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Effect of preservation techniques on the regeneration of gel entrapped nitrifying sludge
PII: S0043-1354(98)00200-0
Wat. Res. Vol. 33, No. 1, pp. 164±168, 1999 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/98 $ - see front matter
EFFECT OF PRESERVATION TECHNIQUES ON THE REGENERATION OF GEL ENTRAPPED NITRIFYING SLUDGE M M C. VOGELSANG** , K. GOLLEMBIEWSKI and K. éSTGAARD*
Department of Biotechnology, NTNU, N-7034 Trondheim, Norway (First received November 1997; accepted in revised form April 1998) AbstractÐGel entrapped nitri®ers may be used in wastewater treatment plants as the main N-removal system, or as an additive to existing bio®lm or sludge systems. In any case, gel entrapped nitri®ers may be stored for safety measures and added in cases of a collapse of the activity in the plant. Dierent preservation techniques; freezing, drying and lyophilization, were evaluated as means to optimize the conservation of the nitri®cation activity immobilized in PVA-SbQ gel beads during storage. Glycerol, sucrose and trehalose were added in various amounts as cryoprotectants. After 2±3 months of storage, the nitri®ers were reactivated in 450 ml CSTR using a synthetic nitri®cation medium with excess ammonium. Beads frozen without any additives or in 15% glycerol maintained 60% and 40% of the original nitri®cation rate they had before conservation, respectively. After preservation by drying and lyophilization, 10% or less of the activity was recovered. However, even those beads reached 100% of the previous activity within 4±9 d of cultivation. In the case of drying, addition of 10 mM trehalose improved the initial activity signi®cantly. When lyophilized, the presence of protectants up to 100 mM seemed to have a positive eect on the reactivation of the nitri®ers. The stress of the drying process apparently altered the structure of the gel beads so that they never fully regained the initial shape and volume, but without any signi®cant loss in the obtainable maximum activity. It is concluded that for long time storage at a wastewater treatment plant, freezing without any additives should be the preferred preservation technique. For transportation, however, when weight and size are important economical factors, drying may be a better alternative. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐnitrogen removal, gel entrapped nitri®ers, preservation techniques
INTRODUCTION
Gel entrapment of the nitri®ers in wastewater treatment may have several advantages compared to a conventional activated sludge system. The simpli®ed separation of water and biocatalyte will give prolonged biomass retention time even at short hydraulic retention times (HRT), thereby increasing the fraction of slow-growing nitri®ers. High biomass concentration and ease of separation may save the treatment plant both space and investments. Also, decreased sensitivity to changes in the external environment is of importance, particularly at low temperatures (Wijels et al., 1995; Leenen, 1997; Vogelsang et al., 1997) due to the mesophilic nature of the nitri®ers (éstgaard et al., 1994). In domestic wastewater, gel forming biopolymers such as alginate are rapidly degraded (Vogelsang and éstgaard, 1996), and therefore useless. However, gel beads formed by the synthetic polymer PVA-SbQ (poly vinyl alcohol with grafted stilbazolium groups) (Ichimura and Watanabe, 1980), have been shown to maintain their integrity *Author to whom all correspondence should be addressed [Tel: +47-7359-4062; Fax: +47-7359-1283]. 164
(Vogelsang et al., 1997). In Japan, another gel entrapment system based on polyethylene glycol cubes has already been tested out in full scale, and is now commercialized by Hitachi in their Pegasus project (Emori et al., 1996). It is directed towards conventional pre-denitri®cation plants, immobilizing both nitri®ers and denitri®ers (Tanaka et al., 1996). Gel entrapped nitri®ers may also be added to update existing bio®lm or activated sludge plants, provided a suitable retention system is included. A grid system may be applied for larger beads, while micro beads would probably require an airlift reactor (Hunik et al., 1994; Leenen, 1997). In both cases, it should also be possible to use gel entrapped nitri®ers as a safety system, having a ready-to-use inoculum whenever there is a loss of nitri®cation activity. For the feasibility of the immobilized system in this case, factors such as cost of transportation and storage, and rate and degree of regeneration of the activity, are of vital importance. Important cost factors of transportation and storage are the weight and volume of the system, as well as additives and energy required for preservation and storage. For those reasons, it would be of particular interest to remove all excess water from the matrix if this can be obtained without losing too much of the activity.
Gel entrapped nitrifying sludge
165
acrylic cylinders (length 0.5 m; inner diameter 0.06 m) with a ®ne net at the ends to prevent the beads from escaping. These beads were ¯uidized and dried for 2.5 h in an air¯ow at 258C. To obtain freeze dried beads, the beads were frozen over dry ice and acetone for 15 min and vacuum dried at room temperature in a Lyovac GT 2 (LeyboldHeraeus, KoÈln, Germany) for 15 h. The dried and lyophilized beads were stored at ÿ208C for 2 and 3 months, respectively, before regeneration.
