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Reduction of suspended biomass in municipal wastewater using bdelloid rotifers
Water Research 37 (2003) 2027–2034
Reduction of suspended biomass in municipal wastewater using bdelloid rotifers J. Lapinski, A. Tunnacliffe* Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK Received 24 April 2002; accepted 2 December 2002
Abstract Clarification of municipal wastewater was shown to be improved significantly by the addition of cultured bdelloid rotifers. The rate and degree of suspended particle removal were correlated with rotifer number. The size range of unsettled particles suspended in wastewater was determined and found to overlap with the size range of particles consumed by rotifers. Rotifers were shown to have two distinct effects on suspended particles: consumption of biomass due to feeding activity; and improved settling, probably due to enhanced aggregation. These experiments demonstrate the potential for the use of bdelloid rotifers in an enhanced wastewater treatment process, with reduced biomass production and improved effluent clarity. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bdelloid rotifers; Biomass reduction; Activated sludge; Metazoa; Total suspended solids; Clarification
1. Introduction One of the most widely used methods of wastewater treatment is the activated sludge process1 [1]. Although initially invented almost a century ago [2], the process has essentially remained the same throughout the intervening period. One of the main by-products of the activated sludge process is waste sludge, whose disposal accounts for B60% of operating costs in the East Anglian region of the United Kingdom and are similarly high elsewhere [3]. However, this cost is rising as disposal routes for sludge are becoming more restricted. Dumping at sea, which previously accounted for 30% of sludge disposed, is now illegal and use of waste sludge on agricultural land is increasingly under threat due to *Corresponding author. Tel.: +44-1223-766549; fax: +441223-334162. E-mail address: [email protected] (A. Tunnacliffe). 1 Griffiths M. AWG plc, Thorpe Wood House, Peterborough, Cambridgeshire, PE3 6WT, UK. E-mail: mailto:mgriffiths@ anglianwater.co.uk
consumer pressure [4]. As a result, the cost of waste sludge disposal is likely to continue to increase significantly. New methods of reducing sludge production, together with new disposal routes, are therefore urgently required. Other wastewater treatment processes, such as the trickling filter or the Kaldnes suspended carrier process [5] also have their attendant problems, e.g. the frequent occurrence of pin flocs, small sludge particles, which do not settle in the clarifier. These pin flocs are carried over into the effluent water, which can result in discharge limits for solids being exceeded [6]. Novel technologies that reduce the production of pin flocs, would therefore be of benefit to the water industry. Bdelloid rotifers (‘‘leech-like wheel-bearers’’) are metazoans of B300 mm length which are ubiquitous in most fresh- and wastewater habitats [7]. Their name stems from the fact that most species possess a corona of cilia around the mouth opening that beat sequentially to produce a vortex of water. The vortex draws particles suspended in water into the rotifer’s mouth [8]. It has been suggested that rotifers play an important role in the activated sludge process, functioning as nuclei for floc
0043-1354/03/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0043-1354(02)00626-7
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formation [9]. Furthermore, it has been stated that rotifer population growth is mainly limited by the amount of food available [10] and that they consume several times their body weight per day [8]. In principle therefore, rotifers might be exploited for removal of suspended bio-solids from wastewater, either by grazing on particulate matter or by enhanced ‘‘bioflocculation’’ or a combination of both. Here we explore the potential for the use of bdelloid rotifers as a biotechnological tool to improve effluent clarity and to decrease biomass yield in municipal wastewater.
2. Materials and methods 2.1. Wastewater Samples were taken from the AWG (formerly Anglian Water) plc. domestic wastewater treatment plant in Cambridge near Milton (UK). Effluent samples from the Kaldnes (K1) ‘‘Monster’’ moving bed reactor plant were taken before the clarifier stage. Activated sludge samples were taken from the aerobic zone of the main activated sludge plant. All samples were autoclaved immediately after sampling at 1361C, 2 bar for 20 min, so that (a) the effect of rotifers can be measured in the absence of other live biological organisms; (b) the sludge is partially disintegrated, which generates many small particles and accentuates the effects being demonstrated; and (3) any associated biohazard is removed. 2.2. Rotifer culture Bdelloid rotifers of the genus Philodina were obtained from Jones Biomedicals & Laboratory. Rotifer clone P1 (Philodina roseola) was established from one individual, selected by micropipette manipulation. Rotifers were cultured in an aerated 10 l plastic container containing filtered (charcoal filter, Liff Industries Ltd., model no. N-SK13, and Nalgene 0.2 mm PES filter) tap water, which was changed weekly. Rotifers were fed Escherichia coli, which had been grown in Luria broth (LB) medium overnight, recovered by centrifugation and resuspended in distilled water. The bacterial suspensions could be stored at 41C for up to a week. Rotifers were harvested by increasing the salt concentration of the tank to 0.1 M NaCl, which causes the animals to detach from the tank walls. The medium was subsequently filtered through a Nitex nylon mesh of 20 mm pore size (Precision Textiles Ltd.), from which rotifers were transferred to tissue culture flasks (Nunc, 1-78883A) after washing several times with distilled water. The total number of rotifers in a flask was estimated by counting animals in areas of known size in five different randomly selected spots of the flask.
2.3. Wastewater particle size analysis Pictures were taken using a Leitz Ortholux II fluorescent microscope, a JVC TK-C1381 video camera and a Sony DSR-20P digital video recorder connected to an Apple Macintosh G4 computer. Pictures were captured using Adobe Premiere 5.1 and were exported to NIH Image 1.62. Particle size was calibrated using 1 mm fluorescent polystyrene beads (micromer blueF plain, Micromod GmbH). The size of particles from turbid supernatants of autoclaved activated sludge was analysed using the calibrated system. 2.4. Rotifer ingested particle size range Individuals of the rotifer clone Philodina roseola P1 were sampled and fed for 3 days on suspensions of fluorescent polystyrene particles (micromer blueF plain, Micromod) of the sizes 0.2, 1, 3, 5, 7 or 10 mm in a multiwell culture plate. Animals were observed and photographed with the Ortholux microscope (see above; filter A, excitation: 340–380 nm, band pass >430 nm). Where particles of a particular size were successfully ingested, fluorescence was clearly visible in the gut of the animal. 2.5. Laboratory-scale mini reactor Wastewater was introduced into Duran bottles, which were sealed with special bottle cap assemblies (Anachem, A-610). The wastewater was aerated, using an aquarium air pump, dispersing the air through a solvent inlet filter (Anachem, A-310). The air was sterile-filtered using Hepa-Vent 0.3 mm glass microfibre filters at the air in- and outlet points. The reactors were operated with wastewater volumes of either 400 or 900 ml and with light aeration. Samples were taken in a flow cabinet to prevent contamination. In reactors operated with activated sludge samples, the antibiotics ampicillin and kanamycin were added at 100 mg/ml to prevent growth of E. coli, on which rotifers are fed. Prevention of bacterial growth was confirmed after plating reactor samples on plain LB agar and incubation at 371C overnight. The addition of antibiotics was not necessary in reactors with Kaldnes effluent, as bacterial growth was extremely limited under these conditions. Rotifers of the genus Philodina were added from tissue culture flasks containing a known number of organisms under sterile conditions. Optical density at 600 nm (OD600), measured in a Jasco 7800 Spectrophotometer, was used to determine wastewater turbidity. Five 1 ml samples were measured and the standard deviation calculated. Total suspended solids (TSS) were collected using 1.2 mm pore size glass microfibre filters (Whatman, Cat. No. 1822 047). The filters were initially heated at 1051C overnight in a Gallenkamp Hotbox oven, then cooled to room
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3. Results
0.200 No rotifer control
0.175
Optical density at 600nm
temperature in a desiccator over dried silica gel. The weight of each filter was determined individually in a four figure Mettler AE 163 precision balance and then used to filter 30 ml of wastewater. Filters were then reincubated at 1051C overnight and the weight redetermined.
