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Importance of bacterial storage polymers in bioprocesses
e>
Pergamon
War. ScL Tech. Vol. 3S, No. I, pp. 41-47,1997. Copyright C 1996IAWQ. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved. 0273-1223J97 SI1'OO + 0-00
Pll: 50273-1223(96)00877-3
IMPORTANCEOFBACTEIDALSTORAGE POLYMERS IN BIOPROCESSES M. C. M. van Loosdrecht, M. A. Pot and J. J. Heijnen Kluyverlaboratory for Biotechnology, Delft University of Technology. Julianalaan 67. 2628 Be Delft. The NetherkJnds
ABSTRACf In waste water treatment processes microorganisms are subjected to • feast and famine regime. For sequencing balch processes this is often even more pronounced. Based on literature reports and own research it is hypothesized that in general microorganisms respond to these feast-famine regimes by accumulating storage polymers (polyhydroxyalkanoates) when substrate is presenL 11le storage polymers are used for growth when the external substrate is depleted. In this manner the organisms are capable to balance their growth. A general hypothesis explaining polymer formation is developed. The advantages and disadvantages of Ibis formation of storage polymers for the operation of SBR processes is discussed. Copyright C 1996 IAWQ. Published by Elsevier Science Ltd
KEYWORDS Storage polymers; PHB; SBR; feast-famine regime; denitrification. INTRODUcrION In general growth and respiration processes in activated sludge systems are assumed to occur on soluble or particulate COD (Gujer and Henze, 1991). On the other hand the capacity of microorganisms to accumulate internal storage polymers (e.g. polyhydroxyalkanoates. lipids and glycogen) is often reported (examples are Stanier et 01. 1959, Zevenhuizen and Ebbink 1974, Chudoba et 01. 1973, Van den Eijnde et 01. 1984). These reports have not resulted in a general acceptance of a significant role of storage processes in activated sludge processes. Only in the process of biological phosphorus elimination it is generally accepted that storage polymers such as glycogen and polyhydroxybutyrate (PHB) are of prime importance in the metabolism of bio-P bacteria (Wentzel et 01. 1986, Satoh et 01. 1992). Recently also the presence of "G-bacteria" accumulating glycogen and PHB has pointed to the importance of storage polymers in activated sludge (Cech et 01. 1994). Although storage polymers are often neglected in activated sludge research they are probably of significant importance. Activated sludge processes are highly dynamic with respect to the feed regime, especially when use is made of SBR processes. Only for relatively short periods of time the microorganisms have external substrate available. Without internally stored substrate the microorganisms will continuously undergo rapid growth and starvation periods. Microorganisms which are capable to quickly store substrate and consume this stored substrate in a more balanced way have a strong competitive advantage over organisms without 41
M. C. M. VAN LOOSDRECHT tt aJ.
42
the capacity of substrate storage. In SBR processes the role of storage polymer formation is even more important than in continuously operated treatment plants. Substrate is fed in a short time to the reactor after which e.g. nitrification-denitrification takes place. Several types of organic storage polymers have been reported in literature: PHB. Glycogen and lipids (Zevenhuizen and Ebbink 1974). Inorganic storage polymers such as polyphosphate are not discussed here. From the organic storage compounds PHB is probably the most dominant polymer as it is directly formed out of the central metabolite Acetyl-CoA. Glycogen is probably only formed when sugars are present in the influent, or when it plays an essential role in the bacterial metabolism as e.g. for bio-P or "G" bacteria. The role of lipids is unclear. There are only few reports on the presence of lipids, and in these reports it is not clear how a distinction is made between stored lipids and celmembrane lipids. Growth of bacteria on storage polymers has hardly been studied in literature. There are almost no reports on studies with pure cultures growing on storage polymers. Documented kinetic relationships for these processes are not available either. Moreover when the accumulation of storage polymers by pure cultures is studied, this is usually done under conditions where growth limitation due to e.g. nitrogen shortage occurs. In activated sludge however storage occurs under conditions without growth limitation. The purpose of this contribution is to discuss. based on a limited literature review and some own experimental results. the role of storage processes in activated sludge systems and discuss the possible advantages and disadvantages for the treatment process. ACTNATED SLUDGE PROCESSES WHERE STORAGE POLYMERS PLAYA SIGNIFICANT ROLE Bjolo~jcal
P-elimjnation
The role of storage polymers is most abundantly studied in the context of biological P-elimination (e.g. Wentzel et al. 1986, Satoh et al. 1992). After it was recognized that fatty acids form the dominant substrate for bio-P bacteria, it was also clear that this substrate is accumulated in the cell during the anaerobic phase of the process. It is now widely accepted that in the anaerobic phase of the process organic substrates are fermented to fatty acids. These are converted by the bio-P bacteria to PHB at the expense of the energy released in the hydrolysis of glycogen and polyphosphate (Smolders et al. 1995). When oxygen or nitrate become available the PHB is oxidized in order to generate energy for growth and restoring the glycogen and polyphosphate levels (Smolders et al. 1995). It is clear that for the bio-P bacteria storage polymers form a crucial part of the bacterial metabolism. This is also true for the recently described "G-bacteria" (Cech et al. 1994, Liu et al. 1994). These organisms are also capable of storing substrates under conditions where an electron acceptor is absent. This storage is based on the energy released in the conversion of sugars (either from glycogen or from external substrates) to polyhydroxyalkanoates (PHA). This energy enables the organism to accumulate fatty acids concomitantly. Again in the presence of an electron acceptor the organisms grow on the stored substrate. Concludingly it can be stated that dynamic conditions with respect to the availability of an electron acceptor clearly selects for bacteria capable of storing substrates under anaerobic conditions. Bulkjn~ slud~e control
The general accepted theory around competition of filamentous and non-filamentous bacteria is based on the difference in substrate affinity. Since filamentous bacteria have a lower Ks value, they are capable to efficiently compete for substrate with floc forming bacteria when the substrate concentration is low. On the other hand floc forming bacteria seem to have a higher maximal substrate uptake velocity. This theory explains why filamentous bacteria can become dominant in processes with a completely mixed reactor.
