Bioaugmentation combined with biofilim process in the treatment of petrochemical wastewater at low temperatures

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J. Water Resource and Protection. 2008. 1: 1-65.

Published Online June 2008 in SciRes (http://www.SRPublishing.org/Journal/jwarp/).

Bioaugmentation combined with biofilim

process in the treatment of petrochemical

wastewater at low temperatures

Jingbo GUO, Fang MA, Kan JIANG, Di CUI

School of Municipal and Environmental Engineering & State Laboratory of Urban Water Resources and Environment

Harbin Institute of Technology,Harbin,China

E-mail: guojingbo99@yahoo.cn.com

Abstract

Three sets of lab-scale reactors, which applied activated sludge process, bioaugmented activated sludge process and

bioagumented biofilm process, respectively, were operated parallel to explore the optimum process for the treatment of

petrochemical wastewater at low temperatures (13-15℃). Though being inoculated twice with enriched specialized

bacteria, the bioaugmented activated sludge reactor (R2) didn’t show significant overall improvement on effluent

quality when compared with the unbioaugmented reactor (R1) (average removal efficiency, COD: R1=65.02%,

R2=70.39%; NH4+-N: R1=42.07%, R2=52.49%), except for increased levels of enzyme activity as described by

dehydrogenase activity (DHA) and slightly better performance at the early stage of inoculation. Microscopic

observation indicated that free-living cells were scarce in R2 and the main explanation was the grazing of protozoa to

the bioaugmented cells. However, the application of porous polyurethane foam as carrier in the bioaugmented biofilm

reactor (R3) could retain sufficient biomass within the reactor, and the COD (75.80%) and NH4+-N (70.13%) removal

efficiencies were enhanced with more stable performances. In conclusion, massive inoculation couldn’t always warrant

successful bioaugmentation due to predation to the inoculated specialized bacteria, and biofilm process was promising

when combined with bioaugmentation technology in the treatment of petrochemical wastewater at low temperatures.

Keywords: Bioaugmentaion; Low temperature; Activated sludge; Bioflim; Specialized bacteria

1. Introduction

Traditionally, activated sludge process is widely used in

dealing with industrial wastewater owing to its simplicity

and relatively low cost. Petrochemical wastewater is

heterogeneous organic compound mixtures, which

contains quantity of organic compounds that possess

some degree of either toxicity or activity inhibition to the

microorganisms in the biological unit [1]. Moreover, the

microbial activity would be further inhibited under low

temperature conditions [2-4], when the adsorption and

settling ability of the activated sludge would be

influenced. Thus, microorganisms in the activated sludge

system, even well acclimatized, are inefficient in dealing

with petrochemical wastewater containing relatively high

concentration of organic compounds due primarily to low

biodegradability and inhibitory effects of these organic

compounds [5], especially under low temperature

conditions.

Bioaugmentation is the application of indigenous or

allochthonous wild-type or genetically modified

organisms to polluted hazardous waste sites or bioreactors

in order to accelerate the removal of undesired pollutants

[6]. It had been widely used in enhancing the removal

ability of biological system to nitrogen [7] and

phosphorous [8] as well as various organic refractory

chemicals contained in industrial wastewater [9,10].

Although bioaugmentation of activated sludge system

with the introduction of specialized bacteria was

successful in some cases with significant improvement to

the removal of target compounds[11,12], it is not yet

widely applied due to several factors concerning

unfavorable environmental conditions and the

competition between the inoculums and other

microorganisms existed in the system[5,11]. However,

these limitations can be solved by replacing the

suspending biomass system by attached biomass process

[13]. Immobilization of mixed populations of

microorganisms, predominantly bacteria, on or within

inert supports has the following advantages [14,15]: (1)

high reactor biomass concentrations, (2) strong capacity

56 J. GUO ET AL.

to handle shock loadings, and (3) low excess sludge

production.

The objective of this research is to investigate the

effectiveness of bioaugmentation technology and to

explore an optimum bioaugmentation strategy for the

treatment of petrochemical wastewater under low

temperatures (13-15℃). Therefore, three lab-scale

reactors, which applied activated sludge process (R1),

bioaugmented activated sludge process (R2) and

bioagumented biofilm process (R3), respectively, were

operated parallel under low temperatures to compare their

performances in treating petrochemical wastewater.