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2,000 rotifers/l 10,000 rotifers/l
0.150
50,000 rotifers/l
0.125 0.100 0.075 0.050 0.025 0.000
3.1. Bdelloid rotifers improve wastewater clarification
0
20
40
60
80
100
Time [h]
Wastewater from a Kaldnes wastewater treatment plant, an aerobic suspended carrier process for increased build-up of bio-film, was collected prior to the clarifier and autoclaved. Autoclaving sterilised the samples and produced a suspension of many small sludge particles similar to pin flocs. The effluent was operated in parallel in two lightly aerated batch mini-reactors. One reactor was used as a control, the other was inoculated with B50,000 rotifer/l of the genus Philodina. The optical density of the suspension was measured at 600 nm (OD600) periodically and the experiment run for 67.5 h (see Fig. 1). In the control reactor without rotifers, OD600 dropped from 0.158 to 0.099, indicating a degree of clarification of B37%, due to particle settling during the incubation period. In comparison, the reactor with 50,000 rotifers/l showed an improved clarification: OD600 dropped from an initial value of 0.154 to a value of 0.006, representing particle removal of 96%. If this improved clarification is due to the rotifers, it should be possible to correlate the degree of clarification with the numbers of rotifers inoculated. Therefore, four identical reactors containing Kaldnes wastewater were run in parallel with rotifers at densities of 0, 2000, 10,000 and 50,000 rotifers/l, respectively, over a period of 88.5 h (Fig. 2). Initial OD600 values were in the range 0.150– 0.183. The density of the suspension in all four reactors
Optical density at 600nm
0.18 0.16
No rotifer control 50,000 rotifers/l
0.14 0.12 0.10 0.08 0.06 0.04 0.02 0 0
20
40
60
80
Time [h]
Fig. 1. Wastewater clarification using bdelloid rotifers. Turbidity of wastewater due to particles in suspension was measured over time in the presence or absence of 50,000 rotifers/l as indicated by the key. 7SD is indicated by vertical bars.
Fig. 2. Clarification depends on rotifer concentration. Wastewater turbidity, as indicated by optical density at 600 nm, was measured over time in the presence of rotifer populations of varying density. 7SD is indicated by vertical bars.
decreased significantly with time, but for the reactor with the highest rotifer density (50,000 rotifers/l), almost all particles were removed from suspension within 48 h. In comparison, OD600 in the control reactor without rotifers dropped by only 39% in 48 h, compared to the starting value. The OD600 in the reactors with 2000 rotifers/l and 10,000 rotifers/l dropped to 0.068 and 0.048, respectively, within 48 h, thus showing corresponding intermediate degrees of clarification. This experiment indicates that rotifers were indeed involved in particle removal and that the effect depends on the numbers of rotifers inoculated. 3.2. Clarification requires live rotifers It is conceivable that rotifers improve clarification of wastewater due to several factors. An experiment was performed to exclude the possibility that rotifers simply function passively as flocculants, without a requirement for an active process. Rotifers were exposed to a brief burst of microwave radiation (10 s, 800 W), resulting in a killing rate of 100%, but without apparent structural damage. Three mini-reactors containing Kaldnes wastewater were set-up, one with 50,000 live rotifers/l, one with 50,000 dead rotifers/l and one without rotifers. OD600 was measured over a period of 44 h (Fig. 3). Initial OD600 in all three reactors was determined to be in the range 0.145–0.157. After 44 h, only the reactor containing live rotifers showed a significantly reduced optical density when compared to the control reactor without rotifers. The absorption was reduced to 0.006 compared to 0.078 in the control and to 0.106 in the dead rotifer reactor. This demonstrates a 96% clarification of particles in suspension in the live rotifer reactor, whereas the control and the dead rotifer reactors only showed clarification of 50% and 27%, respectively, compared with their starting values. This experiment shows that live rotifers are required for effective particle removal.
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3.3. Suspended wastewater particles are ingestible by rotifers
of particles were in the range up to 1.5 mm, although larger particles were occasionally observed (Fig. 4). To confirm that the smaller particles counted in the analysis were the cause of turbidity, filtration experiments were performed on wastewater. An autoclaved Kaldnes wastewater sample was passed through filters with pore size of 11 or 0.2 mm and OD600 of the filtrate was measured. Prior to filtration, the sample had an OD600 of 0.238 (70.002), which was reduced to 0.176 (70.001) by filtration through the 11 mm filter. This showed that at least 74% of the turbidity of the wastewater was caused by particles smaller than 11 mm, which corresponds well with the observation of a high number of small suspended particles in such wastewaters. The OD600 of the filtrate was further reduced to 0.012 (70.001) by filtration through a 0.2 mm filter, representing a reduction of 95%. This demonstrated that the turbidity of the sample was largely caused by suspended particles in the range up to 11 mm and not by particles outside this range. This agrees with the wellcharacterised observed and theoretical size range of particles in colloidal suspension. The size range of particles ingested by rotifers was determined by feeding with a suspension of mono-sized blue fluorescent polystyrene beads of 0.2, 1, 3, 5, 7 or 10 mm in water. The rotifers were then observed under a fluoresence microscope. When rotifers were able to feed upon the particles, the polystyrene beads in the rotifer gut were excited by UV light and the gut fluoresced with a blue colour (Fig. 5). A control experiment without fluorescent beads confirmed that rotifers do not have a natural fluorescence at that wavelength. Rotifers were able to feed comfortably on polystyrene beads of the sizes 0.2, 1 and 3 mm. Furthermore, these rotifers were alive and continued to reproduce, as the egg in the
A question arises as to whether suspended particles removed from wastewaters are actually consumed by the rotifers or whether some other activity leads to particle aggregation and improved settling. One way of answering this is to determine the size of particles that are in suspension in wastewaters and to compare this to the particle size feeding range of rotifers. If rotifers are physically unable to ingest the particles in suspension, removal of particles by this route can be ruled out. The size of unsettled wastewater (activated sludge) particles in suspension was determined, using video microscopy. Images of suspended particles were obtained and analysed by computer, using mono-sized polystyrene particles as a standard. Five different images with a total of 350 particles were analysed. The vast majority (94%)
Optical density at 600nm
0.200 No rotifer control
0.175
Dead rotifers (50,000/l)
0.150
Live rotifers (50,000/l)
0.125 0.100 0.075 0.050 0.025 0.000 0
10
20
30
40
50
Time [h]
Fig. 3. Clarification requires live rotifers. Turbidity of wastewater was measured in the presence of live rotifers (50,000/l) or rotifers which had been killed, by brief exposure to microwaves, at the same density. 7SD is indicated by vertical bars.
