Applied Mechanics and Materials
ISSN: 1662-7482, Vol. 108, pp 217-223
doi:10.4028/www.scientific.net/AMM.108.217
© 2012 Trans Tech Publications, Switzerland
Online: 2011-10-24
An Acetic-Acid Type Climax Community and Its changes Aroused by
COD/SO42- Ratio in An Acidogenic Sulfate-Reducing Reactor
Hongxu BAO1,2, Xiping MA1,2, Jian WANG1,2, Kui JING 1,2，Zhihui CHEN1,2，
Aijie WANG3
1
School of Environmental Science, Liaoning University, Shenyang 110036, China
2
Key Lab of Pollution Control and Environmental Remediation of Liaoning Province, Shenyang
110036, China
3
State Key Laboratory of Urban Water Resources and Environments, Harbin Institute of
Technology, Harbin 150090, China
Baohongxu@ lnu. edu. cn，Cicilia_305@ hotmail.com
Keywords:Acetic-acid Type Microbial Metabolism; Acetic-acid Type Climax Community;
COD/SO42- ratio; ecological succession; sulfate-reducing bacteria (SRB)
Abstract.The objective of this study is to investigate the microbial community and its
characteristics changes aroused by the ratios of COD/SO42- in the acidogenic-phase reactor of twophase anaerobic process. A continuous-flow lab-scale test was conducted in an acidogenic sulfatereducing reactor with molasses wastewater as sole organic carbon source and sodium sulfate as the
electron acceptor. The experimental results showed that Acetic-acid Type Microbial Metabolism
resulted in the formation of An Acetic-acid Type Climax Community. The change of the
COD/SO42- ratio caused an ecological succession from a stable climax community at moderate and
high COD/SO42- ratios to a sub-stable climax community at a lower COD/SO42- ratio. But Aceticacid Type Microbial Metabolism kept unchanged during this course, which indicated the stability of
Acetic-acid Type Climax Community. The Acetic-acid Type Microbial Metabolism and Acetic-acid
Type Climax Community were of typical characteristics in the acidogenic sulfate-reducing reactor.
Introduction
It is well known that acid-producing phase reactor of two-phase anaerobic treatment process has
remarkable advantages for the treatment of sulfate-laden wastewater [1]. In such a reactor, sulfate
could be converted to sulfide (including H2S, HS- and S2-) by the cooperation of sulfate-reducing
bacteria (SRB) [2], acidogenic bacteria (AB), and hydrogen producing acetogens (HPA).
Furthermore, AB could endure higher sulfide concentration than other anaerobes, and the existence
of SRB accelerates the acidification of organic substrates significantly. In addition, most of sulfide
exists as H2S in such a reactor, which is easy to be stripped off by gas stripping or stirring, and
consequently alleviate its toxicity on SRB and AB as much as possible[3,4]. Till now, the main
research achievements on this aspect are shown as follows: Sarner (1988) [5] proved that when
anaerobic filter was employed as acid-producing phase reactor of two-phase anaerobic treatment
process to treat pulp-wastewater, sulfate removal rate could reach 63% under the following
conditions: influent COD concentration was 19300 mg/1, influent sulfate concentration was
5225mg/1and pH value in this reactor was at 6.0~6.3；Gao (1989) [6] substantiated that when
continuous-flow stirred tank reactor (CSTR) was adopted as acid-producing phase reactor of twophase anaerobic treatment process to treat whey wastewater, sulfate removal rate could reach 88%
under the following conditions: influent COD concentration was 8500 mg/1, influent sulfate
concentration was 1000mg/1and pH value in this reactor was at 6.1~6.2; Zuo (1996) and Li (2000)
presented novel process including three units of sulfate-reduction [7,8], sulfide-oxidation and
methane-production for the treatment of sulfate-laden wastewater, which has high sulfate removal
capability; Wang (2001) certified that over 80% sulfate removal rate could be obtained as long as
COD/SO42- ratio was higher than 2.0 and sulfate loading rate was lower than 7.5kg SO42-/m3·d in an
acidogenic sulfate-reducing reactor [9].