2. Materials and methods

2.1. Stand-up and operation of the A/O process

Three identical plexiglass anoxic-oxic (A/O) set-ups

(shown in Fig.1), with effective volumes of anoxic tank,

oxic tank and clarifier were 1.5L, 3.5L and 1.25L,

respectively, were adopted. Each aerobic tank was

inoculated with the same amount (0.5L) of activated

sludge (MLSS=4000mg/L) taken from the aerobic tank of

the petrochemical wastewater treatment plant (WWTP).

Polyurentane foams were added as carrier in R3. Under

steady-state, specialized bacteria were bioaugmened into

the oxic tank of R2 and R3, while R1 was operated under

the similar condition without bioaugmentation.

Wastewater collected from the primary settling basin of

petrochemical WWTP (Table1 shows its quality) was fed

into these systems with flow rate increased stepwise to

0.5m3/h. The activated sludge was recycled at a 100%

ratio and the excess sludge was discharge at a 10% ratio

per day from R1 and R2. The hydraulic retention time

(HRT) for anoxic stage and oxic stage was 3h and 7h,

respectively. The dissolved oxygen (DO) in oxic tank was

4.0-6.0 mg/L. The wastewater was 13-15℃ during the

whole process.

Figure 1. The schematic diagram of the experimental A/O

process

Table 1. Influent quality of the biological systems

Parameters Value Level Ⅰ Criteriaa

COD 400~600 100

200~300 30

NH4+-N 30-50 15

SS 70-200 70

Oil and grease ≤80 10

pH 7-9 6-9

a.Integrated wastewater discharge standard of China [16]; Values are in mg/L except

for pH.

2.2. Bioaugmentation method

Specialized bacteria previously isolated from various

environments, which mainly consisted of Pseudomonas,

Bacillus, Acinetobacter, Flavobacterium and Micrococcus,

were functioned as COD degrading bacteria (mainly

consist of oil and grease, phenol and aniline degrading

bacteria), bioflocculant-producing bacteria and denitrifier.

They were primarily acclimated with petrochemical

wastewater after being taken from the refrigerating

chamber and then inoculated into R2 and R3 with a total

dry mass 150 mg/L 4 days after steady-state was achieved.

The second bioaugmentation in R2 was performed 13

days later in the same way while the addition amount

rising up to 700mg/L. Systems were hermetically isolated

from each other to avoid cross-contamination.

2.3. Immobilization on polyurethane foams

Polyurethane foam is considered as a suitable carrier

for cell immobilization for its easy control of the pore size,

stable maintenance of quantity of cells and large-scale

application at low price. It was widely used as a carrier in

the biodegradation of organic compounds [17,18].

Spontaneous adhesion immobilization strategy was

adopted in the present study as it is simple, cheap and

allows significant biomass immobilization [13]. Physical

characteristics of the polyurethane foam are summarized

in Table2. Strip polyurethane foams were stuffed in

spherical polythene plastic (D=80mm) frame to protect

the carrier from being washed out from the system. The

carrier hold-up was 30% in R3.

Table 2. Physical characteristics of the polyurethane

foam Items Values

Pore size 150-500μm

Specific densiy 0.2-0.95

Specific area 2.0×104m2/m3

Acid and alkali resistence ability 5≦pH≦11

Service life 10 years

2.4. Analytical methods

Daily composite samples of influent and effluent were

obtained by mixing samples collected every 6 hours.

COD, NH4+-N were analyzed according to standard

methods [19]. Biomass attached on the polyurethane foam

was removed by microwave agitation, while the biomass

of the activated sludge process was collected directly.

Biomass concentration was determined by filtering the

samples through 0.45 μm millipore filter and then drying

at 105℃ until constant weight. The free-living bacteria

were counted as colony forming unit (CFU) using culture

method. Protozoa was observed by electronic microscope.