Importance of bacterial storage polymers in bioprocesscs
43
As a remediation for bulking sludge, selectors have been designed (Chudoba el al. 1973). In these plug flow reactors return sludge and influent are mixed. The residence time in these selectors is short (approx. 15 minutes) in comparison to the total residence time. The criteria for the design of a selector is the removal of easily biodegradable COD from the liquid phase. A part of the COD is adorned by the sludge flocs. but the soluble COD has for the main part to be taken up by the sludge. This soluble COD is accumulated in the cells as storage polymer. The energy for this process is derived from the oxidation of a minor fraction of the COD with oxygen or nitrate, or in the case of an anaerobic selector. from the hydrolysis of polyphosphate. The relation between storage polymer formation and bulking sludge control has been studied in detail by Van den Eijnde el al. (1983, 1984) whereas Chudoba et al. (1973, 1985) have been studying this process implicitly. Van den Eijnde et al. (1984) showed that a floc forming bacterium Arthrobacter sp. has a large storage capacity for glucose. whereas the filamentous bacterium Sphaerotilis natans has only a very limited capacity. These observations with pure cultures correlated very well with the observation that continuously fed (completely mixed) activated sludge systems had a high SVI and low storage capacity. Another intermittently (completely mixed) activated sludge system had a low SVI and a high capacity for accumulation of storage components. The experiments of Chudoba et al. (1973. 1985) were aimed at illustrating the Ks theory. Analysis of their results (see below) however reveals that also in their experiments significant amounts of polymers have been formed. AlB and CSAS processes The observation that COD may be very rapidly removed from the liquid by adsorption and absorption or storage processes has led to several processes in which use is made of this characteristic. These are the AlB process and the CSAS type of processes. In both processes sludge and influent are mixed. and after a relative short retention time separated again. The retention time is just long enough to allow the sludge to adIabsorb the COD inside the sludge floc. The non-soluble compounds are likely to be incorporated physically in the sludge floc. The soluble components are accumulated inside the cells as storage polymers. In the CSAS process (Contact Stabilisation Activated Sludge Process. Ulrich and Smith 1951) the return sludge is aerated in a "sludge regeneration" tank. In this tank stored substrate is oxidized and cell growth occurs. In this manner also the storage pools are emptied for a new cycle. In the AlB process (Bohnke 1977) the solids retention time of the A-stage is very short. In this process the majority of the stored material will be removed with the excess sludge and fermented in the sludge digestor. In spite of the relatively wide-spread use of these treatment systems there is hardly any information in the literature which can be used to get more insight in the turnover of storage compounds in this type of sludge. COD conversion In the case of "standard" aerobic or anoxic COD removal processes the formation of storage compounds is not as influential as in the above described processes. This means that it is often ignored or not observed. Only a proper evaluation of e.g. COD balances during the treatment process can give more insight. Until recently there was usually not enough information available to substantiate storage processes. However. recent studies with respirometry and pure substrates point strongly to the occurrence of storage phenomena In general the relation between oxygen and substrate consumed is much lower than found in pure culture studies, indicating storage of the substrate. There is however no direct evidence in literature. Chudoba et al. (1985) studied the conversion of glucose in plug flow and completely mixed reactor types. The theoretical yield for the formation of glycogen is 0.96 gCOD/gCOD, whereas the yield of biomass on glucose for pure cultures is 0.64 gCOD/gCOD. Chudoba et al. also observed a yield of 0.71 or 0.88 gCOD/gCOD for completely mixed or plug flow systems respectively. This yield was calculated from the amount of oxygen consumed until glucose was fully converted. They also observed that the yield increased when more compartments were placed in series (Chudoba et al. 1973) 0.76 versus 0.82 moUmol for 3 or 5 compartments. This deviation between observed and pure culture yields strongly indicates the formation of storage polymers.
M. C. M. VAN LOOSDRECIff d aI.
44
Also indirectly there are several strong indications that storage processes are ubiquitous in activated sludge. Heijnen (1994) bas shown, based on a large literature review, that based on thermodynamic reasoning that biomass synthesis requires a standard amount of energy dissipation. From these finding it can be derived that for aerobic growth the yield is approximately 0.5 gCOD/gCOD for a wide range of aerobic bacteria and substrates. For waste water processes however usually a yield of 0.6-0.7 gCOD/gCOD is used. This yield is again based on respirometric observations. This discrepancy between pure culture observation and mixed culture observations can be explained when a fraction of the substrate is converted into storage polymers. In the IAWQ-model consumption of storage polymers is accounted for in the decay coefficient of the model. Also for denitrification generally the gCOD/gNO)-N needed in activated sludge processes is higher than expected from pure culture studies (4.5 versus 3.5 gCOD/gN), also here the discrepancy points to the occurrence of storage polymers. 1.0
chemostat
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batch operation
operation
~
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S
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I
O~
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-1
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2 1 time alter pulse (h)
3
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Figure \. Concentrations of acetic acid and PHB and the calculated growth rate in an experiment where a continuous culture is switched to batch mode and a pulse of acetate is added.