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218
Mechanical Engineering and Materials Science
However, most of the research work above mentioned focused on the reactor performance and
seldom investigated the microbial behavior and characteristics in acid-producing phase reactor [10].
The main achievements to date in this area is summarized as follows: 1) detecting the ecological
distribution of microbes in bio-film reactors using molecular biological techniques, such as gene
probe and fluorescent in situ hybridization [11]; 2) discussing the effect of ecological factors such as
pH and the COD/SO42- ratio[12] on SRB [13,14]; 3) proving that SRB play an important role of in
the inter-species hydrogen transfer during the course of their cooperation with AB and HPA[1]; 4)
analyzing the nutrition diversity of SRB: four groups of SRB have been classified according to their
substrates, which are HSRB (with hydrogen as substrate) [15,16], ASRB (with acetate as substrate),
FSRB (with VFAs containing three or more carbon as substrates), and the group that utilize
aromatic compounds as substrates[18]. The objective of this study is to investigate the microbial
community in such a system and its changes as in response to variations in the COD/SO42- ratio.
This was conducted in a continuous-flow acidogenic sulfate-reducing reactor [14] with molasses
wastewater as the sole organic carbon source and sodium sulfate as the electron acceptor.
Materials and Methods
A. Wastewater influent
Sodium sulfate was used as electron acceptor and molasses wastewater obtained from a beet
sugar refinery was used as electron donor. The ratios of molasses (COD) to sodium sulfate (SO42-)
in the influent were varied in different tests. Adequate amounts of nitrogen and phosphorus were
added to substrate to ensure a nutritional balance in both the continuous-flow and the batch tests.
For the continuous-flow experiment, the COD/SO42- ratios were varied at 5.0, 3.0, 4.0 and 2.0
sequentially as shown in Table I.
TABLE I THE OPERATIONAL CONDITIONS OF EXPERIMENTS
* Volumetric sulfate loading rate
B. Analytical methods
Parameters such as COD, BOD, MLSS, MLVSS and pH were determined according to
Standard Methods [18]. H2S was analyzed by GC-122 gas chromatograph (Shanghai Analytical
Apparatus Corporation, Shanghai) with a thermal conductivity detector. Ethanol and VFA including
acetate, propionate and butyrate etc. were also analyzed by a GC-122 gas chromatography using a
flame-ionization detector (FID). The separation was accomplished by a 2.0-m stainless steel column
packed with GDX103 (60/80 mesh). Nitrogen was used as carrier gas at a flow rate of 60 mL/min.
The flow rates of hydrogen and oxygen were 50mL/min and 490mL/min, respectively. The
temperatures for detector and oven were 210℃ and 190℃, respectively [19]. Sulfate was determined
by an ion exchange chromatograph (CDD-6A, Shimadzu, ShimpackIC-AI, Column Temperature
40℃, mobile phase 2.5 mM of potassium hydrogen phthalate). Sulfide was determined by the
methylene blue colorimetric method as described by Truper and Schlegel [20]. H2 and CO2 in biogas
were analyzed by a gas chromatography (Model SC-2 Shanghai Analytical Apparatus Corporation,
Shanghai) using a thermal conductivity detector (TCD) and a 2.0-m stainless steel column packed
with TDS-01 (60/80 mesh). Nitrogen was used as the carrier gas at a flow rate of 70mL/min. The
temperatures for detector and oven were both at 150℃[21]. The ORP in the reactor (Eh) was
Applied Mechanics and Materials Vol. 108
219
calculated from the equation of Eh = Ec + 249.1[mV], where Ec was the observed ORP measured
by an acidity voltmeter (pHS-25, Shanghai Analytical Apparatus Corporation, Shanghai) and 249.1
was the potential value of saturated calomel electrode.
C. Enumeration of anaerobes
SRB was enumerated using a serial dilution technique [22]. One liter of culture medium
contained 0.5g K2HPO4, 1.0g NH4Cl, 0.5g Na2SO4, 0.1g CaCl2·2H2O, 2.0g MgSO4.7H2O, 1.0g
yeast extract and 4mL sodium lactic (70% strength), and the medium’s pH was adjusted to 7.4~7.6.