Common preservation techniques involving freezing and/or drying usually require addition of cryoprotectants such as sugars or polymers to help the bacteria to survive the treatment. In this short note, we present the eects these kinds of treatments had on the physical shape of PVA-SbQ gel beads as well as on the regeneration rate of the nitri®cation after several months of storage. The beads had been cultivated in a continuously stirred tank reactor (CSTR) fed with natural domestic wastewater for more than 10 months prior to preservation (Vogelsang et al., 1997).
Shrinking and swelling The total weight and volume of all beads in each batch were determined before and after the regeneration. Data were corrected for experimental losses of beads during handling (less than 5%, except for an accidental 20% loss of the sample frozen with glycerol). The recovery rate of gel bead weight and size during reactivation were studied in more detail by measuring the weight-gain of lyophilized beads (100 mM trehalose) kept in small tubes with medium under gentle stirring. Pictures of single beads were taken along the way.
MATERIALS AND METHODS
Materials The alginate and PVA-SbQ used here are described in detail by Vogelsang et al. (1997). The volume of gel beads were always measured in a measuring cylinder, in wet state as the excluded volume of beads lying in water. The reactivation medium had the following composition: 0.4 g/l K2HPO4, 1.0 g/l NaHCO3, 25 mg/l MgSO47H2O, 15 mg/l CaCl22H2O, 2.0 mg/l FeCl24H2O, 5.5 mg/l MnCl24H2O, 0.68 mg/l ZnCl2, 1.2 mg/l CoCl26H2O, 1.2 mg/l NiCl26H2O, 28 mg/l EDTA, and concentrations of NH4 varying from 25.0 mg to 125.0 mg N/l. The components were dissolved in tap water and the pH was adjusted to 7.3 (éstgaard et al., 1994).
Reactivation of the nitri®ers The reactivation was done in 1 l reactors with 450 ml medium operated as CSTR (approx. 2±4 h hydraulic retention time; 500±600 rpm by magnetic stirring), except for the ®rst 4±24 h, when they were kept as batch reactors to quantify the initial activity. The reactors were kept at 308C and pH 7.3020.03 and aerated (approximately 3 l/ min) through a rectangular glass sintered aquarium ®lter (6 cm3). The ¯ow and the concentration of ammonium in the medium (described above) were increased as the activity increased, keeping the concentration of ammonium in the reactors within 10±40 mg N/l. The frozen beads were transferred directly to the reactors. The dried and the lyophilized beads were kept in bottles with water saturated air (1 atm, 0308C) for 2 h before being transferred to the dierent reactors. Of the dierent lyophilized beads, only 80 ml of the initial 100 ml were used in the regeneration. The reactivation was continued until a steady state nitri®cation with no or low nitrite accumulation was reached. Concentrations of ammonium and nitrite were determined daily using DrLange Cuvette tests LCK 303 and LCK 341 (DrLange, 1992) in accordance with international standards (DIN 38 406-E5-1 and DIN 38 405-D 10, respectively). Calculated activities are normalized per 100 ml original gel volume.
Gel entrapment and lab scale nitri®cation The gel immobilized nitri®ers used in these experiments were all taken from the same 4 l CSTR that had been kept operative for more than 10 months on fresh sewage supplied with excess ammonium (Vogelsang et al., 1997). The ®nal activity in the reactor when it was shut down was 7.4 mg NH4-N/h per 100 ml beads. The gel entrapment procedure and reactor maintenance are described elsewhere (Vogelsang et al., 1997). The freezing, drying and lyophilization processes A set of 13 batches of 100 ml beads (average diameter 3.02 0.1 mm) were kept in solutions with dierent additives, according to Table 1, for 1 h before they were frozen, dried or lyophilized. All beads in each batch were treated together during the whole process. In the two batches that were frozen, water surrounding the beads was removed, one batch given glycerol corresponding to 15% ®nal concentration, and the beads were put directly in the freezer and stored for 2 months at ÿ808C. The drying was performed in a home made ¯uidized bed drier, using
RESULTS AND DISCUSSION
Physical changes of gel beads In principle, the drying and freeze±drying processes should result in a complete loss of the
Table 1. Volume (Vdry) and weight (Wdry) of the beads after preservation and after regeneration (Wregen) relative to the initial volume (V0) and initial weight (W0), respectively Preservation technique Freezing (ÿ808C) Drying
Lyophilization
Protectant
Vdry/V0 (%)
Wdry/W0 (%)
Wregen/W0 (%)
none 15% glycerol none 15% glycerol 10 mM sucrose 100 mM sucrose 10 mM trehalose 100 mM trehalose none 10 mM sucrose 100 mM sucrose 10 mM trehalose 100 mM trehalose
ÿ ÿ 10 11 12 16 13 10 66 46 37 37 21
ÿ ÿ 6 7 7 10 13 11 6 10 7 9 9
100 98 57 56 64 63 62 67 63 60 54 61 57
166
C. Vogelsang et al.
Fig. 1. Beads (a) before preservation, (b) in dried (above) and lyophilized (below) state, (c) after reactivation (dried bead above and lyophilized bead below). Bar is 5.0 mm.
