite, granular active carbon, granulated clay, pottery stone, volcanic ash, and slag, by methane fermentation tests, as reported previously (4). From our results, cristobalite was found to be the best support medium and zeolite was the second best. Both media had rough surfaces under the scanning electron microscope (SEM: JSM-T200, Jeol, Tokyo), but their surface charges were different. Cristobalite was positively charged and zeolite was negatively charged under conditions of methane fermentation. The cristobalite used in that test was treated at 400°C, and it was found that the surface charge of cristobalite changed with different temperatures of treatment. In this report, we describe the importance of surface charge as well as the roughness of the surface, as assessed by measurements of physical properties and a methane fermentation test. First, we measured the components of cristobaiites treated at various temperatures. As shown in Table 1, metal contents were almost the same in every sample of cristobalite, but the SiO2 content increased with increases in
temperature. The increase of SiO2 content was caused by a decrease in ignition loss with increased temperatures, that is, by the decrease in organic matter with treatment at high temperatures. With this exception, the components of the cristobalites were almost identical. Table 2 shows the bulk density (Pd, g/cm3), the wet density (Pw, g/cma), the specific surface area (m2/g), the average pore radius (mm), and the total pore volume (p, cm3/g) of cristobalites. Bulk and wet densities were measured by using a pycnometer, specific surface area by use of Monosorb (Yuasa-Quantachrome, Osaka), average pore radius and total pore volume by use of a Porosimeter (MOD 220, Carlo-Erba Co., Milano, Italy) as reported previously (4). The ratio of total pore volume to total volume of the medium was calculated from the following equation: Ratio of pores to medium (%) ---- { P d ' p / ( 1 --Pw+P~+Pd'P)} x 100
(1)
Bulk and wet densities were almost the same for every temperature of treatment, but specific surface area rapidly decreased above 1,000°C. Since average pore radii increased with decreases in specific surface area, small-sized pores might be destroyed by treatment at high temperature. With treatment at 1,200°C, the ratio of total pore volume to total volume of a medium was reduced to 50% of the ratio after treatment below 1,000°C. Surface structures of media were examined by SEM. Figure 1 shows scanning electron micrographs of surface structures of cristobalites treated at 400, 600, 800, and 1,000°C. Surface structures were rough and almost the same for every temperature. Points of zero charge were also measured by the method reported previously (4). As shown in Fig.2, points of zero charge of cristobalites treated below 600°C numbered about 5 and their surfaces were positively charged in a reactor in which the pH was controlled at 7. In the case of treatment above 800°C, points of zero charge numbered above 8 and surfaces were negatively charged. This distinction might be caused by differences in the numbers of
* Corresponding author. 323
324
KIDA ET AL.
J. FERMENT. BIOENG., TABLE 1. Components of cristobalite treated at various temperatures
Conditions of heat treatment
SiO2 (%)
Na20 (%)
K,O (~)
TiO2 (%)
CaO (%)
MgO (~)
S (%)
AI,O3 (/a~)
Fe.,O3 (o/0)
Ig. loss (%)
Raw material 400°C, 2 h 600°C, 2 h 800°C, 2 h 1000°C, 2 h 1200°C, 2 h
76.5 78.9 80.6 80.8 81.9 82.9
0.39 0.41 0.40 0.40 0.42 0.41
0.94 0.94 0.97 0.98 0.98 0.99
0.24 0.26 0.26 0.26 0.27 0.28
1.80 2.21 1.73 1.88 1.95 1.69
0.89 0.90 0.91 0.92 0.94 0.94
0.21 0.27 0.24 0.22 0.15 0.04
6.75 7.23 7.17 7.24 7.42 7.45
3.91 4.81 3.93 4.16 4.32 4.30
8.30 4.02 3.43 2.62 1.23 0.22
TABLE 2.
Physical properties of cristobalite treated at various temperatures
Conditions of heat treatment
Bulk density (g/cm ~)
Wet density (g/cm 3)
Specific surface area (m2/g)
Average pore radius x ( 10- s mm)
Total pore volume (cm3/g)
Ratio of pores a (%)
Raw material 400°C, 2 h 600°C, 2 h 800°C, 2 h 1000°C, 2 h 1200°C, 2 h
0.82 0.82 0.77 0.78 0.82 0.89
1.33 1.46 1.47 1.44 1.46 1.50
49.2 51.9 49.4 52.4 28.3 1.8
8.8 19.2 7.5 12.9 26.9 130.1
0.299 0.281 0.265 0.276 0.312 0.131
33 39 40 39 42 23
'
This value was calculated from Eq. I.