Percentage of total particles
40 35 30 25 20 15 10 5 0 0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
2.75
3
10
20
Size range of particles [µm] Fig. 4. Size range distribution of particles suspended in wastewater. The figure quoted below each bar indicates the maximum extent of the size range; the minimum extent of the size range is defined by the figure below the previous bar. Thus, ‘‘1.5’’ means the range 1.25– 1.5 mm.
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rotifer shown in Fig. 5 indicates. With the larger fluorescent particles, settling out was more rapid, making feeding by rotifers more difficult. Also, only the larger, mature rotifers were able to ingest particles 5 mm or larger in size. However, together these results demonstrate that rotifers are able to efficiently ingest particles in the range 0.2–3 mm, which correlates with more than 95% of the suspended particles in wastewater (Fig. 4) and have more limited access to particles up to 10 mm in size. It can therefore be concluded that rotifers are able to ingest most particles suspended in Kaldnes wastewater and that this is likely to be a major route for particle removal. However, this does not prove that rotifers actually consume wastewater
Fig. 5. Bdelloid rotifers ingest polystyrene particles. Negative image of Philodina roseola, fed with 1 mm mono-sized polystyrene beads. The coloured stripe represents fluorescing particles in the gut. An egg is visible within the animal, indicating continued reproduction. The rotifer has been outlined for clarity.
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biomass, rather than simply ingesting it. Neither does it answer the question of whether rotifers also affect wastewater clarity by enhancing particle settleability. 3.4. Rotifers remove biomass from suspension by two routes To address these issues, experiments were performed which measured changes in the amount of biomass in the system. The biomass present in wastewaters is usually expressed as TSS. For this work, wastewater from a conventional activated sludge plant, rather than from the Kaldnes process, was used, since TSS of samples from the latter process was too low for accurate weight measurements. If rotifers feed on particles in wastewater, a reduction in biomass of the system, due to partial mineralisation, should be observed. However, the amount of solids in suspension will depend on the degree of agitation of the wastewater sample. In the following experiments, therefore, TSS was measured either with or without stirring at sampling times. This also allowed information to be obtained on whether rotifers influence settling of particles in suspension. Activated sludge was sampled, autoclaved and the supernatant transferred into four mini-reactors. Two of these mini-reactors, one seeded with 100,000 rotifers/l, the other without rotifers, were not disturbed prior to sampling for TSS and OD600 analysis. The two other mini-reactors, again with or without rotifers at 100,000/ l, were mixed vigorously prior to sampling, using a magnetic stirrer. In the latter two reactors, total biomass was measured, including that which had settled out. The experiment was operated for 48 h and results are shown in Table 1.
Table 1 Biomass reduction using 100,000 rotifers/l Optical density at 600 nm Start
End
DOD600
Control
Undisturbed Stirred
0.28870.002 0.29870.002
0.25270.005 0.28470.001
13% 5%
Rotifers
Undisturbed Stirred
0.28670.003 0.30170.004
0.19170.002 0.24270.002
33% 20%
Start
End
Total suspended solids (mg/l) DTSS
Control
Undisturbed Stirred
12872 13172
10673 13174
18% 0%
Rotifers
Undisturbed Stirred
13373 14672
8275 13073
38% 11%
The experiment was run for 48 h: OD600 values are an average of five samples, TSS values are an average of three samples.
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At the start of the experiment, all reactors had an OD600 within the range 0.286–0.301 and TSS ranged from 128 to 146 mg/l (Table 1). During the course of the experiment, OD600 of the control reactors decreased by 13% for the undisturbed and by 5% for the stirred reactor. In comparison, the reactors containing 100,000 rotifers/l showed a reduction in OD600 of 33% for the undisturbed reactor and of 20% for the stirred reactor. A similar result was observed for solids in suspension. The controls showed a decrease in TSS of 18% for the undisturbed reactor and no change in the stirred reactor. In contrast, TSS in the rotifer reactor decreased by 38% in the undisturbed and by 11% in the stirred reactor. As measurements of stirred reactor samples indicate the total biomass in the system, it can be concluded that the total biomass in the reactor containing rotifers was reduced by B11%, most likely due to its consumption as a food source. In addition, rotifers augmented the natural settling process of suspended biomass by B10%, possibly due to a flocculation effect. In a second, similar experiment, 200,000 rotifers/l were added to activated sludge and incubated for 24 h. TSS was decreased by B9% in the stirred reactor containing rotifers, compared with no significant change of TSS in the stirred control reactor. In the unstirred control reactor, TSS was decreased only insignificantly, but was decreased by 34% in the reactor containing rotifers (data not shown). This indicates removal of biomass of B9% due to consumption by rotifers and removal of B25% of biomass, due to the enhanced settleability they confer. Both experiments suggest a rate of consumption of biomass by 100,000 rotifers/l of 9–11% within 48 h and an augmented settling effect on 10–25% of TSS within that time period.
