3rd European Bioremediation Conference
P 032
VARIABILITY OF COMMUNITIES AND PHYSIOLOGICAL
CHARACTERISTICS BETWEEN FREE-LIVING BACTERIA AND
ATTTACHED-BACTERIA DURING THE PAH BIODEGRADATION
IN SOIL/WATER SYSTEM
Yi-Tang Chang1,2, Jiunn-Fwu Lee*,1 and Huan-Ping Chao1
1
Graduate Institute of Environmental Engineering,
National Central University, Chung-Li, 320, Taiwan
2
Department of Safety, Health and Environmental Engineering,
Tung Nan Institute of Technology, Shen-Keng, Taipei, 222, Taiwan
ABSTRACT
Biodegradation of polycyclic aromatic hydrocarbons (PAHs) via attached and
free-living microorganisms in soil/water systems were observed to examine pattern
biodiversities. The predominant free-living species, Brevundimonas (Pseudomonas)
diminuta, Caulobacter sp., Mycoplana bullata, Acidovorax sp. and Pseudomonas
aeruginosa, can be detected by fluorescence in situ hybridization (FISH). Two approaches
including community-level physiological profiling (CLPP) assessed with the Biolog GN
plates and enzymatic activities determined by the API ZYM strip were used to explain
functional diversities between free-living and attached bacteria.
1. INTRODUCTION
PAHs are common organic contaminants in soil and groundwater. Bioremediation is
an effective approach to remove the PAH contamination. PAH bioavailability might be
strikingly affected by their fate and transportation in soil and groundwater [1]. Thus, the
mechanisms of PAH uptake on soils such as absorption and partition can be the key point
for bioremediation. With the different aqueous microenvironments, both free-living and
attached bacteria can biodegrade PAHs in a soil/water system. However, the different
microbial communities and physiological characteristics can be found from the free-living
and attached bacteria. The objective of this study is to differentiate the microbial diversities
between free-living and attached bacteria by FISH. CLPP and enzymatic activities during
PAH biodegradation in soil/water systems are applied to observe the changes in
physiological characteristics. The importance of soil organic matter (SOM) to PAH
biodegradation also are investigated by a clay and a natural soil.
2. MATERIAL AND METHOD
2.1 Chemicals and soils
The selected PAHs, naphthalene (Nap) and phenanthrene (Phe) are purchased from
the Acros and the Fluka companies, respectively. A clay, Ca-montmorillonite (bentonite)
and a natural soil Taichung soil (TCS) which had comparable characteristics such as BET
surface area, SOM and CEC were applied for the biodegradation experiments.
2.2 PAH biodegradation experiments
During the batch experiments, PAH-degrading bacteria of chemostat were transferred
into different sterilized PAH in mineral salts basal (MSB) with the different soil/water
systems. Serum bottles containing the above substrates were incubated using a
reciprocating shaker at 125 rpm under room temperature (28oC averagely) in a dark
environment. Inoculum was sampled regularly. Then inoculum was centrifuged and filtrated
to separate the solution into free and attached aqueous samples.
2.3 PAH analysis
Samples were extracted by CH2Cl2 and then the extracts were injected into HPLC to
determine Nap and Phe concentration. The HPLC equipped with a UV detector at 275 nm
(ECOM, Czech Republic) and C18 column. The operating conditions were as following:
1.0 mL/min mobile phase was composed of 80% acetonitrile and 20% deinoized water.
2.4 Bacterial numbers, community and physiological activities
The numbers of free bacteria were examined using the spreading plate technique on
three different mediums. Total microbial count on TGE-agar and R2A-agar was observed. A
selective medium of Pseudomonas-agar-P (Basis) supplied by the Merck Company
(Germany) were used to count the numbers of the Pseudomonads family. In addition, the
changes in the bacterial community were determined by one of bimolecular tools, FISH.
The detected probes contained EUB338, ALF1b, BET42a, GAM42a, Pdi, ACI208, PseaerA
and NONEUB338. More details on oligonucleotide probes and experiment procedures have
been described in probeBase [2].
Functional similarity of free-living and attached bacteria during PAH biodegradation
were performed by the Biolog GN microplate. The sampled inoculum was added into each
well of the microtiter plates. After incubation at 30oC for 60 hrs, the average absorbance
values of microtiter plates were measured repeatedly at 450 nm and 650 nm with an ELISA
reader (StatFax, U.S.A.). The true purple/blue color produced represents as a positive result
in the Biolog assay. CLPP was developed by the average well color development (AWCD)
and principal component analysis (PCA). Enzymatic activities were determined by the API
ZYM strips (Bio Mérieux SA, Lyon, France). The procedure of the API-ZYM strip in this
study was to inject the inoculum into each microcupule and then to incubate inoculum at
same conditions as CLPP. The reacted activity in each microcapsule was scored from 0 to
40 nmole, depending on the chromogenic substrate intensities of enzymatic reactions.
3. RESULTS
3.1 PAH biodegradation
Table 1 indicates the pseudo-first order rate constants (kT) for the PAH biodegradation
in soil/water systems. kFR (for free-living bacteria) and kAT (for attached bacteria) values
were also obtained individually. It can be reasonably found the kT for Nap-TCS was higher
than that for Phe-TCS according to the difference in the chemical structure of PAHs.