Enumerations of AB and HPA were carried out by the three–tube Most Probable Number
(MPN) technique [18]. The culture medium for AB included 10.0g Glucose, 5.0g peptone, 3.0g
beeves, 3.0g NaCl, 0.5g cysteine and 0.002g resin lazuline in 1000ml distillated water with a final
pH value of 7.2~7.4. The culture medium for HPA included 30mmol CH3CH2COONa, 2.0g yeast
extract, 0.1g MgCl2, 1.0g NH4Cl, 0.4g K2HPO4, 0.5g cysteine and 0.002g resin lazuline in 1000ml
distilled water with a final pH value of 7.0 ~7.3.
D. Experimental apparatus
A continuous-flow reactor [9] was used as the acidogenic sulfate-reducing reactor in this study.
It was a continuous stirred tank reactor (CSTR) equipped with an internal gas-liquid-solid-phase
separator. The reactor had a total volume of 20.7L and an effective volume of 9.6L. It was designed
to maintain an anaerobic condition, yet allow H2S gas to release effectively. Temperature was
maintained at 35±1°C during the entire study.
Results and Discussions
A. An Acetic-acid Type Climax Community and its structure
It is well accepted that the most important indices for an acid-producing phase reactor are
acidification type and acidification extent achieved [23]. Acidification type is referred to the
microbial metabolism pathway according to the proportion of VFA in the end liquid products. Till
now, three acidification types were presented. That is, propionate acid type and butyric acid type
fermentation [23], and ethanol type fermentation [19]. Acidification extent is the percentage of
effluent COD that express with the accumulated VFA divided by influent COD as shown in the
following equation(1):
VFA effluent (in COD) / COD influent ×100%
(1)
A Comparison of end-products distribution under the conditions of supplying sulfate in
influent to that of no sulfate in influent in acidogenic sulfate-reducing reactor is shown in Table II.
Obviously, when there is no sulfate in influent, the microbial acidification is ethanol-type
fermentation according to Ren’s viewpoint [19]. We could deduce that the metabolic pathway of
ethanol-type fermentation was attributed to the function of AB and HPA, because SRB could not
obtain electron donor under this conditions.
However, when supplying sulfate in influent, the proportion of acetic acid in end products is
from 50.6% to 72.2%, which is much higher than that of butyric acid, propionic acid, valeric acid
and ethanol. Therefore, the authors submitted a new concept of Acetic-Acid Type Microbial
Metabolism to explain this kind of microbial acidification type [9]. Consequently, the community
presenting Acetic-Acid Type Microbial Metabolism was named Acetic-acid Type Climax
Community.
TABLE II COMPARISON OF END-PRODUCTS DISTRIBUTION IN DIFFERENT CONDITIONS
Note: Values in the table are statistical averages in different experimental phases. When SO42-=0, 600, 1000, 1000, and
2000mg/L, the numbers of samples were 50, 70, 66, 65, and 50 respectively. The results are averages of five measured
values ± standard deviations
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Mechanical Engineering and Materials Science
B. Ecological succession of Microbial Community aroused by COD/SO42- ratio
It is generally accepted that COD/SO42- ratio is the key affecting the course of Sulfatereduction[24-26] Therefore, the change in the Acetic-acid Type Climax Community as a function of
the COD/SO42- ratio was investigated. The ecological succession of climax community aroused by
COD/SO42- ratio and its characteristics are shown in Table III.
TABLE III ECO-SUCCESSION OF COMMUNITY AROUSED BY COD/SO42- RATIO AND ITS CHARACTERISTICS
Note: the values of pH, ORP ALK, proportion of acetic acid and sulfate-reducing rate in table IV are statistical averages according to different
experimental stage. When COD/SO42-=3.0, 4.0 and 2.0, the amount of samples are 66, 65, and 50 respectively.