unbound water of the gel beads, leading to decreased weight. They may also irreversibly aect the shape and size of the beads. Table 1 shows the relative weight and volume of the beads after drying and lyophilization. All beads lost 87±94% of the weight, as expected when considering the 5% dry matter of PVA-SbQ plus the biomass within the beads. The 2% alginate initially present was most probably degraded and lost long ago (Vogelsang et al., 1997). The ®nal shape and size of the beads, however, diered considerably (Table 1). The drying process gave much smaller and more equalsized beads than lyophilization (10±15% and 20± 65% of the original volume, respectively). This is also visualized in Fig. 1 by pictures taken of one typical lyophilized and one typical dried bead before and after the regeneration. Additives reduced the mean volume of the lyophilized beads, but even within each batch, the size diered signi®cantly, possibly caused by local variations in pre-freezing or drying rate within the freeze±drying ¯asks. When the beads were exposed to water again during regeneration, they started gaining weight rapidly, reaching 90% of their ®nal weight after approximately 1.5 h, as illustrated by the example of the lyophilized beads (100 mM trehalose) in Fig. 2. As seen in Table 1, the drying and lyophilization processes permanently changed the structural integrity of the beads, so that they never regained their initial weight nor shape during the regeneration. See also Fig. 1.
tion curves, exempli®ed by one frozen, one dried and one lyophilized sample. The total set of data are summarized by the calculations shown in Table 2. The initial activity had to be determined before new growth had a signi®cant eect, but preferably after transient adaptive eects. According to éstgaard et al. (1994), a lag of 1.5±2 h may be expected for temperature adaptation. The initial activity was therefore determined while the reactors were run in the batch mode 2 h after inoculation for all reactors, then checked 1±2 times the ®rst 14 h if the activity was low. This activity was compared to the 7.4 mg NH4-N/h per 100 ml beads activity of the nitri®ers before the preservation (Vogelsang et al., 1997). The beads that had been preserved by freezing at ÿ808C showed the highest initial activity, conserving 60% and 40% of the activity without or with 15% glycerol added as protectant, respectively. Even dried beads with 10 mM trehalose, had conserved as
Reactivation of the nitri®ers After 2 to 3 months storage, as described in the Materials and Methods section, the preserved gel entrapped nitri®ers were reactivated in reactors supplied with synthetic wastewater until a stable ammonium removal and a low nitrite accumulation were reached. Figure 3 shows some typical reactiva-
Fig. 2. Weight of lyophilized (in 100 mM trehalose) beads during regeneration, relative to the weight before freeze±drying. Dotted line represents the ®nal weight after regeneration.
Gel entrapped nitrifying sludge
167
Table 2. Reactivation of gel immobilized nitri®ers after preservation by freezing, drying or lyophilization with dierent protectants added: initial activity and ratio of the original activity conserved, estimated from activity measurements during the ®rst 2±14 h of regeneration in batch mode; time before the original activity was regenerated; maximum nitrite accumulation during regeneration relative to the ammonium removal rate Protectant Preservation technique Freezing (ÿ808C) Drying
Lyophilization
None 15% glycerol None 15% glycerol 10 mM sucrose 100 mM sucrose 10 mM trehalose 100 mM trehalose None 10 mM sucrose 100 mM sucrose 10 mM trehalose 100 mM trehalose
Initial activity (mg N/ Ratio of the original Time before the h) activity conserved (%) original activity was regenerated (d) 4.4 3.0 0.5 0.8 0.6 0.03 2.8 0.6 0.1 0.2 0.5 0.3 0.7
much as 40% of the activity. For all the other beads the conserved activity ratios were 10% or less. However, even cultures with low or insigni®cant initial activity could easily be regenerated after some days of cultivation. After a lag period of 0± 2.4 d for dried beads and 1.6±3.6 d for lyophilized beads (when estimated as the time needed to reach 10% of the original activity), the original activity was generally obtained within 4±9 d (Table 2). In the case of lyophilized beads without protectant, an experimental accident causing severe pH shock at day 8 (70% of original activity) made it necessary to estimate the time of full recovery by linear regression and extrapolation. As discussed in detail by Vogelsang et al. (1997), the biomass of the deeper gel layers will be oxygen limited by the overgrowing nitri®ers when the system has reached steady state. The overall steady state activity will therefore