4. Discussion Bdelloid rotifers have been shown here to have a significant impact on wastewater by improving removal of unsettled particles from suspension. It was shown that particles in suspension are mainly in the size range 0.2– 10 mm, which corresponds with the size range of particles consumed by rotifers. These particles are removed from suspension by rotifers through a twofold mechanism. First, rotifers were shown to graze on small, suspended particles and to consume them. The total biomass of a system in which rotifers graze on suspended particles is subsequently reduced by B10% (TSS) within 48 h at the rotifer density used. Consistent with this, a preliminary experiment suggested total solids removal is also significantly reduced by rotifer grazing (data not shown). Second, particles were removed by an apparent ability of rotifers to function as facilitators of floc formation. Small particles were most likely aggregated into larger flocs, which settled out of suspension. This
effect reduced the total biomass in suspension by an additional 10–25%. Thus, rotifers were shown to reduce sludge in the system and to improve the clarity of wastewater through enhanced particle settling. The observation that rotifers feed actively on suspended particles is supported by the experiments of Vadstein et al. [11]. The monogonont rotifer Brachionus plicatilis was shown to be able to remove all tested particles, ranging in size from 0.3 to 3 mm, with an optimum particle feeding size of B2 mm. A similar species, Brachionus calyciflorus, showed active particle removal of silica beads of 3–6 mm [12]. These values are within the confirmed feeding range of Philodina roseola and support the assumption that rotifers feed on all suspended particles within a certain size range. Although the particles which were used in the rotifer clarification experiments were in part created by autoclaving wastewater samples, the unsettled particles are in the same size range of particles observed in natural effluents. Atteia et al. [13] report that suspended particles in samples from a brook which drains peat areas, are in the size range of 0.5–10 mm. More than 90% of the suspended particles in effluents from trickling filters have been reported to be smaller than 30 mm [6]. As rotifers can be found in all aerobic wastewater treatment processes [14], it can be assumed that they naturally feed on suspended particles of these sizes and under these conditions. Increasing rotifer numbers in the system could therefore prove to be an economical and sustainable method for improving effluent clarity. Chemical flocculants are used in wastewater treatment processes to improve clarification of the final effluent. Addition of flocculants increases the operating costs of wastewater treatment facilities due to purchase costs and the resultant increase of waste sludge produced by the system [15]. Other methods of improving effluent clarity, such as sand filters, are costly to build and maintain. Our experiments indicate that rotifers function as a bioflocculant in wastewater. Enhanced floc formation by bdelloid rotifers is probably due to a secreted substance that rotifers use to anchor themselves to the substrate. The foot of bdelloid rotifers contains a series of pedal glands, also referred to as cement glands [8]. These glands secrete a mucous glue, which enables rotifers to attach to surfaces. Cement glands are used by a variety of organisms for substrate anchorage under water. Analysis of the cement composition of Acanthocephalans, the sister phylum of Rotifera, showed it to consists of proteinaceous material, the main protein constituent having a size of 23 kDa [16]. The sticky substance secreted by rotifers may also be proteinaceous and would probably function as a flocculant. Several technologies have been suggested for the purpose of sludge reduction to date. One strategy is to inhibit the build up of sludge flocs. Low and Chase [17] reviewed the use of chemical or biological strategies for
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uncoupling metabolism from growth in wastewater microorganisms. A second approach is sludge disintegration, which will release cell contents and disrupt flocs into smaller particles that are more accessible as food sources to wastewater microorganisms. Wet sludge disintegration techniques used so far include mechanical, chemical, thermo-chemical, biological and oxidative methods. Most uncoupling or disintegration technologies have an unfavourable cost:benefit ratio and are not widely regarded as economically viable (reviewed by Weemaes and Verstraete [18]). A third strategy for sludge reduction is to increase the number of predators within the system. Oligochaete worms (Tubificidae) have been shown to reduce biomass production in trickling filters and activated sludge plants, but increased the numbers of small particles in suspension in the final effluent [19,20]. Welander et al. [21] showed that predator grazing in a two-stage activated sludge process had a marked effect on biomass production. A first stage reactor without biomass recycling produces bacteria, which are consumed in the second stage, where ciliates and metazoa graze on the bacteria. This process is also referred to as the low sludge production (LSP) process. The LSP process has been successfully implemented at a pulp mill in Folla, Sweden, where the sludge production of the wastewater treatment plant was lowered by 70–90% [21]. The LSP process works best with relatively warm wastewater and requires a high concentration of microbial nutrients in the influent, or nutrient addition at the first stage. Municipal wastewater contains low concentrations of soluble, readily biodegradable matter and the effectiveness of a municipal LSP process is probably reduced, compared with a plant treating industrial wastewater, particularly at the low ambient temperatures prevalent in some countries. Inamori and colleagues have used rotifers, together with other bacterial, algal and metazoan species found naturally in wastewater treatment plants, to form a microcosm which provides a laboratory model of naturally occurring ecosystems (e.g. [22]). Like the LSP process, this illustrates the ability of rotifers to thrive in systems where they are able to predate bacteria or algae. The use of rotifers for the treatment of wastewater was suggested previously [23,24]: a mix of agricultural wastes is subjected to an algae/bacteria reactor, where COD is converted to cellular biomass (algae), which is subsequently grazed by rotifers in a second stage predator reactor. In this report, we demonstrate the ability of bdelloid rotifers not only to feed on algae or bacteria suspended in wastewater, but also on the sludge particles themselves. Rotifers inoculated into particulate wastewater survive and multiply over periods of many weeks (J.L. and A.T., unpublished). We envisage the use of rotifers as an adjunct to conventional activated sludge processes, either in a separate predator tank or within an
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existing aeration tank. One problem in encouraging rotifer growth may be the availability of particles in the size range ingestible by rotifers. This might be overcome by partial disintegration of sludge particles prior to a rotifer grazing stage, combining limited sludge disintegration with rotifer predation and might prove an efficient way of reducing sludge production in municipal wastewater treatment plants. Furthermore, rotifers might also be used as ‘‘bioclarifiers’’ to improve the clarity of the final effluent.
5. Conclusions Bdelloid rotifers have been shown to remove wastewater particles from suspension, partially by consumption and partially by improving settleability. Their beneficial effect on biomass yield and wastewater clarity suggests that rotifers might be used as biotechnological tools in wastewater processing, either through encouraging their growth in existing wastewater plants, or through modification to include a specific rotifer predation/clarification module.
Acknowledgements We would like to thank Jonathan Strickland, Jo Callan, Matt Griffiths, Keith Edwards and Steve Kaye of AWG plc for their stimulating discussions and provision of wastewater samples. J.L. holds the AWG Research Studentship; A.T. is the AWG Senior Research Fellow of Pembroke College, Cambridge.