HYPOTHESIS FOR STORAGE POLYMER FORMATION Recent research in our laboratory (pot et al. 19968, b) has brought more insight in the general aspects of storage polymer formation. This research is partly summarized in two figures. In Fig. I the effect of transition from continuous to batch culturing on a pure culture is shown. In steady state continuous culture all substrate was converted to biomass at a constant rate, no biopolymers were formed. When the steady state was disturbed by adding a pulse of substrate the organism took up the substrate at a high rate. The growth rate however did not directly increase to a rate corresponding to the substrate uptake rate. The surplus of acetate taken up was converted to PHB. When the external substrate was depleted the organism started growing on the stored PHB. The growth rate on PHB is clearly lower then on the original substrate. This kind of behaviour has been reported more often in literature (Pagni el al. 1992, van Niel 1995). Obviously formation of PHB allows the organisms to maintain a balanced metabolism when sudden changes in substrate addition occur. When organisms are growing under limited substrate conditions they need relatively large amount of substrate uptake enzymes because the uptake is limited by the substrate concentration. If the substrate concentration suddenly rises this results in a rapid uptake of the substrate. This amount of substrate cannot directly be converted by the growth processes in the cell. If this substrate could not be converted to a polymer the whole cell metabolism would have the risk of getting out of balance. When the substrate concentration remains high the organisms can adjust their growth rate (see Fig. I). However in pulse-wise fed SBR systems or in plug-flow reactors only during a limited period high substrate concentrations are experienced by the bacteria and storage processes can playa dominant role. In a second range of experiments the effect of pulsed or continuous substrate addition to an anoxic-aerobic SBR with a mixed, open, sludge culture has been studied. The substrate (influent) was always fed in the anoxic phase. The sludge age and volumetric loading rate were identical in both cases. Figure 2 shows that
Importance of bacterial storage polymers in bioprocesses
4S
in both modes of substrate addition PHB accumulation occurs. As expected accumulation was larger in the pulse fed SBR than in the continuous fed SBR. Also in a mixed microbial population a feast-famine regime enriches for organisms which use storage polymers to balance their growth. Also here it seems that the difference between substrate uptake and substrate need for growth is balanced by the PHB formation. This has a direct negative affect for denitrification, since a fraction of the added COD is transferred as PHB to the aerobic phase where it is oxidized. Remarkably the COD/N ratio observed for the whole process was almost identical for the two SBR systems. This formation of storage polymers could be the main reason for the discrepancy between microbial and activated sludge data on the COD requirement for denitrification. 0.25
anoxc phase
,
aero
IC
p ase
1
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~0.20
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o 80.15
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~
i'
~0.10
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~-
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_----4.. . .-_.. . .. ,
HAc-continuoUi addition 0.00 . . . .~~~.-....... -;;.;;;-~-;;;.....-.:: =-4i-i--"""".....
o
60
120 time [minI
180
240
Figure 2. Profiles of Acetic acid and PHB in a cycle in a SBR which is fed pulse wise at the start of the cycle, and a SBR fed continuously throughout the anoxic phase. Nitrate was always present during the cycle.
Based on scarce literature data and our own research we have postulated a hypothesis for the occurrence of storage polymer formation under non-growth limiting conditions. If microorganisms observe regularly periods with low or no substrate and periods with abundant substrate (feast-famine regime) bacteria will be enriched that are capable of balancing their growth independent of the external substrate concentration. Bacteria not capable of substrate storage will have to invest extra energy to very rapid growth in the short periods with substrate, and deteriorate in the periods without. Since substrate is not available they will encounter problems in maintaining their cell structure in proper shape for when new substrate is fed to the system. Bacteria capable of storing e.g. PHB or similar compounds can maintain a more or less constant relatively low growth rate, and can keep all cell systems viable when the external substrate is depleted. In this manner these bacteria have a strong competitive advantage. PHB is especially interesting as storage polymer since many substrates are degraded in the cell with acetyl-COA as intermediate. This substance is also the precursor for PHB formation. IMPLICATION OF STORAGE POLYMER FORMATION FOR PROCESS DESIGN Storage polymers are obviously an intrinsic part of the metabolism of activated sludge processes and certainly in SBR processes one is capable of manipulating these processes by the way the influent is added to the process. Formation of storage polymers influences the following processes. Sludge production: Polymer formation leads to a high sludge provided the polymer is not oxidized. This has the advantage of minimizing the aeration energy and maximizing sludge production. When this is coupled to methane generation the overall energy balance of a treatment plant can be made positive. When for several reasons a low sludge production is needed storage polymer formation and subsequent growth on it leads to a lower sludge yield, compared to direct growth on the soluble COD.