unavoidably be limited by the oxygen transfer of the reactor system, including factors such as air¯ow and stirring rate (Vogelsang et al., 1997). To obtain exactly the same quantitative oxygen limitation in a dierent reactor system is a most dicult experimental task. Reactivation was therefore performed in reactors with somewhat better oxygen transfer than the original, so that the initial activities should not be restricted by this factor. Due to the improved growth conditions, the activity continued to increase above the original level before a new steady state was reached (Fig. 3). The ®nal steady state maximal activities were within 13±25 mg N/h. These variations did not correlate to changes in bead surface area, and is probably largely due to experimental variations in the aeration system applied. In cases of adaptation to improved growth conditions, nitrite build-up due to a delayed response of Nitrobacter sp. is frequently observed, as illustrated in Fig. 3. The maximal recorded values are summarized in Table 2, relative to the ammonium removal rate at the same time. The samples that
59 41 7 11 8 0.4 38 8 2 3 7 4 9
1.5 3.0 4.3 5.3 5.0 8.7 5.5 7.2 9 6.7 5.8 6.5 7.5
Maximum relative nitrite accumulation (%) 2.8 3.2 22 24 27 14 17 20 n.d. 36 26 30 16
Fig. 3. Ammonium removal rate (.) and nitrite accumulation rate (w) during the regeneration of (a) frozen beads without any protectant added, (b) dried beads in 10 mM sucrose, and (c) lyophilized in 10 mM sucrose. Dotted lines represent the original activity before preservation.
168
C. Vogelsang et al.
had been frozen showed less than 3% nitrite accumulation, whereas the others experienced a 20± 30% maximal accumulation of nitrite. Concluding remarks Gel entrapped nitri®ers stored by freezing performed excellently, with high initial activity and insigni®cant nitrite accumulation during regeneration. Due to this high survival, addition of glycerol had no additional positive eect on the reactivation rate. Both drying and lyophilization led to loss of 90% or more in original activity. However, this activity could be regenerated after 4±9 d of cultivation. In the case of drying, addition of 10 mM trehalose improved the initial activity signi®cantly. When lyophilized, the presence of protectants up to 100 mM seemed to have a positive eect on the reactivation of the nitri®ers. Both drying and lyophilization led to irreversible changes in gel structure, so that the original shape and volume could not be fully regained. These changes did not aect the functional properties of the beads, but could be of importance for the reactivation rate. REFERENCES
DrLange (1992) Handbook of Photometrical Operation Analysis. Dr. Bruno Lange, Berlin. Emori H., Mikawa K., Hamaya M., Yamaguchi T., Tanaka K. and Takeshima T. (1996) PEGASUS Innovative biological nitrogen removal process using entrapped nitri®ers. In Immobilized Cells: Basics and Applications, eds R. H. Wijels, R. M. Buitelaar, C.
Bucke and J. Tramper, pp. 546±555. Elsevier, Amsterdam. Hunik J. H., Tramper J. and Wijels R. H. (1994) A strategy to scale-up nitri®cation processes with immobilized cells of Nitrosomonas europaea and Nitrobacter agilis. Bioproc. Eng. 11, 73±82. Ichimura K. and Watanabe S. (1980) Highly photosensitive polymers with anomalously low content of stilbazolium groups. J. Polym. Sci. Polym. Chem. Ed. 18, 613± 617. Leenen E. J. T. M. (1997) Nitri®cation by arti®cially immobilized cells. Model and practical system. Ph.D. thesis, Wageningen Agricultural University, The Netherlands. éstgaard K., Lee N. and Welander T. (1994) Nitri®cation at low temperatures. In Second International Symposium on Environmental Biotechnology, 4±6 July 1994, Brighton, pp. 134±137. Institution of Chemical Engineers, Rugby, U.K. Tanaka K., Sumino T., Nakamura H., Ogasawara T. and Emori H. (1996) Application of nitri®cation by cells immobilized in polyethylene glycol. In Immobilized Cells: Basics and Applications, eds R. H. Wijels, R. M. Buitelaar, C. Bucke and J. Tramper, pp. 622±632. Elsevier, Amsterdam. Vogelsang C. and éstgaard K. (1996) Stability of alginate gels applied for cell entrapment in open systems. In Immobilized Cells: Basics and Applications, eds R. H. Wijels, R. M. Buitelaar, C. Bucke and J. Tramper, pp. 213±220. Elsevier, Amsterdam. Vogelsang C., Husby A. and éstgaard K. (1997) Functional stability of temperature-compensated nitri®cation in domestic wastewater treatment obtained with PVA-SbQ/alginate gel entrapment. Wat. Res. 31(7), 1659±1664. Wijels R. H., Englund G., Hunik J. H., Leenen E. J. T. M., Bakketun AÊ., GuÈnther A., OboÂn de Castro J. M. and Tramper J. (1995) Eects of diusion limitation on immobilized nitrifying microorganisms at low temperatures. Biotechnol. Bioeng. 45, 1±9.