However, the relatively higher kT of Phe in bentonite/water system indicates that
PAH-biodegarders prefer utilizing Phe to Nap under a specific condition. This might be
ascribed to presence of specific enzymes in the system to increase Phe degradation. The kT
of Nap-TCS is higher than that of NAP-bentonite but Phe exhibits an opposite trend. The
different mechanism of PAH uptake between TCS and bentonite is thought as the possible
reason. Nap biodegradation might be accelerated by the “priming effect” due to the
contained 1.883% natural organic matters in TCS. On the other hand, the major mechanism
of PAH uptake on TCS is partitioning to the SOM. Phe with the relatively higher Kow has a
greater partitioning amount. The difficult Phe desorption from SOM therefore restricted an
effective biodegradation. Another explanation mentioned for the “priming effect” on Phe
biodegradation was so insignificant as to inhibit Phe bioavailability. Moreover, the kFR
value is always higher than the kAT value in the same soil/water system. It can be inferred
that biodegradation of PAH uptake on the soils is limited because attached bacteria move
poorly. As expected, the higher kFR values were found, which demonstrates free-living
bacteria possess relatively higher PAH-biodegrading activities.
Table 1 Pseudo-first order degradation rate constants of PAHs in soil/water systems, kT =
total degradation rate constant, FR = free PAHs, AT = attached PAHs
Run No.
1
2
3
4
PAHs in different
Nap-bentonite
Phe-bentonite
Nap-TCS
Phe-TCS
soil/water systems
kT (day-1)
0.149
0.382
0.842
0.139
FR
AT
FR
AT
FR
AT
FR
AT
kFR or kAT (day-1)
0.038
0.031
0.110 0.096 0.482 0.348 0.118 0.053
3.2 Bacteria population
The changes in microbial numbers and communities were examined by total plate
count and FISH. The SOM obviously affected the microbial numbers. There is a significant
total count increase in the free-living bacterial population of 103-106 CFU/ml in the
bentonite/water systems but a slight increase of 101-102 CFU/ml in the TCS/water system.
The approximate numbers and identical increasing trend between Pseudomonas species and
total count furthermore demonstrate that Pseudomonas species are dominant species in all
systems. To examine different bacterial communities, the percentages of Domain Bacteria
in free-living bacteria and attached bacteria reached to the range from 43.31±7.67% to
52.83±7.84% and the range from 43.58±6.60% to 51.23±6.15%, respectively. Three phyla,
α-, β- and γ-Proteobacteria were mainly included in Bacteria. The percentages of
γ-Proteobacteria in attached bacteria populations were obviously changed from
15.54±4.84% to 29.32±6.02% for Nap biodegradation and from 11.14±2.86% to
13.26±2.25% for Phe biodegradation. Especially, there are the distinguished differences in
specific bacterial communities during biodegradation as detected by Pdi, ACI208 and
PseaerA probes. The percentage of Brevundimonas (Pseudomonas) diminuta, Caulobacter
sp., Mycoplana bullata, Acidovorax sp. and Pseudomonas aeruginosa were dominated in
free-living bacteria, ranged from 20.65% to 29.67%. Attached bacteria merely contained a
small ratio of these specific bacteria, ranged from 5.18% to 12.53%.
3.3 Physiological characteristics
3.3.1 CLPP
Figure 1 illustrates that the CLPP variation for free-living and attached bacterial
communities in different soil/water systems. The attached bacteria sorbed on
bentonite are considered to inhibit their PAH biodegradation to demonstrate a
comparable variation in physiological characteristics. For TCS, free-living and
attached bacterial communities of CLPPs were similar. This result needs to be
discussed furthermore.
Figure 1 CLPP of PAH biodegradation in different soil/water systems: (A) Nap and (B) Phe.
Free-living bacteria on bentonite of biodegrading Nap and Phe were presented by
Arean and Areaq, respectively. Attached Bacteria on bentonite of biodegrading
Nap and Phe were presented by Areao and Arear, respectively. Bacteria in TCS
systems were presented by Area p for Nap and Area s for Phe.
3.3.2 Enzymatic activities
The enzymatic activities of the API ZYM system for Nap and Phe
biodegradation in soil/water systems were determined by the PCA method. Esterases,
aminopeptidases and some enzymes related to glycolysis and gluconeogenesis have
an identifiable correlation to PAH biodegradation with free-living or attached
microorganisms. In contrast, Nap biodegradations for the enzymatic activities is
influenced by soil properties, but attached or free-living bacteria only lead to slight
changes. For phe, an obvious variety of enzymatic activities in free-living and
attached bacteria was detected. Few differences in enzymatic activities were acquired
from the two soil/water systems.
4. CONCLUSION
PAH biodegradation in soil/water systems obviously was affected by the chemical
structure of PAHs and the SOM content. The free-living or attached microorganisms
possibly lead to the differences in the biodegrading rates of PAHs, the variety of bacterial
community, physiological roles and enzymatic activities. PAHs in the different soil/water
systems may also generate a disparate result. It is necessary that the complete evaluation for
biodegrading organic pollutant should be performed before in situ bioremediation.
REFERENCES
[1] Lehman, R.M., F.S. Colwell and G.A. Bala (2001) "Attached and Unattached Microbial
Communities in a Simulated Basalt Aquifer under Fracture- and Porous-Flow
Conditions", Appl. Environ. Microbiol. 67:2799-2809.
[2] Loy, A., M. Horn and M. Wagner, (2003) "probeBase - An Online Resource for rRNAtargeted Oligonucleotide Probes", Nucleic Acids Res. 31:514-516.