M. C. M. VAN LOOSDRECHT et al.
46
Denitrification: In principle there is no large difference between COD conversion to storage compounds
under aerobic or denitrifying conditions. However if storage processes play an important role this leads to less efficient use of COD for denitrification. Fatty acids or other COD-sources added for denitrification can be partially stored inside the cells, and in this way be transferred to the aerobic, nitrification, stage. The result is a relatively high CODIN ratio needed for the denitrification process. Since microorganisms tend to balance their growth rate substrate will always be transferred from the denitrification zone to the nitrification zone in the form of storage compounds. The larger the aerobic zone the more pronounced this transfer will be. To overcome this effect substrates leading to low polymer formation should be found. Another possibility is to limit the aerobic retention as much as possible by adequate process control or eventually growing nitrifiers in biofilms in the aerobic phase. CONCLUSIONS
Storage processes play a dominant role in the activated sludge process. It seems that bacteria use storage capacity to provide substrate for growth when the extracellular substrate is depleted. In this manner these bacteria can balance their growth rate in dynamic processes. In some processes such as biological P-removal the significance of storage polymers is very obvious. Also in cases where it is less obvious ignoring the occurrence of storage polymers can lead to misinterpretation of e.g. respiration measurements. Recognition of the fact that storage processes are an intrinsic aspect of microbial physiology and ecology of activated sludge processes makes it possible to optimize existing processes or design new processes. Especially in SBR processes where the substrate can be added in a pulse-wise mode, accumulation of polymers can be enhanced. REFERENCES Bohnke. B. (1977) Das Adsorptions-Belebungsverfahren. Korrespondenz abwasser. 24. 121-127. Cech, J.S., Hartman. P.• Macek. M. (1994) Bacteria and protozoa population dynamics in biological phosphate removal systems. War.Sci.Tech. 29(7).109-117. Chudoba, J.S., Orau. P. Ouova. V. (1973) Control of activated sludge filamentous bulking-II Selection of microorganisms by means ora selector. Wat.Res. 7. 1389-1406. Chudoba, J., Cech. J.S., Farkac, J.• Orau. P. (1985) Control of activated sludge filamentous bulking-experimental verification of a kinetic selection theory. War.Res. /9. 191-196. Gujer, W., Henze, M. (1991) Activated sludge modelling and simulation. War.Sci.Tech.• 23(4-6), 1011-1023. Heijnen. J.J. (1994) Thermodynamics of microbial growth and its implications for process design. Tibrech. 12. 483-492. Liu. W.T., Mino, T.• Nakamura. K.• Matsuo. T. (1994) Role of glycogen in acetate uptake and polyhydroxybutyrate synthesis in anaerobic aerobic activated sludge with minimized polyphosphate content. J. Ferment.Bioeng.• 77. 535-540. Pagni. M., Beffa, T.• Isch. C.• Aragno, M. (1992) Linear growth and po!yhydroxybutyrate synthesis in response to pulse wise addition of the growth limiting substrate to steady state heterotrophic continuous cultures of Aquaspirillum aurorrophicum. J.Gen.Microb.• 138, 429-436. Pot, M., Van Leeuwen, M.A., Van Loosdrecht, M.C.M., Heijnen. 1.1. (l996a) Kinetic modelling of polyhydroxybutyrate production and consumption by Thiosphaera pantotropha under dynamic substrate supply. Biotech.Bioeng. (submitted). Pot, M., Van Loosdrecht, M.C.M., Heijnen. J.J., (1996b) Effect of substrate addition on polymer storage in nitrification/denitrification SBR processes. War. Res. (submitted). Satoh, H.• Mino. T., Matsuo. T. (1992) Uptake of organic substrates and accumulation of polyhydroxyalkanoates linked with glycolysis of intercellular carbohydrates under anaerobic conditions in the biological excess phosphorus removal process. War.Sci.Tech.• 26. 933-942. Smolders, OJ.F., Van Loosdrecht, M.C.M., Heijnen, 1.1. (1995) A metabolic model for the biological phosphorus removal process. War.Sci.Tech. 31(2), 79-93. Stanier, R.Y., Doudoroff. M.• Kunisawa, R., Contopoulou, R. (1959) The role of organic substrates in bacterial photosynthesis. Proc.Natl.Acad.Sci. U.S.A.• 45, 1246-1249. Ulrich. A.H., Smith. M.W. (1951) The biosorption process of sewage and waste treatment. Sewage and Industrial Wastes. 23, 1248-1255. Van den Eijde E. (1983) Influence of feeding pattem on the glucose metabolism of Arthrobacter sp. and Sphaeroti/us natans, growing in chemostat culture, simulating activated sludge bulking. Eur. J. Appl. Microbiol. Biotechnol. 17, 35-43.
Importance of bacterial storage polymen in bioprocesses
47
Van den Eijnde E.• L. Vriens. M. Wynants. and H. Verachten (1984) Transient behaviour and time aspects of intennittently and continously fed bacterial cultures with regard to filamentous bulking of activated sludge. Appl. MicrobioL Bioluhnol. /9. 44-52. Van Niel. E.WJ. (1995) Rapid short tenn uptake polyhydtoxybutyrate production by Thiosphaera panlolropha in the presence of excess acetate. AppLEnv.Microbiol. 17. Wentzel. M.e.• Lotter. L.. Loewenthal. R.E.• Marais. G.v.R. (1986) Metabolic behaviour of Acintlobacltr spp. in enhanced biological phosphorus removal-a biochemical model. Water 5.14.. 12. 209-224. Zevenhuizen. L.P.T.M.• Ebbink. A.G. (1974) Interrelations between glycogen. poly-B-hydtoxybutyrate and lipids during accumulation and subsequent utilization in a Pseudomonas. AnI. van Luuwtnhotlc, 40. 103-120.