Wat. Res. Vol. 33, No. 1, pp. 164±168, 1999 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/98 $ - see front matter
EFFECT OF PRESERVATION TECHNIQUES ON THE REGENERATION OF GEL ENTRAPPED NITRIFYING SLUDGE M M C. VOGELSANG** , K. GOLLEMBIEWSKI and K. éSTGAARD*
Department of Biotechnology, NTNU, N-7034 Trondheim, Norway (First received November 1997; accepted in revised form April 1998) AbstractÐGel entrapped nitri®ers may be used in wastewater treatment plants as the main N-removal system, or as an additive to existing bio®lm or sludge systems. In any case, gel entrapped nitri®ers may be stored for safety measures and added in cases of a collapse of the activity in the plant. Dierent preservation techniques; freezing, drying and lyophilization, were evaluated as means to optimize the conservation of the nitri®cation activity immobilized in PVA-SbQ gel beads during storage. Glycerol, sucrose and trehalose were added in various amounts as cryoprotectants. After 2±3 months of storage, the nitri®ers were reactivated in 450 ml CSTR using a synthetic nitri®cation medium with excess ammonium. Beads frozen without any additives or in 15% glycerol maintained 60% and 40% of the original nitri®cation rate they had before conservation, respectively. After preservation by drying and lyophilization, 10% or less of the activity was recovered. However, even those beads reached 100% of the previous activity within 4±9 d of cultivation. In the case of drying, addition of 10 mM trehalose improved the initial activity signi®cantly. When lyophilized, the presence of protectants up to 100 mM seemed to have a positive eect on the reactivation of the nitri®ers. The stress of the drying process apparently altered the structure of the gel beads so that they never fully regained the initial shape and volume, but without any signi®cant loss in the obtainable maximum activity. It is concluded that for long time storage at a wastewater treatment plant, freezing without any additives should be the preferred preservation technique. For transportation, however, when weight and size are important economical factors, drying may be a better alternative. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐnitrogen removal, gel entrapped nitri®ers, preservation techniques
INTRODUCTION
Gel entrapment of the nitri®ers in wastewater treatment may have several advantages compared to a conventional activated sludge system. The simpli®ed separation of water and biocatalyte will give prolonged biomass retention time even at short hydraulic retention times (HRT), thereby increasing the fraction of slow-growing nitri®ers. High biomass concentration and ease of separation may save the treatment plant both space and investments. Also, decreased sensitivity to changes in the external environment is of importance, particularly at low temperatures (Wijels et al., 1995; Leenen, 1997; Vogelsang et al., 1997) due to the mesophilic nature of the nitri®ers (éstgaard et al., 1994). In domestic wastewater, gel forming biopolymers such as alginate are rapidly degraded (Vogelsang and éstgaard, 1996), and therefore useless. However, gel beads formed by the synthetic polymer PVA-SbQ (poly vinyl alcohol with grafted stilbazolium groups) (Ichimura and Watanabe, 1980), have been shown to maintain their integrity *Author to whom all correspondence should be addressed [Tel: +47-7359-4062; Fax: +47-7359-1283]. 164
(Vogelsang et al., 1997). In Japan, another gel entrapment system based on polyethylene glycol cubes has already been tested out in full scale, and is now commercialized by Hitachi in their Pegasus project (Emori et al., 1996). It is directed towards conventional pre-denitri®cation plants, immobilizing both nitri®ers and denitri®ers (Tanaka et al., 1996). Gel entrapped nitri®ers may also be added to update existing bio®lm or activated sludge plants, provided a suitable retention system is included. A grid system may be applied for larger beads, while micro beads would probably require an airlift reactor (Hunik et al., 1994; Leenen, 1997). In both cases, it should also be possible to use gel entrapped nitri®ers as a safety system, having a ready-to-use inoculum whenever there is a loss of nitri®cation activity. For the feasibility of the immobilized system in this case, factors such as cost of transportation and storage, and rate and degree of regeneration of the activity, are of vital importance. Important cost factors of transportation and storage are the weight and volume of the system, as well as additives and energy required for preservation and storage. For those reasons, it would be of particular interest to remove all excess water from the matrix if this can be obtained without losing too much of the activity.
Gel entrapped nitrifying sludge
165
acrylic cylinders (length 0.5 m; inner diameter 0.06 m) with a ®ne net at the ends to prevent the beads from escaping. These beads were ¯uidized and dried for 2.5 h in an air¯ow at 258C. To obtain freeze dried beads, the beads were frozen over dry ice and acetone for 15 min and vacuum dried at room temperature in a Lyovac GT 2 (LeyboldHeraeus, KoÈln, Germany) for 15 h. The dried and lyophilized beads were stored at ÿ208C for 2 and 3 months, respectively, before regeneration.
