Biochemical Engineering Journal 50 (2010) 150–151
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Biochemical Engineering Journal
journal homepage: www.elsevier.com/locate/bej
Letter to the Editor
Technical comments on “Oxic-settling-anoxic (OSA) pro-
cess combined with 3,3 ,4 ,5-tetrachlorosalicylanilide (TCS) to
reduce excess sludge production in the activated sludge system”
by Fenxia Ye and Ying Li, Biochem. Eng. J. 49 (2010) 229–234
(doi:10.1016/j.bej.2010.01.001)
Keywords:
Metabolic uncoupling
OSA
TCS
Energy wastage
Dear Editor,
Ye and Li [1] in a recent article discussed the combined applica-
tion of oxic-settling-anoxic (OSA) system and a chemical uncoupler,
3,3 ,4 ,5-tetrachlorosalicylanilide (TCS) to reduce the excess sludge
production as compared to a control system representing the con-
ventional activated sludge process (ASP) [1]. The application of
uncoupler to wastewater treatment, especially ASPs, is known
for over 15 years and a number of chemicals have been tested
for their efficacy in biomass yield reduction. This is an attractive
option as significant sludge volume reduction is observed for a low
dosage of these chemicals. In the current study, chemical uncou-
pling was coupled with oxic-settling-anoxic system to enhance the
“metabolic uncoupling” of excess sludge.
However, the manuscript does not offer any novel information
as the effects of chemical uncouplers, OSA system and combined
(uncoupler and OSA) systems are already known [2–4]. Previ-
ous works have already described the variations in performance
parameters (TN, TP, TOC and COD removal, etc.) for above men-
tioned modifications in conventional ASP systems [3–5]. The main
strength of the current work, as compared to previous studies by
the same group [3–4], is the application of multiple uncoupler
doses to the combined system. While progressively increasing TCS
doses will help in determining the incremental uncoupling effect
on the performance of combined (OSA + TCS) system; the study
still has much room for improvement. Some suggestions are listed
for enhancing the novelty of the current work and similar future
studies.
1. Does the chemical uncoupling complement energy wastage by
OSA process?
In other words, is it beneficial to combine the two processes?
If the effects are additive, then a combined system is useful; oth-
erwise the better modification of the two should be identified
(on the basis of efficiency as well as practicality). The necessary
investigation would have required minimal changes to the current
1369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2010.03.009
experimental setup. An additional experiment without TCS addi-
tion in the modified system would have acted as a control OSA
system. The observations from this additional experiment com-
bined with the current data could have provided a definitive answer
to the inter-relationship between chemical uncoupling and OSA
process vis-à-vis excess sludge reduction.
2. What is the effect of these modifications on the microbial com-
munity?
The study presented DGGE profiles of samples from control and
modified systems (for all the different TCS doses). It is not clear if
the samples were extracted from aeration tank or settling tank. It is
important to note that the sludge is exposed to aerobic and anaero-
bic conditions as well as a chemical uncoupler. And hence changes
in microbial community distribution and activity are expected. An
ideal place to study such changes is the anoxic sludge holding tank
and should be conducted in combination with additional exper-
iment suggested above (0 mg TCS addition to modified system).
Such a microbial examination will yield new information about the
changes in microbial population and associated long-term effects
of chemical uncoupling and OSA system.
Further improvements could be made by studying the micro-
bial community changes at different time points during the
experimental run. Detailed study of microbial community during
the experiment will also resolve some of the conflicting results
obtained in the current study. An example is the effect of TCS dose
on microbial community. The authors state that at low doses (0.05
and 0.10 g) TCS coupled with anoxic settling reduced the sludge
yield and caused no significant impacts to microbial community.
However at the highest TCS dose of 0.15 g, negative effects in terms
of toxicity and cell death were suspected [1]. Interestingly, Fig. 3
did not reveal significant differences between the control and three
modified systems and lack of any statistical treatment (error bars,
etc.) makes it difficult to draw any concrete conclusions about the
TCS dosage effects on microbial community. This is further sup-
ported by lack of significant differences between the DGGE profiles
for the three modified systems (Fig. 7). Surprisingly, the DGGE
profiles corresponding to 0.05 and 0.15 g TCS are very similar to
each other [1]. Thus authors’ conclusions of TCS toxicity and resul-
tant cell death are not supported by microbial community analysis.