Pergamon
War. ScL Tech. Vol. 3S, No. I, pp. 41-47,1997. Copyright C 1996IAWQ. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved. 0273-1223J97 SI1'OO + 0-00
Pll: 50273-1223(96)00877-3
IMPORTANCEOFBACTEIDALSTORAGE POLYMERS IN BIOPROCESSES M. C. M. van Loosdrecht, M. A. Pot and J. J. Heijnen Kluyverlaboratory for Biotechnology, Delft University of Technology. Julianalaan 67. 2628 Be Delft. The NetherkJnds
ABSTRACf In waste water treatment processes microorganisms are subjected to • feast and famine regime. For sequencing balch processes this is often even more pronounced. Based on literature reports and own research it is hypothesized that in general microorganisms respond to these feast-famine regimes by accumulating storage polymers (polyhydroxyalkanoates) when substrate is presenL 11le storage polymers are used for growth when the external substrate is depleted. In this manner the organisms are capable to balance their growth. A general hypothesis explaining polymer formation is developed. The advantages and disadvantages of Ibis formation of storage polymers for the operation of SBR processes is discussed. Copyright C 1996 IAWQ. Published by Elsevier Science Ltd
KEYWORDS Storage polymers; PHB; SBR; feast-famine regime; denitrification. INTRODUcrION In general growth and respiration processes in activated sludge systems are assumed to occur on soluble or particulate COD (Gujer and Henze, 1991). On the other hand the capacity of microorganisms to accumulate internal storage polymers (e.g. polyhydroxyalkanoates. lipids and glycogen) is often reported (examples are Stanier et 01. 1959, Zevenhuizen and Ebbink 1974, Chudoba et 01. 1973, Van den Eijnde et 01. 1984). These reports have not resulted in a general acceptance of a significant role of storage processes in activated sludge processes. Only in the process of biological phosphorus elimination it is generally accepted that storage polymers such as glycogen and polyhydroxybutyrate (PHB) are of prime importance in the metabolism of bio-P bacteria (Wentzel et 01. 1986, Satoh et 01. 1992). Recently also the presence of "G-bacteria" accumulating glycogen and PHB has pointed to the importance of storage polymers in activated sludge (Cech et 01. 1994). Although storage polymers are often neglected in activated sludge research they are probably of significant importance. Activated sludge processes are highly dynamic with respect to the feed regime, especially when use is made of SBR processes. Only for relatively short periods of time the microorganisms have external substrate available. Without internally stored substrate the microorganisms will continuously undergo rapid growth and starvation periods. Microorganisms which are capable to quickly store substrate and consume this stored substrate in a more balanced way have a strong competitive advantage over organisms without 41
M. C. M. VAN LOOSDRECHT tt aJ.
42
the capacity of substrate storage. In SBR processes the role of storage polymer formation is even more important than in continuously operated treatment plants. Substrate is fed in a short time to the reactor after which e.g. nitrification-denitrification takes place. Several types of organic storage polymers have been reported in literature: PHB. Glycogen and lipids (Zevenhuizen and Ebbink 1974). Inorganic storage polymers such as polyphosphate are not discussed here. From the organic storage compounds PHB is probably the most dominant polymer as it is directly formed out of the central metabolite Acetyl-CoA. Glycogen is probably only formed when sugars are present in the influent, or when it plays an essential role in the bacterial metabolism as e.g. for bio-P or "G" bacteria. The role of lipids is unclear. There are only few reports on the presence of lipids, and in these reports it is not clear how a distinction is made between stored lipids and celmembrane lipids. Growth of bacteria on storage polymers has hardly been studied in literature. There are almost no reports on studies with pure cultures growing on storage polymers. Documented kinetic relationships for these processes are not available either. Moreover when the accumulation of storage polymers by pure cultures is studied, this is usually done under conditions where growth limitation due to e.g. nitrogen shortage occurs. In activated sludge however storage occurs under conditions without growth limitation. The purpose of this contribution is to discuss. based on a limited literature review and some own experimental results. the role of storage processes in activated sludge systems and discuss the possible advantages and disadvantages for the treatment process. ACTNATED SLUDGE PROCESSES WHERE STORAGE POLYMERS PLAYA SIGNIFICANT ROLE Bjolo~jcal
P-elimjnation
The role of storage polymers is most abundantly studied in the context of biological P-elimination (e.g. Wentzel et al. 1986, Satoh et al. 1992). After it was recognized that fatty acids form the dominant substrate for bio-P bacteria, it was also clear that this substrate is accumulated in the cell during the anaerobic phase of the process. It is now widely accepted that in the anaerobic phase of the process organic substrates are fermented to fatty acids. These are converted by the bio-P bacteria to PHB at the expense of the energy released in the hydrolysis of glycogen and polyphosphate (Smolders et al. 1995). When oxygen or nitrate become available the PHB is oxidized in order to generate energy for growth and restoring the glycogen and polyphosphate levels (Smolders et al. 1995). It is clear that for the bio-P bacteria storage polymers form a crucial part of the bacterial metabolism. This is also true for the recently described "G-bacteria" (Cech et al. 1994, Liu et al. 1994). These organisms are also capable of storing substrates under conditions where an electron acceptor is absent. This storage is based on the energy released in the conversion of sugars (either from glycogen or from external substrates) to polyhydroxyalkanoates (PHA). This energy enables the organism to accumulate fatty acids concomitantly. Again in the presence of an electron acceptor the organisms grow on the stored substrate. Concludingly it can be stated that dynamic conditions with respect to the availability of an electron acceptor clearly selects for bacteria capable of storing substrates under anaerobic conditions. Bulkjn~ slud~e control
The general accepted theory around competition of filamentous and non-filamentous bacteria is based on the difference in substrate affinity. Since filamentous bacteria have a lower Ks value, they are capable to efficiently compete for substrate with floc forming bacteria when the substrate concentration is low. On the other hand floc forming bacteria seem to have a higher maximal substrate uptake velocity. This theory explains why filamentous bacteria can become dominant in processes with a completely mixed reactor.
