Metabolic uncouplers reduce excess sludge

Process Biochemistry 38 (2003) 1373 /1377 www.elsevier.com/locate/procbio

Metabolic uncouplers reduce excess sludge production in an activated sludge process Xue-Fu Yang a, Min-Li Xie a, Yu Liu a,b,* a

b

School of Chemical and Environmental Engineering, Beijing Technology and Business University, 11 ucheng Road, 100037 Beijing, PR China Environmental Engineering Research Centre, School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore Received 20 September 2002; received in revised form 25 November 2002; accepted 19 December 2002

Abstract This study compared the effectiveness of four metabolic uncouplers (p -chlorophenol, m -chlorophenol, m -nitrophenol and o nitrophenol) in reducing sludge production from an activated sludge process. A series of batch experiments fed with different concentrations of individual metabolic uncouplers were conducted at constant initial biomass and glucose concentrations. Results showed that sludge production was reduced with the increase of metabolic uncoupler concentration from 0 to 20 mg l 1. Among four metabolic uncouplers studied, m -chlorophenol was the most effective in reducing sludge production, and had less effect on the process performance, e.g. 86.9% of sludge reduction was achieved at a m -chlorophenol concentration of 20 mg l1, while the COD removal efficiency was lowered only by 13.2% as compared to the control test. The acidity constant (pKa) of a metabolic uncoupler highly influenced its effectiveness in sludge reduction, i.e. metabolic uncoupler with a lower pKa value has a higher potential to reduce sludge production. It appears that selection of metabolic uncoupler and optimization of its dosage should be based on a compromise of the desired sludge reduction and system performance. # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Metabolic uncoupler; Sludge reduction; Growth yield; COD removal efficiency

1. Introduction The basic function of a wastewater biological treatment process is to convert soluble organics to carbon dioxide, water and bacterial cells. The produced cells need to be separated from the purified water and disposed of in a concentrated form called excess sludge. The excess sludge generated from the biological treatment process is a secondary solid waste that must be disposed of in a safe and cost-effective way. So far, the ultimate disposal of excess sludge has been and continues to be one of the most expensive problems faced by wastewater utilities, e.g. the treatment of the excess sludge may account for up to 65% of the total plant operation cost [1]. The requirements of new European Union laws on municipal wastewater treatment will create a doubling in sludge production by the end of * Corresponding author. Tel.: /65-67905254; fax: /65-67910676. E-mail address: [email protected] (Y. Liu).

2005, while at the same time placing additional restrictions on sludge disposal [2]. It can be anticipated that with the expansion of population and industry the increased excess sludge production will generate a real challenge in the field of environmental engineering technology. It must be realized that sludge disposal to all the established outlets could become increasingly difficult or, in the case of sea disposal, will become illegal by this year, while the regulations of food safety and agriculture in most countries are becoming more and more stringent in relation to application of biosolids in agriculture. In order to solve excess sludge-associated environmental and economic problems, three major technical approaches have been seriously considered. These are (i) to convert the excess sludge to value-added construction materials [3]; (ii) to manage existing outlets of sludge disposal [2], and (iii) to reduce sludge production from the wastewater biological treatment process rather than the post-treatment or disposal of the sludge generated.

0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00019-0

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X.-F. Yang et al. / Process Biochemistry 38 (2003) 1373 /1377

Chemical oxidation methods such as sludge alkaline heat treatment, ozonation and chlorination has received intensive attention in the past years [4 /6]. Recent research looked into the feasibility of using metabolic uncouplers to reduce excessive sludge production from the wastewater biological treatment process [7 /9]. However, previous works only focused on one individual metabolic uncoupler, and information on how to select a more effective metabolic uncoupler is lacking. Thus, the main objectives of this study were to compare effectiveness of four metabolic uncouplers in reducing sludge reduction as well as to investigate their effects on process performance. It is expected that the present work will be helpful for selection of metabolic uncoupler and optimization of its dosage for sludge reduction.

2. Materials and methods The biomass used for batch experiments was taken from a laboratory scale fill-and-draw system (4.5 l) with a mean sludge retention time of 5 days. The system was fed with a mixture of glucose as a sole carbon source for bacterial growth, KH2PO4, Na2HPO4, NH4Cl, CaCl2 and FeCl3. The same synthetic substrate was used in the following batch experiments. For all batch tests, initial biomass and substrate concentrations were fixed at 2000 mg mixed liquor suspended solids (MLSS) l 1 and 400 mg chemical oxygen demand (COD) l 1, respectively. Four metabolic uncouplers (p -chlorophenol, m -chlorophenol, m -nitrophenol and o-nitrophenol) were added to batch cultures in a concentration range of 0 to 20 mg l 1. Batch tests were carried out at 259/1 8C and pH 7.0, while dissolved oxygen concentration was greater than 2.0 mg l 1. Soluble glucose-COD and MLSS concentrations were determined by standard methods [10]. The observed growth yield (Yobs) was calculated as the maximum increase in biomass concentration, divided by the corresponding decrease of soluble glucose-COD concentration during batch experiment.