Common preservation techniques involving freezing and/or drying usually require addition of cryoprotectants such as sugars or polymers to help the bacteria to survive the treatment. In this short note, we present the eects these kinds of treatments had on the physical shape of PVA-SbQ gel beads as well as on the regeneration rate of the nitri®cation after several months of storage. The beads had been cultivated in a continuously stirred tank reactor (CSTR) fed with natural domestic wastewater for more than 10 months prior to preservation (Vogelsang et al., 1997).
Shrinking and swelling The total weight and volume of all beads in each batch were determined before and after the regeneration. Data were corrected for experimental losses of beads during handling (less than 5%, except for an accidental 20% loss of the sample frozen with glycerol). The recovery rate of gel bead weight and size during reactivation were studied in more detail by measuring the weight-gain of lyophilized beads (100 mM trehalose) kept in small tubes with medium under gentle stirring. Pictures of single beads were taken along the way.
MATERIALS AND METHODS
Materials The alginate and PVA-SbQ used here are described in detail by Vogelsang et al. (1997). The volume of gel beads were always measured in a measuring cylinder, in wet state as the excluded volume of beads lying in water. The reactivation medium had the following composition: 0.4 g/l K2HPO4, 1.0 g/l NaHCO3, 25 mg/l MgSO47H2O, 15 mg/l CaCl22H2O, 2.0 mg/l FeCl24H2O, 5.5 mg/l MnCl24H2O, 0.68 mg/l ZnCl2, 1.2 mg/l CoCl26H2O, 1.2 mg/l NiCl26H2O, 28 mg/l EDTA, and concentrations of NH4 varying from 25.0 mg to 125.0 mg N/l. The components were dissolved in tap water and the pH was adjusted to 7.3 (éstgaard et al., 1994).
Reactivation of the nitri®ers The reactivation was done in 1 l reactors with 450 ml medium operated as CSTR (approx. 2±4 h hydraulic retention time; 500±600 rpm by magnetic stirring), except for the ®rst 4±24 h, when they were kept as batch reactors to quantify the initial activity. The reactors were kept at 308C and pH 7.3020.03 and aerated (approximately 3 l/ min) through a rectangular glass sintered aquarium ®lter (6 cm3). The ¯ow and the concentration of ammonium in the medium (described above) were increased as the activity increased, keeping the concentration of ammonium in the reactors within 10±40 mg N/l. The frozen beads were transferred directly to the reactors. The dried and the lyophilized beads were kept in bottles with water saturated air (1 atm, 0308C) for 2 h before being transferred to the dierent reactors. Of the dierent lyophilized beads, only 80 ml of the initial 100 ml were used in the regeneration. The reactivation was continued until a steady state nitri®cation with no or low nitrite accumulation was reached. Concentrations of ammonium and nitrite were determined daily using DrLange Cuvette tests LCK 303 and LCK 341 (DrLange, 1992) in accordance with international standards (DIN 38 406-E5-1 and DIN 38 405-D 10, respectively). Calculated activities are normalized per 100 ml original gel volume.
Gel entrapment and lab scale nitri®cation The gel immobilized nitri®ers used in these experiments were all taken from the same 4 l CSTR that had been kept operative for more than 10 months on fresh sewage supplied with excess ammonium (Vogelsang et al., 1997). The ®nal activity in the reactor when it was shut down was 7.4 mg NH4-N/h per 100 ml beads. The gel entrapment procedure and reactor maintenance are described elsewhere (Vogelsang et al., 1997). The freezing, drying and lyophilization processes A set of 13 batches of 100 ml beads (average diameter 3.02 0.1 mm) were kept in solutions with dierent additives, according to Table 1, for 1 h before they were frozen, dried or lyophilized. All beads in each batch were treated together during the whole process. In the two batches that were frozen, water surrounding the beads was removed, one batch given glycerol corresponding to 15% ®nal concentration, and the beads were put directly in the freezer and stored for 2 months at ÿ808C. The drying was performed in a home made ¯uidized bed drier, using
RESULTS AND DISCUSSION
Physical changes of gel beads In principle, the drying and freeze±drying processes should result in a complete loss of the
Table 1. Volume (Vdry) and weight (Wdry) of the beads after preservation and after regeneration (Wregen) relative to the initial volume (V0) and initial weight (W0), respectively Preservation technique Freezing (ÿ808C) Drying
Lyophilization
Protectant
Vdry/V0 (%)
Wdry/W0 (%)
Wregen/W0 (%)
none 15% glycerol none 15% glycerol 10 mM sucrose 100 mM sucrose 10 mM trehalose 100 mM trehalose none 10 mM sucrose 100 mM sucrose 10 mM trehalose 100 mM trehalose
ÿ ÿ 10 11 12 16 13 10 66 46 37 37 21
ÿ ÿ 6 7 7 10 13 11 6 10 7 9 9
100 98 57 56 64 63 62 67 63 60 54 61 57
166
C. Vogelsang et al.
Fig. 1. Beads (a) before preservation, (b) in dried (above) and lyophilized (below) state, (c) after reactivation (dried bead above and lyophilized bead below). Bar is 5.0 mm.