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[17] Low EW, Chase HA. Reducing production of excess biomass during wastewater treatment. Water Res 1999; 33:1119–32. [18] Weemaes MPJ, Verstraete WH. Evaluation of current wet sludge disintegration techniques. J Chem Technol Biotechnol 1998;73:83–92. [19] Rensink JH, Rulkens WH. Using metazoa to reduce sludge production. Water Sci Technol 1997;36:171–9. [20] Lee NM, Welander T. Reducing sludge production in aerobic wastewater treatment through manipulation of the ecosystem. Water Res 1996;30:1781–90. [21] Welander T, Alexandersson T, Ericsson T, Gunnarsson L, Storlie A. Reducing sludge production in biological effluent treatment by applying the LSP process. Proceedings of the 2000 TAPPI International Environment Conference Exhibition, 6–10 May 2000, Denver, Book 2, 2000. p. 757–64. [22] Tanaka N, Inamori Y, Murakami K, Akamatsu T, Kurihara Y. Effect of species composition on stability and reproducibility of a small-scale microcosm system. Water Sci Technol 1994;30:125–31. [23] Groeneweg J, Schluter . M. Method of treating liquid agricultural wastes. United States Patent 4,348,285, 1982. [24] Groeneweg J, Schluter . M. Method of treating agricultural wastes. United States Patent, 4,432,869, 1984.
Reduction of suspended biomass in municipal wastewater using bdelloid rotifers J. Lapinski, A. Tunnacliffe* Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK Received 24 April 2002; accepted 2 December 2002
Abstract Clarification of municipal wastewater was shown to be improved significantly by the addition of cultured bdelloid rotifers. The rate and degree of suspended particle removal were correlated with rotifer number. The size range of unsettled particles suspended in wastewater was determined and found to overlap with the size range of particles consumed by rotifers. Rotifers were shown to have two distinct effects on suspended particles: consumption of biomass due to feeding activity; and improved settling, probably due to enhanced aggregation. These experiments demonstrate the potential for the use of bdelloid rotifers in an enhanced wastewater treatment process, with reduced biomass production and improved effluent clarity. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bdelloid rotifers; Biomass reduction; Activated sludge; Metazoa; Total suspended solids; Clarification
1. Introduction One of the most widely used methods of wastewater treatment is the activated sludge process1 [1]. Although initially invented almost a century ago [2], the process has essentially remained the same throughout the intervening period. One of the main by-products of the activated sludge process is waste sludge, whose disposal accounts for B60% of operating costs in the East Anglian region of the United Kingdom and are similarly high elsewhere [3]. However, this cost is rising as disposal routes for sludge are becoming more restricted. Dumping at sea, which previously accounted for 30% of sludge disposed, is now illegal and use of waste sludge on agricultural land is increasingly under threat due to *Corresponding author. Tel.: +44-1223-766549; fax: +441223-334162. E-mail address: [email protected] (A. Tunnacliffe). 1 Griffiths M. AWG plc, Thorpe Wood House, Peterborough, Cambridgeshire, PE3 6WT, UK. E-mail: mailto:mgriffiths@ anglianwater.co.uk
consumer pressure [4]. As a result, the cost of waste sludge disposal is likely to continue to increase significantly. New methods of reducing sludge production, together with new disposal routes, are therefore urgently required. Other wastewater treatment processes, such as the trickling filter or the Kaldnes suspended carrier process [5] also have their attendant problems, e.g. the frequent occurrence of pin flocs, small sludge particles, which do not settle in the clarifier. These pin flocs are carried over into the effluent water, which can result in discharge limits for solids being exceeded [6]. Novel technologies that reduce the production of pin flocs, would therefore be of benefit to the water industry. Bdelloid rotifers (‘‘leech-like wheel-bearers’’) are metazoans of B300 mm length which are ubiquitous in most fresh- and wastewater habitats [7]. Their name stems from the fact that most species possess a corona of cilia around the mouth opening that beat sequentially to produce a vortex of water. The vortex draws particles suspended in water into the rotifer’s mouth [8]. It has been suggested that rotifers play an important role in the activated sludge process, functioning as nuclei for floc
0043-1354/03/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0043-1354(02)00626-7
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formation [9]. Furthermore, it has been stated that rotifer population growth is mainly limited by the amount of food available [10] and that they consume several times their body weight per day [8]. In principle therefore, rotifers might be exploited for removal of suspended bio-solids from wastewater, either by grazing on particulate matter or by enhanced ‘‘bioflocculation’’ or a combination of both. Here we explore the potential for the use of bdelloid rotifers as a biotechnological tool to improve effluent clarity and to decrease biomass yield in municipal wastewater.
2. Materials and methods 2.1. Wastewater Samples were taken from the AWG (formerly Anglian Water) plc. domestic wastewater treatment plant in Cambridge near Milton (UK). Effluent samples from the Kaldnes (K1) ‘‘Monster’’ moving bed reactor plant were taken before the clarifier stage. Activated sludge samples were taken from the aerobic zone of the main activated sludge plant. All samples were autoclaved immediately after sampling at 1361C, 2 bar for 20 min, so that (a) the effect of rotifers can be measured in the absence of other live biological organisms; (b) the sludge is partially disintegrated, which generates many small particles and accentuates the effects being demonstrated; and (3) any associated biohazard is removed. 2.2. Rotifer culture Bdelloid rotifers of the genus Philodina were obtained from Jones Biomedicals & Laboratory. Rotifer clone P1 (Philodina roseola) was established from one individual, selected by micropipette manipulation. Rotifers were cultured in an aerated 10 l plastic container containing filtered (charcoal filter, Liff Industries Ltd., model no. N-SK13, and Nalgene 0.2 mm PES filter) tap water, which was changed weekly. Rotifers were fed Escherichia coli, which had been grown in Luria broth (LB) medium overnight, recovered by centrifugation and resuspended in distilled water. The bacterial suspensions could be stored at 41C for up to a week. Rotifers were harvested by increasing the salt concentration of the tank to 0.1 M NaCl, which causes the animals to detach from the tank walls. The medium was subsequently filtered through a Nitex nylon mesh of 20 mm pore size (Precision Textiles Ltd.), from which rotifers were transferred to tissue culture flasks (Nunc, 1-78883A) after washing several times with distilled water. The total number of rotifers in a flask was estimated by counting animals in areas of known size in five different randomly selected spots of the flask.