Importance of bacterial storage polymers in bioprocesscs
43
As a remediation for bulking sludge, selectors have been designed (Chudoba el al. 1973). In these plug flow reactors return sludge and influent are mixed. The residence time in these selectors is short (approx. 15 minutes) in comparison to the total residence time. The criteria for the design of a selector is the removal of easily biodegradable COD from the liquid phase. A part of the COD is adorned by the sludge flocs. but the soluble COD has for the main part to be taken up by the sludge. This soluble COD is accumulated in the cells as storage polymer. The energy for this process is derived from the oxidation of a minor fraction of the COD with oxygen or nitrate, or in the case of an anaerobic selector. from the hydrolysis of polyphosphate. The relation between storage polymer formation and bulking sludge control has been studied in detail by Van den Eijnde el al. (1983, 1984) whereas Chudoba et al. (1973, 1985) have been studying this process implicitly. Van den Eijnde et al. (1984) showed that a floc forming bacterium Arthrobacter sp. has a large storage capacity for glucose. whereas the filamentous bacterium Sphaerotilis natans has only a very limited capacity. These observations with pure cultures correlated very well with the observation that continuously fed (completely mixed) activated sludge systems had a high SVI and low storage capacity. Another intermittently (completely mixed) activated sludge system had a low SVI and a high capacity for accumulation of storage components. The experiments of Chudoba et al. (1973. 1985) were aimed at illustrating the Ks theory. Analysis of their results (see below) however reveals that also in their experiments significant amounts of polymers have been formed. AlB and CSAS processes The observation that COD may be very rapidly removed from the liquid by adsorption and absorption or storage processes has led to several processes in which use is made of this characteristic. These are the AlB process and the CSAS type of processes. In both processes sludge and influent are mixed. and after a relative short retention time separated again. The retention time is just long enough to allow the sludge to adIabsorb the COD inside the sludge floc. The non-soluble compounds are likely to be incorporated physically in the sludge floc. The soluble components are accumulated inside the cells as storage polymers. In the CSAS process (Contact Stabilisation Activated Sludge Process. Ulrich and Smith 1951) the return sludge is aerated in a "sludge regeneration" tank. In this tank stored substrate is oxidized and cell growth occurs. In this manner also the storage pools are emptied for a new cycle. In the AlB process (Bohnke 1977) the solids retention time of the A-stage is very short. In this process the majority of the stored material will be removed with the excess sludge and fermented in the sludge digestor. In spite of the relatively wide-spread use of these treatment systems there is hardly any information in the literature which can be used to get more insight in the turnover of storage compounds in this type of sludge. COD conversion In the case of "standard" aerobic or anoxic COD removal processes the formation of storage compounds is not as influential as in the above described processes. This means that it is often ignored or not observed. Only a proper evaluation of e.g. COD balances during the treatment process can give more insight. Until recently there was usually not enough information available to substantiate storage processes. However. recent studies with respirometry and pure substrates point strongly to the occurrence of storage phenomena In general the relation between oxygen and substrate consumed is much lower than found in pure culture studies, indicating storage of the substrate. There is however no direct evidence in literature. Chudoba et al. (1985) studied the conversion of glucose in plug flow and completely mixed reactor types. The theoretical yield for the formation of glycogen is 0.96 gCOD/gCOD, whereas the yield of biomass on glucose for pure cultures is 0.64 gCOD/gCOD. Chudoba et al. also observed a yield of 0.71 or 0.88 gCOD/gCOD for completely mixed or plug flow systems respectively. This yield was calculated from the amount of oxygen consumed until glucose was fully converted. They also observed that the yield increased when more compartments were placed in series (Chudoba et al. 1973) 0.76 versus 0.82 moUmol for 3 or 5 compartments. This deviation between observed and pure culture yields strongly indicates the formation of storage polymers.