Detailed microbial investigations as suggested above would clearly
reveal the effects of TCS and OSA process on microbial community.
3. Mechanism of sludge reduction in the combined (OSA + TCS) sys-
tem
There is still ambiguity over the exact mechanisms of metabolic
uncoupling and/or energy spilling by chemical uncouplers, OSA sys-
 Letter to the Editor / Biochemical Engineering Journal 50 (2010) 150–151
tem and other metabolic modifications. While multiple processes
can lead to sludge volume reduction, not all of them could be con-
sidered as metabolic uncoupling. An example is the current system,
where authors referred to the OSA system as an uncoupling modi-
fication to the ASP, although the introduction of an anaerobic unit
causes cell starvation.
The idea of “metabolic uncoupling” refers to partial dissociation
between catabolism and anabolism in such a way that the catabolic
efficiency remains unaltered, while anabolism decreases. In other
words, substrate utilization levels are maintained while biomass
yield (biomass growth per unit substrate consumed) reduces. Addi-
tion of chemical uncouplers like TCS is one such example and its
action is usually attributed to leaky cellular membrane that leads
to dissipation of proton motive force (PMF) and resultant decrease
in conversion of ADP to ATP. OSA system, on the other hand, works
on a different mechanism by inducing starvation conditions and
thus microbial metabolism is in itself modified from substrate-rich
to starvation-survival conditions. In fact, Chen et al. [2] ruled out
“energy uncoupling” and reported sludge decay under anaerobic
conditions to be the major cause of excess sludge reduction in
an OSA system [2]. Thus “metabolic uncoupling” is somewhat of
a misnomer for OSA associated sludge (or biomass yield) reduc-
tion.
Further studies are required to determine the exact mechanism
of sludge reduction by TCS addition and OSA system. Changes in
average cellular protein content were observed as a consequence
of TCS addition to a pure culture of Shewanella oneidensis MR-1 [6].
Whether those changes correspond to a build-up of metabolites
as a pathway of energy spilling or are associated with membrane
protein making it leaky would be an interesting study for mixed
ASP microbial community. Similarly, the OSA process is suspected
to lead to energy wastage through anoxic condition induced cell
starvation. A detailed study of how that affects the microbial activ-
151
ity as well as community structure would be a step forward in
understanding the mechanism of these modifications.
References
[1] F. Ye, Y. Li, Oxic-settling-anoxic (OSA) process combined with 3,3 ,4 ,5-
tetrachlorosalicylanilide (TCS) to reduce excess sludge production in the
activated sludge system, Biochem. Eng. J. 49 (2010) 229–234.
[2] G.H. Chen, K.J. An, S. Saby, E. Brois, M. Djafer, Possible cause of excess sludge
reduction in an oxic-settling-anaerobic activated sludge process (OSA process),
Water Res. 39 (2003) 3855–3866.
[3] F. Ye, Y. Li, Uncoupled metabolism stimulated by chemical uncoupler and oxic-
settling-anerobic combined process to reduce excess sludge production, Appl.
Biochem. Biotechnol. 127 (2005) 187–199.
[4] F.X. Ye, Y. Li, Reduction of excess sludge production by 3,3 ,4 ,5-
tetrachlorosalicylanilide in an activated sludge process, Appl. Microbiol.
Biotechnol. 67 (2005) 269–274.
[5] F. Ye, R.-F. Zhu, Y. Li, Effect of sludge retention time in sludge holding tank on
excess sludge in the oxic-settling-anoxic (OSA) activated sludge process, J. Chem.
Technol. Biotechnol. 83 (2008) 109–114.
[6] G. Saini, B.D. Wood, Metabolic uncoupling of Shewanella oneidensis MR-1, under
the presence of excess-substrate and 3,3 ,4 ,5tetrachlorosalisylanilide (TCS),
Biotechnol. Bioeng. 99 (2008) 1352–1360.
Gaurav Saini ∗
103 Gleeson Hall, School of Chemical,
Biological and Environmental Engineering,
Oregon State University, Corvallis, OR 97330,
United States
∗ Tel.:
+1 541 223 2550;
fax: +1 541 737 3099.
E-mail addresses: sainig@onid.orst.edu,
gauravsaini@gmail.com
13 February 2010