2.3. Wastewater particle size analysis Pictures were taken using a Leitz Ortholux II fluorescent microscope, a JVC TK-C1381 video camera and a Sony DSR-20P digital video recorder connected to an Apple Macintosh G4 computer. Pictures were captured using Adobe Premiere 5.1 and were exported to NIH Image 1.62. Particle size was calibrated using 1 mm fluorescent polystyrene beads (micromer blueF plain, Micromod GmbH). The size of particles from turbid supernatants of autoclaved activated sludge was analysed using the calibrated system. 2.4. Rotifer ingested particle size range Individuals of the rotifer clone Philodina roseola P1 were sampled and fed for 3 days on suspensions of fluorescent polystyrene particles (micromer blueF plain, Micromod) of the sizes 0.2, 1, 3, 5, 7 or 10 mm in a multiwell culture plate. Animals were observed and photographed with the Ortholux microscope (see above; filter A, excitation: 340–380 nm, band pass >430 nm). Where particles of a particular size were successfully ingested, fluorescence was clearly visible in the gut of the animal. 2.5. Laboratory-scale mini reactor Wastewater was introduced into Duran bottles, which were sealed with special bottle cap assemblies (Anachem, A-610). The wastewater was aerated, using an aquarium air pump, dispersing the air through a solvent inlet filter (Anachem, A-310). The air was sterile-filtered using Hepa-Vent 0.3 mm glass microfibre filters at the air in- and outlet points. The reactors were operated with wastewater volumes of either 400 or 900 ml and with light aeration. Samples were taken in a flow cabinet to prevent contamination. In reactors operated with activated sludge samples, the antibiotics ampicillin and kanamycin were added at 100 mg/ml to prevent growth of E. coli, on which rotifers are fed. Prevention of bacterial growth was confirmed after plating reactor samples on plain LB agar and incubation at 371C overnight. The addition of antibiotics was not necessary in reactors with Kaldnes effluent, as bacterial growth was extremely limited under these conditions. Rotifers of the genus Philodina were added from tissue culture flasks containing a known number of organisms under sterile conditions. Optical density at 600 nm (OD600), measured in a Jasco 7800 Spectrophotometer, was used to determine wastewater turbidity. Five 1 ml samples were measured and the standard deviation calculated. Total suspended solids (TSS) were collected using 1.2 mm pore size glass microfibre filters (Whatman, Cat. No. 1822 047). The filters were initially heated at 1051C overnight in a Gallenkamp Hotbox oven, then cooled to room
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3. Results
0.200 No rotifer control
0.175
Optical density at 600nm
temperature in a desiccator over dried silica gel. The weight of each filter was determined individually in a four figure Mettler AE 163 precision balance and then used to filter 30 ml of wastewater. Filters were then reincubated at 1051C overnight and the weight redetermined.
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2,000 rotifers/l 10,000 rotifers/l
0.150
50,000 rotifers/l
0.125 0.100 0.075 0.050 0.025 0.000
3.1. Bdelloid rotifers improve wastewater clarification
0
20
40
60
80
100
Time [h]
Wastewater from a Kaldnes wastewater treatment plant, an aerobic suspended carrier process for increased build-up of bio-film, was collected prior to the clarifier and autoclaved. Autoclaving sterilised the samples and produced a suspension of many small sludge particles similar to pin flocs. The effluent was operated in parallel in two lightly aerated batch mini-reactors. One reactor was used as a control, the other was inoculated with B50,000 rotifer/l of the genus Philodina. The optical density of the suspension was measured at 600 nm (OD600) periodically and the experiment run for 67.5 h (see Fig. 1). In the control reactor without rotifers, OD600 dropped from 0.158 to 0.099, indicating a degree of clarification of B37%, due to particle settling during the incubation period. In comparison, the reactor with 50,000 rotifers/l showed an improved clarification: OD600 dropped from an initial value of 0.154 to a value of 0.006, representing particle removal of 96%. If this improved clarification is due to the rotifers, it should be possible to correlate the degree of clarification with the numbers of rotifers inoculated. Therefore, four identical reactors containing Kaldnes wastewater were run in parallel with rotifers at densities of 0, 2000, 10,000 and 50,000 rotifers/l, respectively, over a period of 88.5 h (Fig. 2). Initial OD600 values were in the range 0.150– 0.183. The density of the suspension in all four reactors
Optical density at 600nm
0.18 0.16
No rotifer control 50,000 rotifers/l
0.14 0.12 0.10 0.08 0.06 0.04 0.02 0 0
20
40
60
80
Time [h]
Fig. 1. Wastewater clarification using bdelloid rotifers. Turbidity of wastewater due to particles in suspension was measured over time in the presence or absence of 50,000 rotifers/l as indicated by the key. 7SD is indicated by vertical bars.
Fig. 2. Clarification depends on rotifer concentration. Wastewater turbidity, as indicated by optical density at 600 nm, was measured over time in the presence of rotifer populations of varying density. 7SD is indicated by vertical bars.
decreased significantly with time, but for the reactor with the highest rotifer density (50,000 rotifers/l), almost all particles were removed from suspension within 48 h. In comparison, OD600 in the control reactor without rotifers dropped by only 39% in 48 h, compared to the starting value. The OD600 in the reactors with 2000 rotifers/l and 10,000 rotifers/l dropped to 0.068 and 0.048, respectively, within 48 h, thus showing corresponding intermediate degrees of clarification. This experiment indicates that rotifers were indeed involved in particle removal and that the effect depends on the numbers of rotifers inoculated. 3.2. Clarification requires live rotifers It is conceivable that rotifers improve clarification of wastewater due to several factors. An experiment was performed to exclude the possibility that rotifers simply function passively as flocculants, without a requirement for an active process. Rotifers were exposed to a brief burst of microwave radiation (10 s, 800 W), resulting in a killing rate of 100%, but without apparent structural damage. Three mini-reactors containing Kaldnes wastewater were set-up, one with 50,000 live rotifers/l, one with 50,000 dead rotifers/l and one without rotifers. OD600 was measured over a period of 44 h (Fig. 3). Initial OD600 in all three reactors was determined to be in the range 0.145–0.157. After 44 h, only the reactor containing live rotifers showed a significantly reduced optical density when compared to the control reactor without rotifers. The absorption was reduced to 0.006 compared to 0.078 in the control and to 0.106 in the dead rotifer reactor. This demonstrates a 96% clarification of particles in suspension in the live rotifer reactor, whereas the control and the dead rotifer reactors only showed clarification of 50% and 27%, respectively, compared with their starting values. This experiment shows that live rotifers are required for effective particle removal.