M. C. M. VAN LOOSDRECIff d aI.
44
Also indirectly there are several strong indications that storage processes are ubiquitous in activated sludge. Heijnen (1994) bas shown, based on a large literature review, that based on thermodynamic reasoning that biomass synthesis requires a standard amount of energy dissipation. From these finding it can be derived that for aerobic growth the yield is approximately 0.5 gCOD/gCOD for a wide range of aerobic bacteria and substrates. For waste water processes however usually a yield of 0.6-0.7 gCOD/gCOD is used. This yield is again based on respirometric observations. This discrepancy between pure culture observation and mixed culture observations can be explained when a fraction of the substrate is converted into storage polymers. In the IAWQ-model consumption of storage polymers is accounted for in the decay coefficient of the model. Also for denitrification generally the gCOD/gNO)-N needed in activated sludge processes is higher than expected from pure culture studies (4.5 versus 3.5 gCOD/gN), also here the discrepancy points to the occurrence of storage polymers. 1.0
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operation
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Figure \. Concentrations of acetic acid and PHB and the calculated growth rate in an experiment where a continuous culture is switched to batch mode and a pulse of acetate is added.
HYPOTHESIS FOR STORAGE POLYMER FORMATION Recent research in our laboratory (pot et al. 19968, b) has brought more insight in the general aspects of storage polymer formation. This research is partly summarized in two figures. In Fig. I the effect of transition from continuous to batch culturing on a pure culture is shown. In steady state continuous culture all substrate was converted to biomass at a constant rate, no biopolymers were formed. When the steady state was disturbed by adding a pulse of substrate the organism took up the substrate at a high rate. The growth rate however did not directly increase to a rate corresponding to the substrate uptake rate. The surplus of acetate taken up was converted to PHB. When the external substrate was depleted the organism started growing on the stored PHB. The growth rate on PHB is clearly lower then on the original substrate. This kind of behaviour has been reported more often in literature (Pagni el al. 1992, van Niel 1995). Obviously formation of PHB allows the organisms to maintain a balanced metabolism when sudden changes in substrate addition occur. When organisms are growing under limited substrate conditions they need relatively large amount of substrate uptake enzymes because the uptake is limited by the substrate concentration. If the substrate concentration suddenly rises this results in a rapid uptake of the substrate. This amount of substrate cannot directly be converted by the growth processes in the cell. If this substrate could not be converted to a polymer the whole cell metabolism would have the risk of getting out of balance. When the substrate concentration remains high the organisms can adjust their growth rate (see Fig. I). However in pulse-wise fed SBR systems or in plug-flow reactors only during a limited period high substrate concentrations are experienced by the bacteria and storage processes can playa dominant role. In a second range of experiments the effect of pulsed or continuous substrate addition to an anoxic-aerobic SBR with a mixed, open, sludge culture has been studied. The substrate (influent) was always fed in the anoxic phase. The sludge age and volumetric loading rate were identical in both cases. Figure 2 shows that
Importance of bacterial storage polymers in bioprocesses
4S
in both modes of substrate addition PHB accumulation occurs. As expected accumulation was larger in the pulse fed SBR than in the continuous fed SBR. Also in a mixed microbial population a feast-famine regime enriches for organisms which use storage polymers to balance their growth. Also here it seems that the difference between substrate uptake and substrate need for growth is balanced by the PHB formation. This has a direct negative affect for denitrification, since a fraction of the added COD is transferred as PHB to the aerobic phase where it is oxidized. Remarkably the COD/N ratio observed for the whole process was almost identical for the two SBR systems. This formation of storage polymers could be the main reason for the discrepancy between microbial and activated sludge data on the COD requirement for denitrification. 0.25
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Figure 2. Profiles of Acetic acid and PHB in a cycle in a SBR which is fed pulse wise at the start of the cycle, and a SBR fed continuously throughout the anoxic phase. Nitrate was always present during the cycle.
Based on scarce literature data and our own research we have postulated a hypothesis for the occurrence of storage polymer formation under non-growth limiting conditions. If microorganisms observe regularly periods with low or no substrate and periods with abundant substrate (feast-famine regime) bacteria will be enriched that are capable of balancing their growth independent of the external substrate concentration. Bacteria not capable of substrate storage will have to invest extra energy to very rapid growth in the short periods with substrate, and deteriorate in the periods without. Since substrate is not available they will encounter problems in maintaining their cell structure in proper shape for when new substrate is fed to the system. Bacteria capable of storing e.g. PHB or similar compounds can maintain a more or less constant relatively low growth rate, and can keep all cell systems viable when the external substrate is depleted. In this manner these bacteria have a strong competitive advantage. PHB is especially interesting as storage polymer since many substrates are degraded in the cell with acetyl-COA as intermediate. This substance is also the precursor for PHB formation. IMPLICATION OF STORAGE POLYMER FORMATION FOR PROCESS DESIGN Storage polymers are obviously an intrinsic part of the metabolism of activated sludge processes and certainly in SBR processes one is capable of manipulating these processes by the way the influent is added to the process. Formation of storage polymers influences the following processes. Sludge production: Polymer formation leads to a high sludge provided the polymer is not oxidized. This has the advantage of minimizing the aeration energy and maximizing sludge production. When this is coupled to methane generation the overall energy balance of a treatment plant can be made positive. When for several reasons a low sludge production is needed storage polymer formation and subsequent growth on it leads to a lower sludge yield, compared to direct growth on the soluble COD.