silanol groups, so the n u m b e r s o f silanol groups were measured by the lithium h y d r i d e - a l u m i n i u m m e t h o d . The re-
(a)
(b)
suits are s h o w n in Fig.2. The n u m b e r s o f silanol groups decreased with increases in the t r e a t m e n t t e m p e r a t u r e . Thus, the difference in the n u m b e r s o f points o f zero charge on cristobalites treated at various t e m p e r a t u r e s d e p e n d e d on the change o f silanol groups f r o m [Si-OH] to [Si-O-Si]. While the surface o f cristobalite which was not heated was positively charged, the a b r a s i o n loss was as high as 7 . 5 % (see Fig. 2). T h e r e f o r e , m e t h a n e f e r m e n t a t i o n tests were carried out using cristobalites treated at 400 and 1,000°C. T h e particle sizes, the m i n i m u m fluidization velocities, and the linear velocities to allow twice the exp a n d e d v o l u m e , which were the s a m e a m o n g b o t h cristobalits, were 0.1-0.3 r o m e , 0.02 c m / s , and 0.28 c m / s , respectively. A total o f 2 0 % ( w / v ) cristobalite was put in a digestor, m a d e o f acrylic resin with a w o r k i n g v o l u m e o f 0.45 l, and the test was started at 53°C, at p H 7.0, with a circulation rate o f culture m e d i u m o f 100 m l / m i n (4). Synthetic wastewater c o n t a i n i n g (g//): P o l y p e p t o n , 2.6; m e a t extract, 2.0; urea, 0.5; N a C l , 0.15; KC1, 0.07; MgSO4. 7 H 2 0 , 0.05; Na2HPO4, 0.5; CaCI2, 0.07, was used for the test. T o t a l organic c a r b o n ( T O C ) in the wastewater was 1,900 mg/l. As s h o w n in Fig.3, the T O C r e m o v a l efficiencies were a l m o s t the s a m e with b o t h cristobalites at T O C A
10
"7"
g
,6- o "6
(c)
(d)
FIG. 1. Scanning electron micrographs of surface structures of cristobalite treated at various temperatures. (a) Treated at 400oc; (b) at 600°C; (c) at 800°C; (d) at t,000°C.
gg
,¢
]F, I, ~ 0
200
400
,' /, 600
800
n~ 1000
1200
Temperatures of heat treatment (~C)
FIG. 2. Effects of heat treatment on points of zero charge (z~), abrasion losses ( o ), and numbers of silanol groups ( • ).
VOL. 73, 1992
NOTES
100 m m
8o
"6 60 r-
o
m
~ 20 --
I
o
0
I 2
I
I 4
I
I 6
I
I 8
I
[
I
10
Volumetric loadlng rate of TOC ( g l l.d)
FIG. 3. Effects of surface charges on cristobalite on removal efficiency of TOC. Symbols: O, positive charge; o, negative charge. loading rates below 4 g/l.d, but above 4 g / l - d , the TOC removal efficiency with cristobalite treated at 400°C was higher than in the case o f that treated at 1,000°C. A t the T O C loading rate o f 8 g/l. d, the T O C removal efficiencies with cristobalites treated at 400°C and 1,000°C were 78% and 63%, respectively. The increase in TOC removal efficiency with increased T O C loading rate probably depends on the increase in numbers of attached microorganisms on the support medium. A n d the difference in the increased numbers o f microorganisms between cristobalites treated at 400°C and 1,000°C seemed to be caused by the difference o f charge on the surface. After completion o f the methane fermentation test, the volume o f attached microorganisms was measured as follows: all the culture medium was poured into a 500-ml cylinder and the support medium was separated from the liquid medium by decantation. The liquid medium containing floating microorganisms and granules was centrifuged at 1 0 , 0 0 0 x g for 10rain. Volatile suspended solids (VSS) in the precipitate were measured. The VSS o f attached microorganisms on the support medium were also measured after washing. The ratios of floated VSS to attached VSS for cristobalites treated at 4000C and 1,000°C were 13.8% and 10.5%, respectively. F r o m this data, the re-
325
moval o f T O C in methane fermentation seems to be dependent on the numbers o f attached microorganisms. Since attached VSS per weight o f cristobalites were 100.6mg/g for treatments at 400°C and 8 4 . 9 m g / g at 1,000°C, respectively, it seems that the support medium o f which the surface was positively charged allowed microorganisms to attach more easily than the negatively charged support medium. F r o m the results mentioned above, it was confirmed that differences in surface charge affect the efficiency o f anaerobic treatment even if the medium and its surface structure are almost identical, and that surface charge is the most important property of the support medium. There are many factors that effect the attachment o f microorganisms to support medium, such as electrostatic forces, Van der Waals forces, and polymers produced by microorganisms. It is likely that electrostatic forces are necessary for the initial adsorption to the surface of the support medium and to prevent the detachment o f microorganisms from the support medium. Silanol groups are very effective for the adsorption if inorganic materials are used for the support medium. The authors wish to thank K. Fujita and Y. Nakano for their excellent technical assistance. We also thank Prof. Dr. T. Nonaka of the Faculty of Engineering, Kumamoto University for measurements of specific surface area and distribution of pore radii, and Y. Hayashida of Department of Applied Microbiology, Kumamoto Industrial Research Institute for operating the scanning electron microscope. REFERENCES I. Lettinga, G., van Velsen, A. F. M., Homba, S.W. de Zeeum, W., and Klapwijk, A.: Use of the upflow anaerobic sludge
blanket CLIASB)reactor concept for biological wastewater treatment especially for anaerobic treatment. Biotechnol. Bioang., 22, 699-734 (1980). 2. Kida, K. and Nakata, T.: Treatment of distillery wastewater by a series of two anaerobic fluidization methods (in Japanese). Bioscience and Industry, 45, 107-116 (1987). 3. Carrondo, M. J. T., Silva, J. M. C., Figueira, M. I. I., Ganho, R. M.B., and Oliveira, J. F. S.: Anaerobic filter treatment of
molasses fermentation wastewater. Wat. Sci. Technol., 15, 117126 (1983). 4. Kida, K., Morimura, S., Sonoda, Y., Obe, M., and Kondo, T.:
Support media for microbial adhesion in an anaerobic fluidizedbed reactor. J. Ferment. Bioeng., 69, 354-359 (1990).