For dehydrogenase activity (DHA) quantification, TTC-

DHA method [20] was used. Polyurethane foam with

Influent Anoxic tankOxic tankClarifier

Effluent

Sludge return

BIOAUGMENTATION COMBINED WITH BIOFILIM PROCESS IN THE TREATMENT OF

PETROCHEMICAL WASTEWATER AT LOW TEMPERATURES 57

100

200

300

400

500

600

161116 21 26

Time(d)

COD(mg/L

)

Influent Effluent of R1

Effluent of R2Effluent of R3

1611 162126

Time(d)

-N(mg/L)

biofilm for scanning electron microscope (SEM)

observation was fixed in 4% glutaraldehyde buffer (2.5%,

pH=6.8) for 1.5h at 4℃, then rinsed three times in 0.1M

phosphate buffer (pH=6.8), dehydrated using an ethanol

series (50%, 70%, 80%, 90% once for 10-15min and

100% thrice for 10-15min), died overnight in the

desiccator and then were fixed on metal supports and

sputter coated with gold (10nm) (k550x, EMITECH,

England). Finally, biofilm samples were observed with a

Philips XL30 SEM (Quanta 200, FEI, the Netherlands)

and photographed.

3. Resluts and disscusions

3.1. Performances of each reactor

The daily influent and effluent COD and NH4+-N of

each reactor were shown in Fig.2. When the influent

COD and NH4+-N were 309.00-548.71mg/L and 32.08-

49.26mg/L, respectively, the performances of R2 were

slightly better than that of R1 (average removal efficiency,

COD: R1=65.02%, R2=70.39%; NH4+-N: R1=42.07%,

R2=52.49%). However, the average effluent COD and

NH4+-N of R2 was up to 133.97mg/L and 20.52mg/L

respectively, and the improvement on pollutants removal

efficiency only lasted for about 2 days after

bioaugmentation. The nitrification behavior of R1 was

even better than R2 before the second inoculation, while

slightly better performance was detected in R2 after the

second bioaugmentation. Thus, even R2 was inoculated

twice with the specialized consortia, improvment induced

by bioaugmentation was still unfavorable.

Therefore, under low temperatures, by inoculating

specialized bacteria into the activated sludge set-up,

bioaugmentation technology failed for the treatment of

petrochemical wastewater as the effluent quality couldn’t

meet the national wastewater discharge standards [16].

The previous literatures indicated that bioaugmentation

would be a useful tool for the removal of recalcitrant

organic compounds and the enhancement of the

wastewater treatment systems’ stability under extreme

environments [21], such as low and high temperatures,

saline environments, acidic and alkaline environments as

well as deep-sea environments. Results herein may due to

the difficulty in the ecological control of the added

specialized strains and the other microorganisms involved

in the activated sludge system, thus the availability of the

specialized consortia added was reduced.

With the application of polyurethane foam as carrier,

the average effluent COD and NH4+-N of R3 were

91.69mg/L and 20.52 mg/L, and it’s quite promising

since the average removing efficiency to COD and

NH4+-N reached to 75.80% and 70.13% respectively.

Meanwhile, in the later period, the effluent concentration

COD and NH4+-N were below 90mg/L and 12 mg/L,

respectively. The results suggested that bioaugmented

system with inert support performed better than both the

conventional and the bioaugmented activated sludge

system. The environment created by polyurethane foam

provided a favorable condition for the growth and

proliferation of the inoculums, i.e, the degrading

capability of microorganisms had a better chances to

display with their immobilization on polyurethane foams.

Moreover, compared to R2, since high concentration of

biomass and high cellular retention time were achieved by

biofilm, only one inoculation was conducted..

Figure 2. Influent and effluent characteristics of each

reactor

3.2. Biomass Concentrations and Enzyme

Activities

The average DHA and biomass in each reactor during

one month operation were detected The DHA of the

activated sludge in R2 (1.83mgTF/gMLSS.h) was much

higher than that of the R1 (0.99mgTF/gMLSS.h), while

the average concentration of MLSS in R1 (1100mg/L)

was slightly higher than that of R2 (900mg/L), which

demonstrated that with the addition of specialized

bacteria, the activity of the activated sludge increased

compared to the unbioaugmented one, however, the

inoculums failed to exhibit their capability to decompose

the target pollutants and there was no notable correlation

between pollutants removal efficiency and enzyme

activity, which differed from the conventional concept

that the higher the enzyme activity, the lower organics

remaining in the effluent[20]. This phenomenon may be

particular to low temperature as the microorganisms

needs long period for its lag phase until the degradation

began to display. For biofilm reactor, sufficient biomass

(2000mg/L) was retained with even higher activity

(2.08mgTF/gMLSS.h), which performed best with the

microorganisms bioaugmented in the system. These

results indicated that massive inoculation didn’t always

coupled with favorable performances, and the availability

of inoculums was crucial for successful bioaugmentation.