Fig. 1. Effects of p -chlorophenol (p -CP) on Yobs and COD removal efficiency. (m) Yobs; (k) COD.

removal efficiency versus initial m -chlorophenol concentration are presented in Fig. 2 as in Fig. 1, sludge production was proportionally reduced on increasing the m -chlorophenol concentration ranging from 0 to 20 mg l 1, while the COD removal efficiency showed a declined trend. At m -chlorophenol concentration of 20 mg l 1, Yobs was reduced by 86.8%, and COD removal efficiency was decreased by 13.5% as compared to the control test without addition of m -chlorophenol. It appears from Figs. 1 and 2 that m -chlorophenol is more effective than p-chlorophenol in reducing sludge production. 3.2. Effects of nitrophenol on Yobs and COD removal efficiency The effects of m -nitrophenol on sludge production and COD removal efficiency are shown in Fig. 3. Both sludge production and COD removal efficiency exhibited a decreasing trend with an increase of m -nitrophenol concentration in a range of 0/20 mg l1. At m nitrophenol concentration of 20 mg l 1, the sludge yield was decreased by 65.5% companied with a 13.2% reduction in COD removal efficiency. Fig. 4 shows

3. Results 3.1. Effects of chlorophenol on Yobs and COD removal efficiency Fig. 1 shows the effects of p -chlorophenol concentration on the observed growth yield (Yobs) and COD removal efficiency. Increased p -chlorophenol concentration results in a decreased Yobs and COD removal efficiency. At a p-chlorophenol concentration of 20 mg l 1, sludge production in terms of Yobs was decreased by 58 and 8.9% reduction in COD removal efficiency was also observed as compared to the control test without addition of p-chlorophenol. Yobs and COD

Fig. 2. Effects of m -chlorophenol (m -CP) on Yobs and COD removal efficiency. (m) Yobs; (k) COD.


Fig. 3. Effects of m -nitrophenol (m -NP) on Yobs and COD removal efficiency. (m) Yobs; (k) COD.

Fig. 4. Effects of o -nitrophenol (o -NP) on Yobs and COD removal efficiency. (m) Yobs; (k) COD.

changing trends of Yobs and COD removal efficiency against initial o -nitrophenol concentration. When the o nitrophenol concentration was increased from 0 to 20 mg l 1, the observed sludge yield declined from 0.65 mg MLSS mg 1 COD to 0.091 mg MLSS mg 1 COD accordingly, and COD removal efficiency was reduced by 26%, indicating a sludge reduction of 86.1% compared to the control test free of metabolic uncoupler. Comparison of m -nitrophenol and o -nitrophenol suggests that o -nitrophenol is a stronger metabolic uncoupler in terms of sludge reduction.

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(ATP). In aerobic bacteria, ATP is generated by oxidative phosphorylation, in which process electrons are transported through the electron transport system from a source of electrons at elevated energy levels (substrate) to a final electron acceptor (oxygen). The chemiosmotic theory shows that oxidative phosphorylation is driven by a proton gradient built up across the cell membrane [11]. However, metabolic uncouplers known as protonophores are able to diminish the proton gradient across cell membrane. As a result, the energy generated from the oxidation of organic substrate would be lost as heat rather being captured in ATP. Consequently, growth efficiency is reduced in uncouplercontaining microbial culture as observed in Figs. 1 /4. The results presented in Figs. 1 /4 exhibit a quasilinear relationship between Yobs and metabolic uncoupler concentration added to the batch cultures. This seems to suggest that dissociation of energy metabolism is proportionally related to the concentration of metabolic uncoupler. It is known that metabolic uncouplers can diffuse relatively freely through the phospholipid bilayer [14], hence the net rate of transport of metabolic uncoupler should be proportional to the concentration difference across the membrane. Once inside the membrane, the deprotonation of the phenolic hydroxyl would dissipate the proton gradient that is a driving force for ATP production. As a result, higher concentration of metabolic uncoupler favour energy dissociation leading to reduced sludge production. It should be pointed out that addition of metabolic uncoupler does not block electron transport along the respiratory chain to oxygen, this is a reason why over 80% of glucose can still be utilized by activated sludge microorganisms (Figs. 1 /4). Fig. 5 summarizes the effects of four metabolic uncouplers on sludge reduction as well as on the system performance compared to the control test without addition of metabolic uncoupler. It can be seen that at the same individual uncoupler concentration (20 mg l 1), m -chlorophenol and o -nitrophenol are more effective than the others in sludge reduction, however o-

4. Discussion Figs. 1 /4 show that addition of metabolic uncoupler to an activated sludge batch culture can significantly reduce sludge production, and uncoupler-induced reduction in COD removal efficiency is much less important compared to lowered sludge production. The metabolic network can be regarded as the sums of biochemical transformations that include interrelated catabolic and anabolic reactions, and biosynthesis is strongly dependent upon the availability of bioenergy

Fig. 5. Comparison of four metabolic uncouplers at a concentration of 20 mg l 1 in terms of sludge reduction (j) and reduction in COD removal efficiency (I).