M. C. M. VAN LOOSDRECHT et al.
46
Denitrification: In principle there is no large difference between COD conversion to storage compounds
under aerobic or denitrifying conditions. However if storage processes play an important role this leads to less efficient use of COD for denitrification. Fatty acids or other COD-sources added for denitrification can be partially stored inside the cells, and in this way be transferred to the aerobic, nitrification, stage. The result is a relatively high CODIN ratio needed for the denitrification process. Since microorganisms tend to balance their growth rate substrate will always be transferred from the denitrification zone to the nitrification zone in the form of storage compounds. The larger the aerobic zone the more pronounced this transfer will be. To overcome this effect substrates leading to low polymer formation should be found. Another possibility is to limit the aerobic retention as much as possible by adequate process control or eventually growing nitrifiers in biofilms in the aerobic phase. CONCLUSIONS
Storage processes play a dominant role in the activated sludge process. It seems that bacteria use storage capacity to provide substrate for growth when the extracellular substrate is depleted. In this manner these bacteria can balance their growth rate in dynamic processes. In some processes such as biological P-removal the significance of storage polymers is very obvious. Also in cases where it is less obvious ignoring the occurrence of storage polymers can lead to misinterpretation of e.g. respiration measurements. Recognition of the fact that storage processes are an intrinsic aspect of microbial physiology and ecology of activated sludge processes makes it possible to optimize existing processes or design new processes. Especially in SBR processes where the substrate can be added in a pulse-wise mode, accumulation of polymers can be enhanced. REFERENCES Bohnke. B. (1977) Das Adsorptions-Belebungsverfahren. Korrespondenz abwasser. 24. 121-127. Cech, J.S., Hartman. P.• Macek. M. (1994) Bacteria and protozoa population dynamics in biological phosphate removal systems. War.Sci.Tech. 29(7).109-117. Chudoba, J.S., Orau. P. Ouova. V. (1973) Control of activated sludge filamentous bulking-II Selection of microorganisms by means ora selector. Wat.Res. 7. 1389-1406. Chudoba, J., Cech. J.S., Farkac, J.• Orau. P. (1985) Control of activated sludge filamentous bulking-experimental verification of a kinetic selection theory. War.Res. /9. 191-196. Gujer, W., Henze, M. (1991) Activated sludge modelling and simulation. War.Sci.Tech.• 23(4-6), 1011-1023. Heijnen. J.J. (1994) Thermodynamics of microbial growth and its implications for process design. Tibrech. 12. 483-492. Liu. W.T., Mino, T.• Nakamura. K.• Matsuo. T. (1994) Role of glycogen in acetate uptake and polyhydroxybutyrate synthesis in anaerobic aerobic activated sludge with minimized polyphosphate content. J. Ferment.Bioeng.• 77. 535-540. Pagni. M., Beffa, T.• Isch. C.• Aragno, M. (1992) Linear growth and po!yhydroxybutyrate synthesis in response to pulse wise addition of the growth limiting substrate to steady state heterotrophic continuous cultures of Aquaspirillum aurorrophicum. J.Gen.Microb.• 138, 429-436. Pot, M., Van Leeuwen, M.A., Van Loosdrecht, M.C.M., Heijnen. 1.1. (l996a) Kinetic modelling of polyhydroxybutyrate production and consumption by Thiosphaera pantotropha under dynamic substrate supply. Biotech.Bioeng. (submitted). Pot, M., Van Loosdrecht, M.C.M., Heijnen. J.J., (1996b) Effect of substrate addition on polymer storage in nitrification/denitrification SBR processes. War. Res. (submitted). Satoh, H.• Mino. T., Matsuo. T. (1992) Uptake of organic substrates and accumulation of polyhydroxyalkanoates linked with glycolysis of intercellular carbohydrates under anaerobic conditions in the biological excess phosphorus removal process. War.Sci.Tech.• 26. 933-942. Smolders, OJ.F., Van Loosdrecht, M.C.M., Heijnen, 1.1. (1995) A metabolic model for the biological phosphorus removal process. War.Sci.Tech. 31(2), 79-93. Stanier, R.Y., Doudoroff. M.• Kunisawa, R., Contopoulou, R. (1959) The role of organic substrates in bacterial photosynthesis. Proc.Natl.Acad.Sci. U.S.A.• 45, 1246-1249. Ulrich. A.H., Smith. M.W. (1951) The biosorption process of sewage and waste treatment. Sewage and Industrial Wastes. 23, 1248-1255. Van den Eijde E. (1983) Influence of feeding pattem on the glucose metabolism of Arthrobacter sp. and Sphaeroti/us natans, growing in chemostat culture, simulating activated sludge bulking. Eur. J. Appl. Microbiol. Biotechnol. 17, 35-43.
Importance of bacterial storage polymen in bioprocesses
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Van den Eijnde E.• L. Vriens. M. Wynants. and H. Verachten (1984) Transient behaviour and time aspects of intennittently and continously fed bacterial cultures with regard to filamentous bulking of activated sludge. Appl. MicrobioL Bioluhnol. /9. 44-52. Van Niel. E.WJ. (1995) Rapid short tenn uptake polyhydtoxybutyrate production by Thiosphaera panlolropha in the presence of excess acetate. AppLEnv.Microbiol. 17. Wentzel. M.e.• Lotter. L.. Loewenthal. R.E.• Marais. G.v.R. (1986) Metabolic behaviour of Acintlobacltr spp. in enhanced biological phosphorus removal-a biochemical model. Water 5.14.. 12. 209-224. Zevenhuizen. L.P.T.M.• Ebbink. A.G. (1974) Interrelations between glycogen. poly-B-hydtoxybutyrate and lipids during accumulation and subsequent utilization in a Pseudomonas. AnI. van Luuwtnhotlc, 40. 103-120.