unbound water of the gel beads, leading to decreased weight. They may also irreversibly aect the shape and size of the beads. Table 1 shows the relative weight and volume of the beads after drying and lyophilization. All beads lost 87±94% of the weight, as expected when considering the 5% dry matter of PVA-SbQ plus the biomass within the beads. The 2% alginate initially present was most probably degraded and lost long ago (Vogelsang et al., 1997). The ®nal shape and size of the beads, however, diered considerably (Table 1). The drying process gave much smaller and more equalsized beads than lyophilization (10±15% and 20± 65% of the original volume, respectively). This is also visualized in Fig. 1 by pictures taken of one typical lyophilized and one typical dried bead before and after the regeneration. Additives reduced the mean volume of the lyophilized beads, but even within each batch, the size diered signi®cantly, possibly caused by local variations in pre-freezing or drying rate within the freeze±drying ¯asks. When the beads were exposed to water again during regeneration, they started gaining weight rapidly, reaching 90% of their ®nal weight after approximately 1.5 h, as illustrated by the example of the lyophilized beads (100 mM trehalose) in Fig. 2. As seen in Table 1, the drying and lyophilization processes permanently changed the structural integrity of the beads, so that they never regained their initial weight nor shape during the regeneration. See also Fig. 1.
tion curves, exempli®ed by one frozen, one dried and one lyophilized sample. The total set of data are summarized by the calculations shown in Table 2. The initial activity had to be determined before new growth had a signi®cant eect, but preferably after transient adaptive eects. According to éstgaard et al. (1994), a lag of 1.5±2 h may be expected for temperature adaptation. The initial activity was therefore determined while the reactors were run in the batch mode 2 h after inoculation for all reactors, then checked 1±2 times the ®rst 14 h if the activity was low. This activity was compared to the 7.4 mg NH4-N/h per 100 ml beads activity of the nitri®ers before the preservation (Vogelsang et al., 1997). The beads that had been preserved by freezing at ÿ808C showed the highest initial activity, conserving 60% and 40% of the activity without or with 15% glycerol added as protectant, respectively. Even dried beads with 10 mM trehalose, had conserved as
Reactivation of the nitri®ers After 2 to 3 months storage, as described in the Materials and Methods section, the preserved gel entrapped nitri®ers were reactivated in reactors supplied with synthetic wastewater until a stable ammonium removal and a low nitrite accumulation were reached. Figure 3 shows some typical reactiva-
Fig. 2. Weight of lyophilized (in 100 mM trehalose) beads during regeneration, relative to the weight before freeze±drying. Dotted line represents the ®nal weight after regeneration.
Gel entrapped nitrifying sludge
167
Table 2. Reactivation of gel immobilized nitri®ers after preservation by freezing, drying or lyophilization with dierent protectants added: initial activity and ratio of the original activity conserved, estimated from activity measurements during the ®rst 2±14 h of regeneration in batch mode; time before the original activity was regenerated; maximum nitrite accumulation during regeneration relative to the ammonium removal rate Protectant Preservation technique Freezing (ÿ808C) Drying
Lyophilization
None 15% glycerol None 15% glycerol 10 mM sucrose 100 mM sucrose 10 mM trehalose 100 mM trehalose None 10 mM sucrose 100 mM sucrose 10 mM trehalose 100 mM trehalose
Initial activity (mg N/ Ratio of the original Time before the h) activity conserved (%) original activity was regenerated (d) 4.4 3.0 0.5 0.8 0.6 0.03 2.8 0.6 0.1 0.2 0.5 0.3 0.7
much as 40% of the activity. For all the other beads the conserved activity ratios were 10% or less. However, even cultures with low or insigni®cant initial activity could easily be regenerated after some days of cultivation. After a lag period of 0± 2.4 d for dried beads and 1.6±3.6 d for lyophilized beads (when estimated as the time needed to reach 10% of the original activity), the original activity was generally obtained within 4±9 d (Table 2). In the case of lyophilized beads without protectant, an experimental accident causing severe pH shock at day 8 (70% of original activity) made it necessary to estimate the time of full recovery by linear regression and extrapolation. As discussed in detail by Vogelsang et al. (1997), the biomass of the deeper gel layers