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nitrophenol resulted in a more significant reduction in the COD removal efficiency (26%), which is nearly twofold higher than that observed in the presence of m chlorophenol (13.2%). These results seem to indicate that m -chlorophenol is a good uncoupler candidate for sludge reduction if a compromise between sludge reduction and system performance in terms of COD removal is taken into account. In fact, p -nitrophenol and 3,3?,4?,5-tetrachlorosalicylanilide have been used to reduce sludge production [7,12]. A shift in the predominant microbial species that occurred upon introduction of p-nitrophenol was observed [16]. Thus, it seems reasonable to consider that the decreased degree of COD removal efficiency is the result of combined effect of species shift, reduced biomass production and potential inhibition induced by chemical uncoupler. Fig. 6 displays the relationship between the acidity constant (pKa) of individual metabolic uncouplers and sludge reduction percentage at an uncoupler concentration of 20 mg l 1. The pKa values used in Fig. 6 are taken from Solomons and Fryhle [13]. It should be realized that the four metabolic uncouplers studied can be classified into two groups, chlorophenols and nitrophenols. The stronger the acid, the smaller is its value of pKa and for the respective chlorophenolic and nitrophenolic uncouplers, Fig. 6 shows that a lower pKa leads to a higher sludge reduction, i.e. metabolic uncoupler with a low pKa value are more effective in dissociating energy metabolism, favouring sludge reduction. In fact, metabolic uncouplers include a diverse group of molecules structurally, but they are all lipophilic weak acids. All organic metabolic uncouplers are protonophores. They can diffuse relatively freely through cell membrane and release a proton to the solution on one side of the membrane and then diffuse across the membrane to fetch another proton [14]. In a normal microbial culture free of metabolic uncoupler, the proton motive force (pmf) for ATP generation can be quantified as follows:

pmf /DC/2.3RT DpH/F [15]. In this equation, DC is called the membrane potential, and DpH is the pH gradient measured from inside to outside, which has a negative value. However, when a metabolic uncoupler is added to the culture, DpH will shift from negative to positive value due to releasing a proton ion of metabolic uncoupler inside the membrane. In this case, the pmf equation shows that the pmf would be diminished significantly, even be zero. Low pKa values favour deprotonation of the phenolic hydroxyl group in chlorophenolic and nitrophenolic uncouplers. Thus, it is reasonable to consider that in the metabolic uncoupler-containing culture, DpH is highly influenced by the pKa value of metabolic uncoupler, i.e. a lower pKa would result in a weaker pmf, and further a reduced sludge production. These provide a plausible explanation for the phenomena observed in Fig. 6. It appears from the above discussion that there would exist a critical metabolic uncoupler concentration at which the pmf for ATP generation disappears completely, i.e. at that critical concentration, a zero sludge production would be expected. Such a theoretical analysis is supported by experimental findings. In a 2,4-aminophenol-containing activated sludge batch culture, zero sludge production was achieved when the 2,4-aminophenol concentration was higher than 20 mg l1 (data no shown). Similarly, it has been reported that at a pnitrophenol concentration of 120 mg l 1, no excess sludge was produced [12]. These results obtained suggest that addition of metabolic uncouplers to biological wastewater treatment systems can significantly reduce sludge production, without serious perturbation on the system performance, but reduced sludge production is associated with a lowered COD removal efficiency. Figs. 1/4 imply that metabolic uncoupler-induced sludge reduction is at the cost of sacrificing the system performance. In a sense of industrial application, when attempts are made to select a metabolic uncoupler and further to optimize its dosage, a compromise between sludge reduction and system performance should be seriously considered.

5. Conclusions

Fig. 6. Relationship between the acidity constant of metabolic uncoupler (pKa) and sludge reduction at the uncoupler concentration of 20 mg l 1.

Metabolic uncouplers are very effective in reducing sludge production from activated sludge process. Compared to the control test, sludge production can be reduced by over 85% at 20 mg l 1 m -chlorophenol and o -nitrophenol, respectively. The addition of metabolic uncoupler up to a concentration of 20 mg l 1 slightly perturbed the system performance, such that the COD removal efficiency was lowered by about 10% as compared to the control test. By compromising sludge reduction and COD removal efficiencies, the results


showed that m -chlorophenol is the most effective among four metabolic uncouplers tested. It was found that stronger lipophilic weak acids may result in a lower sludge production, and this can be used as criteria of selecting more effective metabolic uncoupler. Consequently, metabolic uncoupler-enhanced sludge reduction technique would have great potential in future industrial applications.

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