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3.3. Suspended wastewater particles are ingestible by rotifers
of particles were in the range up to 1.5 mm, although larger particles were occasionally observed (Fig. 4). To confirm that the smaller particles counted in the analysis were the cause of turbidity, filtration experiments were performed on wastewater. An autoclaved Kaldnes wastewater sample was passed through filters with pore size of 11 or 0.2 mm and OD600 of the filtrate was measured. Prior to filtration, the sample had an OD600 of 0.238 (70.002), which was reduced to 0.176 (70.001) by filtration through the 11 mm filter. This showed that at least 74% of the turbidity of the wastewater was caused by particles smaller than 11 mm, which corresponds well with the observation of a high number of small suspended particles in such wastewaters. The OD600 of the filtrate was further reduced to 0.012 (70.001) by filtration through a 0.2 mm filter, representing a reduction of 95%. This demonstrated that the turbidity of the sample was largely caused by suspended particles in the range up to 11 mm and not by particles outside this range. This agrees with the wellcharacterised observed and theoretical size range of particles in colloidal suspension. The size range of particles ingested by rotifers was determined by feeding with a suspension of mono-sized blue fluorescent polystyrene beads of 0.2, 1, 3, 5, 7 or 10 mm in water. The rotifers were then observed under a fluoresence microscope. When rotifers were able to feed upon the particles, the polystyrene beads in the rotifer gut were excited by UV light and the gut fluoresced with a blue colour (Fig. 5). A control experiment without fluorescent beads confirmed that rotifers do not have a natural fluorescence at that wavelength. Rotifers were able to feed comfortably on polystyrene beads of the sizes 0.2, 1 and 3 mm. Furthermore, these rotifers were alive and continued to reproduce, as the egg in the
A question arises as to whether suspended particles removed from wastewaters are actually consumed by the rotifers or whether some other activity leads to particle aggregation and improved settling. One way of answering this is to determine the size of particles that are in suspension in wastewaters and to compare this to the particle size feeding range of rotifers. If rotifers are physically unable to ingest the particles in suspension, removal of particles by this route can be ruled out. The size of unsettled wastewater (activated sludge) particles in suspension was determined, using video microscopy. Images of suspended particles were obtained and analysed by computer, using mono-sized polystyrene particles as a standard. Five different images with a total of 350 particles were analysed. The vast majority (94%)
Optical density at 600nm
0.200 No rotifer control
0.175
Dead rotifers (50,000/l)
0.150
Live rotifers (50,000/l)
0.125 0.100 0.075 0.050 0.025 0.000 0
10
20
30
40
50
Time [h]
Fig. 3. Clarification requires live rotifers. Turbidity of wastewater was measured in the presence of live rotifers (50,000/l) or rotifers which had been killed, by brief exposure to microwaves, at the same density. 7SD is indicated by vertical bars.
Percentage of total particles
40 35 30 25 20 15 10 5 0 0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
2.75
3
10
20
Size range of particles [µm] Fig. 4. Size range distribution of particles suspended in wastewater. The figure quoted below each bar indicates the maximum extent of the size range; the minimum extent of the size range is defined by the figure below the previous bar. Thus, ‘‘1.5’’ means the range 1.25– 1.5 mm.
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rotifer shown in Fig. 5 indicates. With the larger fluorescent particles, settling out was more rapid, making feeding by rotifers more difficult. Also, only the larger, mature rotifers were able to ingest particles 5 mm or larger in size. However, together these results demonstrate that rotifers are able to efficiently ingest particles in the range 0.2–3 mm, which correlates with more than 95% of the suspended particles in wastewater (Fig. 4) and have more limited access to particles up to 10 mm in size. It can therefore be concluded that rotifers are able to ingest most particles suspended in Kaldnes wastewater and that this is likely to be a major route for particle removal. However, this does not prove that rotifers actually consume wastewater
Fig. 5. Bdelloid rotifers ingest polystyrene particles. Negative image of Philodina roseola, fed with 1 mm mono-sized polystyrene beads. The coloured stripe represents fluorescing particles in the gut. An egg is visible within the animal, indicating continued reproduction. The rotifer has been outlined for clarity.
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biomass, rather than simply ingesting it. Neither does it answer the question of whether rotifers also affect wastewater clarity by enhancing particle settleability. 3.4. Rotifers remove biomass from suspension by two routes To address these issues, experiments were performed which measured changes in the amount of biomass in the system. The biomass present in wastewaters is usually expressed as TSS. For this work, wastewater from a conventional activated sludge plant, rather than from the Kaldnes process, was used, since TSS of samples from the latter process was too low for accurate weight measurements. If rotifers feed on particles in wastewater, a reduction in biomass of the system, due to partial mineralisation, should be observed. However, the amount of solids in suspension will depend on the degree of agitation of the wastewater sample. In the following experiments, therefore, TSS was measured either with or without stirring at sampling times. This also allowed information to be obtained on whether rotifers influence settling of particles in suspension. Activated sludge was sampled, autoclaved and the supernatant transferred into four mini-reactors. Two of these mini-reactors, one seeded with 100,000 rotifers/l, the other without rotifers, were not disturbed prior to sampling for TSS and OD600 analysis. The two other mini-reactors, again with or without rotifers at 100,000/ l, were mixed vigorously prior to sampling, using a magnetic stirrer. In the latter two reactors, total biomass was measured, including that which had settled out. The experiment was operated for 48 h and results are shown in Table 1.
Table 1 Biomass reduction using 100,000 rotifers/l Optical density at 600 nm Start
End
DOD600
Control
Undisturbed Stirred
0.28870.002 0.29870.002
0.25270.005 0.28470.001
13% 5%
Rotifers
Undisturbed Stirred
0.28670.003 0.30170.004
0.19170.002 0.24270.002
33% 20%
Start
End
Total suspended solids (mg/l) DTSS
Control
Undisturbed Stirred
12872 13172
10673 13174
18% 0%
Rotifers
Undisturbed Stirred
13373 14672
8275 13073
38% 11%
The experiment was run for 48 h: OD600 values are an average of five samples, TSS values are an average of three samples.
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At the start of the experiment, all reactors had an OD600 within the range 0.286–0.301 and TSS ranged from 128 to 146 mg/l (Table 1). During the course of the experiment, OD600 of the control reactors decreased by 13% for the undisturbed and by 5% for the stirred reactor. In comparison, the reactors containing 100,000 rotifers/l showed a reduction in OD600 of 33% for the undisturbed reactor and of 20% for the stirred reactor. A similar result was observed for solids in suspension. The controls showed a decrease in TSS of 18% for the undisturbed reactor and no change in the stirred reactor. In contrast, TSS in the rotifer reactor decreased by 38% in the undisturbed and by 11% in the stirred reactor. As measurements of stirred reactor samples indicate the total biomass in the system, it can be concluded that the total biomass in the reactor containing rotifers was reduced by B11%, most likely due to its consumption as a food source. In addition, rotifers augmented the natural settling process of suspended biomass by B10%, possibly due to a flocculation effect. In a second, similar experiment, 200,000 rotifers/l were added to activated sludge and incubated for 24 h. TSS was decreased by B9% in the stirred reactor containing rotifers, compared with no significant change of TSS in the stirred control reactor. In the unstirred control reactor, TSS was decreased only insignificantly, but was decreased by 34% in the reactor containing rotifers (data not shown). This indicates removal of biomass of B9% due to consumption by rotifers and removal of B25% of biomass, due to the enhanced settleability they confer. Both experiments suggest a rate of consumption of biomass by 100,000 rotifers/l of 9–11% within 48 h and an augmented settling effect on 10–25% of TSS within that time period.