3.3. Microscope observations

58 J. GUO ET AL.

Obvious proliferation of protozoa after the inoculation

of specialized consortia was observed in R2, while free-

living bacteria were scare. In order to determine whether

the lack of free bacteria was mainly caused by washout or

by protozoa, average free-living bacteria in aeration tank

and secondary tank was counted as colony forming unit

(CFU). Under the same operational conditions, free-living

bacteria in R1 was higher than that of the R2, while the

proportion of free-living cells that lost along with the

effluent were 75% and 30% for R1 and R2, respectively.

It could be inferred that the main reason for the rapid

disappearance of the numerous bacteria inoculated in R2

was not washout but the grazing of the protozoa which

overgrew with the addition of large amount of specialized

bacteria. Conventionally, the proliferation of protozoa

was considered to be an indication for favorable water

quality, however, it was not the case after

bioaugmentation with massive specialized bacteria in the

system, where the ecosystem equilibrium was disturbed.

Thus, it would be advisable to take measures to prevent

the inoculated bacteria from being phagocytized by

overgrowing protozoa.

3.4. SEM observation of biofilm

Microorganisms immobilized on polyurethane foam

were observed by SEM. Images are showed in Fig.3.

There were three kinds of immobilization forms for the

bacteria on polyurethane foam, that is: (a) cells entrapped

in the pores; (b) individual cells distributed randomly on

the on the surface of polyurethane foam; (c) and (d) Cells

congregated together on the surface or in the pores of

polyurethane The numerous microorganisms immobilized

on the porous carrier performed well in the pollution

degradation and were against from the predation of

protozoa and the washing out along with the effluent.

Many previous study pointed out that compared to

conventional free cell systems, the bioreactors with

immobilized cells showed better results including greatly

improved reactor productivity and enhanced withstand

ability to extreme environment such as low temperature

due to its high cell density and optimum microbial

community structure [13,18,19].

However, from Fig.3(a), it was obvious that there was

large percentage polyurethane foam hadn’t been utilized

both for the pores and the surface, possible reasons are

the relatively short operational time or the washing force

of the flow or the unfavorable condition for cells’

immobilization. Thus, certain modification may be

required for the wide application of porous polyurethane

foam s as a carrier.

The results obtained above demonstrated that under

low temperatures, bioaugmentation of the activated

sludge process with domesticated specialized bacteria

induced a slightly better performance in treating

petrochemical wastewater than the system without

bioaugmentation, while bioaugmentation combined with

biofilm process performed effectively in pollutants

removal with high microbial activity. Thus,

bioaugmentation was optimal when combined with

biofilm process.

(a) (b)

Figure3. SEM images of the biofim

4. Conclusion

Under low temperatures, compared to bioaugmened

activated sludge process, since it could retain sufficient

biomass, bioaugmentation combined with biofilm process

performed more effectively in removing pollutants

contained in petrochemical wastewater with high

microbial activity, while bacterial species were

disappeared due to the strong increase in the grazing

pressure exerted on the inoculums in the activated sludge

system. Thus, the application of polyurethane foam as

carrier in the bioaugmentation practice is promising for

the retention of sufficient biomass and prevention

mechanisms to the immobilization cells. Further

researches on the ecological relationships in

bioaugmented system by adopting advanced molecular

microbial techniques are necessary for better

understanding to the bioaugmentation mechanism.

5. Acknowledgment

We gratefully acknowledge the National Basic Research

Program of China (973 Program) (Granted No.

2004CB418505) and Heilongjiang Provincial Science and

Technology Development Program (Granted No.

CC05S301) for their financial supports.

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