It is indicated that a stable climax community at moderate COD/SO42- ratio formed when
decreased COD/SO42- ratio from 5.0 to 3.0. The proportion of acetic acid increased from 50.6% to
64.7%, indicating the formation of the Acetic-acid Type Microbial Metabolism. Correspondingly,
the predominant populations changed significantly. Those of Streptococus, Aeromonas,
Fosobucterium, Clostridium, and Sporosarcina substituted Acetogens genera of Dialister,
Zymomonas. Meanwhile, those of Desulfobacterium, Desulfotomaculum and Desulfococcus
substituted the SRB genera of Desulfobactor. The genera differentiation was based on feature exams
and biochemical tests of the isolated pure cultures. In addition, the level of sulfate removal
increased from 70% to 86% (shown in Table III). The enumeration of the dominant populations
shown in Table IV indicate that the amount of SRB population was as high as (7.5±0.2)×1015 per ml,
that of AB population was a order of magnitude higher than SRB population, and that of HPA
population was a order of magnitude lower than SRB population.
TABLE IV ENUMERATION OF THE DIFFERENT MICROBIAL POPULATIONS IN DIFFERENT EXPERIMENT PHASES
Generally, increasing Ns would reduce pH value in the acidogenic reactor [27]. But in the
course of decreasing COD/SO42- ratio from 5.0 to 3.0, Ns was increased from 3.0 to 4.0 kg SO42(m3·d)-1 by the way of extending HRT from 4.8h to 6.0h, which became the key causing substantial
changes in pH, ORP, ALK and percentage of acetic acid. pH value increased from 5.1 to 6.0, ORP,
being negatively related to the pH value, decreased from -280mV to -380mV. Since sulfate
reduction is an alkalinity-adding course, the value of ALK increased dramatically with sulfate
removal level being increased from 70% to 88%. Gas products increased greatly because genera of
Aeromonas and Aerobacter became dominant, but genus of Desulfobacter with acetic acid as
substrate declined[28-30].
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221
Stable climax community at high COD/SO42- ratio formed when COD/SO42- ratio was
increased from 3.0 to 4.0. During this period, the percentage of acetic acid to end products increased
from 64.7% to 72.2%, and the ecological characteristics of the community changed significantly
with the dominant role of Streptococus and Sporosarcina being substituted by that of Leptotrichia.
Analysis of this phenomenon indicated that higher influent COD concentration (from 3000mg/L to
4000mg/L) supplied more available carbon sources, which accelerated the microbial metabolism
and increased the amount of AB population. Thus, the amount of VFA produced by AB increased
dramatically and the FSRB might accept more electron donor[31]. Consequently, the sulfate
removal level increased to over 90%. As indicated in Table IV, the amount of SRB, AB and HPA
population all increased by an order of magnitude. In the same time, AB and SRB produced more
alkaline substances to maintain the stability of such an ecosystem. With the increase of ALK, pH
was elevated and thereby the ORP decreased. In particular, the dominant role of genus Desulfonema
with acetic acid as its substrate was substituted by genus of Desulfococcus with propionate as its
substrate. This resulted in more acetic acid accumulated in end products[32].
Conclusion
Based on the experimental results and above discussion, conclusions could be drawn as
follows.
Acetic-acid Type Microbial Metabolism resulted in the formation of An Acetic-acid Type
Climax Community. Acetic acid Type Microbial Metabolism and Acetic-acid Type Climax
Community were of typical characteristics of acidogenic sulfate-reducing reactor.
Based on the relationships among microbes, authors presented a structure of the Acetic-acid
Type Climax Community, in which, AB, HSRB and ASRB reside at or close to outside surface of
the sludge floc, while HPA and FSRB inhabit inside of the flocs.
During the course of ecological succession from the stable climax community at moderate and
high COD/SO42- ratios to sub-stable climax community at low COD/SO42- ratio, the amount and
composition of dominant populations changed orderly and showed distinct characteristics.
However, Acetic-acid Type Microbial Metabolism kept unchanged, which indicated the stability of
Acetic-acid Type Climax Community.
Acknowledgment
The authors would like to thank the National Natural Science Foundation of China for financial
supports (No. 50208006).
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Mechanical Engineering and Materials Science
10.4028/www.scientific.net/AMM.108
An Acetic-Acid Type Climax Community and its Changes Aroused by COD/SO42- Ratio in an
Acidogenic Sulfate-Reducing Reactor
10.4028/www.scientific.net/AMM.108.217
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