4. Discussion Bdelloid rotifers have been shown here to have a significant impact on wastewater by improving removal of unsettled particles from suspension. It was shown that particles in suspension are mainly in the size range 0.2– 10 mm, which corresponds with the size range of particles consumed by rotifers. These particles are removed from suspension by rotifers through a twofold mechanism. First, rotifers were shown to graze on small, suspended particles and to consume them. The total biomass of a system in which rotifers graze on suspended particles is subsequently reduced by B10% (TSS) within 48 h at the rotifer density used. Consistent with this, a preliminary experiment suggested total solids removal is also significantly reduced by rotifer grazing (data not shown). Second, particles were removed by an apparent ability of rotifers to function as facilitators of floc formation. Small particles were most likely aggregated into larger flocs, which settled out of suspension. This
effect reduced the total biomass in suspension by an additional 10–25%. Thus, rotifers were shown to reduce sludge in the system and to improve the clarity of wastewater through enhanced particle settling. The observation that rotifers feed actively on suspended particles is supported by the experiments of Vadstein et al. [11]. The monogonont rotifer Brachionus plicatilis was shown to be able to remove all tested particles, ranging in size from 0.3 to 3 mm, with an optimum particle feeding size of B2 mm. A similar species, Brachionus calyciflorus, showed active particle removal of silica beads of 3–6 mm [12]. These values are within the confirmed feeding range of Philodina roseola and support the assumption that rotifers feed on all suspended particles within a certain size range. Although the particles which were used in the rotifer clarification experiments were in part created by autoclaving wastewater samples, the unsettled particles are in the same size range of particles observed in natural effluents. Atteia et al. [13] report that suspended particles in samples from a brook which drains peat areas, are in the size range of 0.5–10 mm. More than 90% of the suspended particles in effluents from trickling filters have been reported to be smaller than 30 mm [6]. As rotifers can be found in all aerobic wastewater treatment processes [14], it can be assumed that they naturally feed on suspended particles of these sizes and under these conditions. Increasing rotifer numbers in the system could therefore prove to be an economical and sustainable method for improving effluent clarity. Chemical flocculants are used in wastewater treatment processes to improve clarification of the final effluent. Addition of flocculants increases the operating costs of wastewater treatment facilities due to purchase costs and the resultant increase of waste sludge produced by the system [15]. Other methods of improving effluent clarity, such as sand filters, are costly to build and maintain. Our experiments indicate that rotifers function as a bioflocculant in wastewater. Enhanced floc formation by bdelloid rotifers is probably due to a secreted substance that rotifers use to anchor themselves to the substrate. The foot of bdelloid rotifers contains a series of pedal glands, also referred to as cement glands [8]. These glands secrete a mucous glue, which enables rotifers to attach to surfaces. Cement glands are used by a variety of organisms for substrate anchorage under water. Analysis of the cement composition of Acanthocephalans, the sister phylum of Rotifera, showed it to consists of proteinaceous material, the main protein constituent having a size of 23 kDa [16]. The sticky substance secreted by rotifers may also be proteinaceous and would probably function as a flocculant. Several technologies have been suggested for the purpose of sludge reduction to date. One strategy is to inhibit the build up of sludge flocs. Low and Chase [17] reviewed the use of chemical or biological strategies for
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uncoupling metabolism from growth in wastewater microorganisms. A second approach is sludge disintegration, which will release cell contents and disrupt flocs into smaller particles that are more accessible as food sources to wastewater microorganisms. Wet sludge disintegration techniques used so far include mechanical, chemical, thermo-chemical, biological and oxidative methods. Most uncoupling or disintegration technologies have an unfavourable cost:benefit ratio and are not widely regarded as economically viable (reviewed by Weemaes and Verstraete [18]). A third strategy for sludge reduction is to increase the number of predators within the system. Oligochaete worms (Tubificidae) have been shown to reduce biomass production in trickling filters and activated sludge plants, but increased the numbers of small particles in suspension in the final effluent [19,20]. Welander et al. [21] showed that predator grazing in a two-stage activated sludge process had a marked effect on biomass production. A first stage reactor without biomass recycling produces bacteria, which are consumed in the second stage, where ciliates and metazoa graze on the bacteria. This process is also referred to as the low sludge production (LSP) process. The LSP process has been successfully implemented at a pulp mill in Folla, Sweden, where the sludge production of the wastewater treatment plant was lowered by 70–90% [21]. The LSP process works best with relatively warm wastewater and requires a high concentration of microbial nutrients in the influent, or nutrient addition at the first stage. Municipal wastewater contains low concentrations of soluble, readily biodegradable matter and the effectiveness of a municipal LSP process is probably reduced, compared with a plant treating industrial wastewater, particularly at the low ambient temperatures prevalent in some countries. Inamori and colleagues have used rotifers, together with other bacterial, algal and metazoan species found naturally in wastewater treatment plants, to form a microcosm which provides a laboratory model of naturally occurring ecosystems (e.g. [22]). Like the LSP process, this illustrates the ability of rotifers to thrive in systems where they are able to predate bacteria or algae. The use of rotifers for the treatment of wastewater was suggested previously [23,24]: a mix of agricultural wastes is subjected to an algae/bacteria reactor, where COD is converted to cellular biomass (algae), which is subsequently grazed by rotifers in a second stage predator reactor. In this report, we demonstrate the ability of bdelloid rotifers not only to feed on algae or bacteria suspended in wastewater, but also on the sludge particles themselves. Rotifers inoculated into particulate wastewater survive and multiply over periods of many weeks (J.L. and A.T., unpublished). We envisage the use of rotifers as an adjunct to conventional activated sludge processes, either in a separate predator tank or within an
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existing aeration tank. One problem in encouraging rotifer growth may be the availability of particles in the size range ingestible by rotifers. This might be overcome by partial disintegration of sludge particles prior to a rotifer grazing stage, combining limited sludge disintegration with rotifer predation and might prove an efficient way of reducing sludge production in municipal wastewater treatment plants. Furthermore, rotifers might also be used as ‘‘bioclarifiers’’ to improve the clarity of the final effluent.
5. Conclusions Bdelloid rotifers have been shown to remove wastewater particles from suspension, partially by consumption and partially by improving settleability. Their beneficial effect on biomass yield and wastewater clarity suggests that rotifers might be used as biotechnological tools in wastewater processing, either through encouraging their growth in existing wastewater plants, or through modification to include a specific rotifer predation/clarification module.
Acknowledgements We would like to thank Jonathan Strickland, Jo Callan, Matt Griffiths, Keith Edwards and Steve Kaye of AWG plc for their stimulating discussions and provision of wastewater samples. J.L. holds the AWG Research Studentship; A.T. is the AWG Senior Research Fellow of Pembroke College, Cambridge.
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