will be oxygen limited by the overgrowing nitri®ers when the system has reached steady state. The overall steady state activity will therefore unavoidably be limited by the oxygen transfer of the reactor system, including factors such as air¯ow and stirring rate (Vogelsang et al., 1997). To obtain exactly the same quantitative oxygen limitation in a dierent reactor system is a most dicult experimental task. Reactivation was therefore performed in reactors with somewhat better oxygen transfer than the original, so that the initial activities should not be restricted by this factor. Due to the improved growth conditions, the activity continued to increase above the original level before a new steady state was reached (Fig. 3). The ®nal steady state maximal activities were within 13±25 mg N/h. These variations did not correlate to changes in bead surface area, and is probably largely due to experimental variations in the aeration system applied. In cases of adaptation to improved growth conditions, nitrite build-up due to a delayed response of Nitrobacter sp. is frequently observed, as illustrated in Fig. 3. The maximal recorded values are summarized in Table 2, relative to the ammonium removal rate at the same time. The samples that
59 41 7 11 8 0.4 38 8 2 3 7 4 9
1.5 3.0 4.3 5.3 5.0 8.7 5.5 7.2 9 6.7 5.8 6.5 7.5
Maximum relative nitrite accumulation (%) 2.8 3.2 22 24 27 14 17 20 n.d. 36 26 30 16
Fig. 3. Ammonium removal rate (.) and nitrite accumulation rate (w) during the regeneration of (a) frozen beads without any protectant added, (b) dried beads in 10 mM sucrose, and (c) lyophilized in 10 mM sucrose. Dotted lines represent the original activity before preservation.
168
C. Vogelsang et al.
had been frozen showed less than 3% nitrite accumulation, whereas the others experienced a 20± 30% maximal accumulation of nitrite. Concluding remarks Gel entrapped nitri®ers stored by freezing performed excellently, with high initial activity and insigni®cant nitrite accumulation during regeneration. Due to this high survival, addition of glycerol had no additional positive eect on the reactivation rate. Both drying and lyophilization led to loss of 90% or more in original activity. However, this activity could be regenerated after 4±9 d of cultivation. In the case of drying, addition of 10 mM trehalose improved the initial activity signi®cantly. When lyophilized, the presence of protectants up to 100 mM seemed to have a positive eect on the reactivation of the nitri®ers. Both drying and lyophilization led to irreversible changes in gel structure, so that the original shape and volume could not be fully regained. These changes did not aect the functional properties of the beads, but could be of importance for the reactivation rate. REFERENCES
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Bucke and J. Tramper, pp. 546±555. Elsevier, Amsterdam. Hunik J. H., Tramper J. and Wijels R. H. (1994) A strategy to scale-up nitri®cation processes with immobilized cells of Nitrosomonas europaea and Nitrobacter agilis. Bioproc. Eng. 11, 73±82. Ichimura K. and Watanabe S. (1980) Highly photosensitive polymers with anomalously low content of stilbazolium groups. J. Polym. Sci. Polym. Chem. Ed. 18, 613± 617. Leenen E. J. T. M. (1997) Nitri®cation by arti®cially immobilized cells. Model and practical system. Ph.D. thesis, Wageningen Agricultural University, The Netherlands. éstgaard K., Lee N. and Welander T. (1994) Nitri®cation at low temperatures. In Second International Symposium on Environmental Biotechnology, 4±6 July 1994, Brighton, pp. 134±137. Institution of Chemical Engineers, Rugby, U.K. Tanaka K., Sumino T., Nakamura H., Ogasawara T. and Emori H. (1996) Application of nitri®cation by cells immobilized in polyethylene glycol. In Immobilized Cells: Basics and Applications, eds R. H. Wijels, R. M. Buitelaar, C. Bucke and J. Tramper, pp. 622±632. Elsevier, Amsterdam. Vogelsang C. and éstgaard K. (1996) Stability of alginate gels applied for cell entrapment in open systems. In Immobilized Cells: Basics and Applications, eds R. H. Wijels, R. M. Buitelaar, C. Bucke and J. Tramper, pp. 213±220. Elsevier, Amsterdam. Vogelsang C., Husby A. and éstgaard K. (1997) Functional stability of temperature-compensated nitri®cation in domestic wastewater treatment obtained with PVA-SbQ/alginate gel entrapment. Wat. Res. 31(7), 1659±1664. Wijels R. H., Englund G., Hunik J. H., Leenen E. J. T. M., Bakketun AÊ., GuÈnther A., OboÂn de Castro J. M. and Tramper J. (1995) Eects of diusion limitation on immobilized nitrifying microorganisms at low temperatures. Biotechnol. Bioeng. 45, 1±9.