Microbial community structure in anaerobic degradation of terephthalate and phenol 3

Since the late 1980s, various types of full-scale anaerobic reactors including the downflow stationary fixed film DSFF, hybrid, anaerobic contact AC, upflow anaerobic sludge bed UASB, do

Chapter 1

Introduction

1.1 Background

With the advancement of technology in the 20th century, synthetic products such as petrochemical-related materials have gained their popularity due to their durability in physical properties and aesthetics in appearance As a result, the demand of raw materials such as terephthalate and phenol for the manufacturing of plastic or petrochemical-related products has increased, and the production of terephthalate- and phenol-containing wastewaters has also increased over the years

Purified terephthalate (PTA, or 1,4-benzenedicarboxylic acid) is an important raw material used in the manufacture of various plastic products The most well-known application of PTA is the production of polyethylene terephthalate (PET) bottles for carbonated drinks (Park & Sheehan, 1996) Other applications include polyester films, textile fibers, adhesive, coatings and packing materials (Franck & Stadelhofer, 1988) PTA is mainly produced in Asia (South Korea, Taiwan, China, Indonesia, Japan, Thailand and Malaysia), and the amount accounts for approximately 66% of total PTA production worldwide The amount produced was approximately 35 million tons in 2006, and is

increased at an annual rate of 5 to 10% (Razo-Flores et al., 2006). During the production of PTA, high-strength wastewater containing terephthalate as the main

component and other organic compounds like acetate, benzoate and p-toluate (para-toluate) is produced and discharged Typically, one ton of PTA produced is

accompanied by about 2.5−4.5 m3 of wastewater at a concentration equivalent to 20−40

kg chemical oxygen demand (COD)/m3 (Bushway & Gilman, 1986; Kleerebezem et al.,

1999c; Shelley, 1991; Vanduffel, 1993)

Like PTA, phenol is used as the raw material for the production of various resins like phenolic, epoxy, polycarbonate and polyamide for plywood adhesive, construction, automotive and appliance industries (Kirk-Othmer, 1978) The amount produced was estimated to be 7 million tons in 2001 and is increased at an annual rate of 6% Phenol

is also a common constituent in the wastewaters generated from phenol manufacturing processes, petrochemical-related processes, coal gasification, coke ovens and oil refining processes The concentration of phenol and phenolic compounds in wastewaters is approximately 1.0−1.7 x 104 mg/L, and the COD contributed by phenol and phenolic

compounds ranges from 40 to 80% of the total COD in the wastewaters (Veeresh et al.,

2005)

With the advantages of cost-saving (low energy and nutrients requirements), effective removal, and energy recovery (methane), anaerobic biological treatments processes have gradually replaced aerobic processes as an attractive alternative for treating toxic and poorly biodegradable wastewaters Since the late 1980s, various types of full-scale anaerobic reactors including the downflow stationary fixed film (DSFF), hybrid, anaerobic contact (AC), upflow anaerobic sludge bed (UASB), downflow anaerobic filter (DAF), internal circulation (IC), expanded granular sludge bed (EGSB) and upflow anaerobic filter (UAF) systems have been demonstrated to effectively treat PTA wastewater (Macarie, 2000) During these anaerobic treatment, degradation of

terephthalate has been suggested as the rate limiting step (Kleerebezem et al., 1997; Kleerebezem et al., 2005) Phenol is highly toxic and a strong growth inhibitor for

microorganisms, but it has been successfully treated by a full-scale and a number of

laboratory-scale anaerobic reactors (Lao, 2002; Suidan et al., 1983a; Veeresh et al.,

2005)

During the anaerobic methanogenic treatment, terephthalate and phenol are degraded through methanogenic metabolism in the absence of alternative electron acceptors (e.g., sulfates, nitrates and oxidized forms metals) This process requires the interaction between syntrophic fermentative bacteria and methanogenic archaea (methanogens) so that the degradation of terephthalate and phenol can be coupled with the hydrogen- and acetate-consuming reactions to overcome the thermodynamic barrier in the initial step (positive Gibbs free energy, ∆Go') (Fig 1.1), and the intermediates can be converted to the final gaseous products (CH4 and CO2)

Figure 1.1 Proposed terephthalate and phenol degradation under methanogenic

conditions

1.2 Problem statements

Due to the recent awareness of environmental protection, more stringent environmental legislation has been implemented to control the discharge of potentially harmful compounds in many countries Phenol is a carcinogenic compound, and can affect

aquatic life at concentrations >1 mg/L (Autenrieth et al., 1991), whereas the toxicity of

terephthalate remains unclear Thus, the release of these compounds in environments has attracted great environmental and public health concerns in recent years, and there is

a strong need to effectively treat these terephthalate- and phenol-containing wastewaters before discharge Over the last 20 years, although empirical know-how of anaerobic treatment technologies utilimately reaches the good performance on the treatments of these wastewaters, these still face a number of challenges, as described below, for improvement

(A) Limited application of treatment processes for PTA wastewater at temperatures above 37°C

Anaerobic processes for treating PTA wastewater have been successfully achieved under mesophilic (30−37ºC) conditions As the production of PTA is carried out under high pressure and temperature, the PTA wastewaters are usually generated at high temperatures (40−60ºC) Therefore, additional time to store them in an equalization tank to cool down the wastewater before the mesophilic anaerobic treatment processes is necessary It further suggests that anaerobic treatment processes under high temperatures (>37ºC) conditions can be an attractive alternative So far, the development of thermophilic anaerobic treatment is relatively limited Thermophilic treatments are reported to generally have much higher specific organic removal rates than

the mesophilic treatments (van Lier et al., 1997), but it is relatively difficult to establish a

stable microbial consortium in the thermophilic (55°C) terephthalate-degrading reactor

(Kleerebezem et al., 1999c) This is attributed to a low number of thermophilic

terephthalate-degrading microorganisms in the original seed sludge, and inappropriate

operational conditions to enrich the microbial consortia (Kleerebezem et al., 1999c)

Until now, the biodegradability of terephthalate and the responsible microorganisms at temperatures above 37°C is poorly known and further studies are necessary

(B) Limited information on phenol-degrading microbial consortia

Anaerobic biological processes have been demonstrated to efficiently treat phenol-containing wastewaters at organic loadings (6−8 kg COD/m3 • day) in the

laboratory-scale bioreactors (Veeresh et al., 2005) Prior to reaching the extraordinary

performance (>90% removal of phenol), it requires a long start-up time (~300 days) to acclimatize the suitable microbial populations from seed sludge, to develop efficient phenol-degrading capability as well as particular microbial interaction and spatial

orientation that can facilitate the rapid exchange of nutrients and products (Veeresh et al.,

2005) Like the terephthalate-degrading methanogenic consortium, phenol degradation under thermophilic conditions is reported to be relatively difficult and unstable as

compared to that under mesophilic conditions (Fang et al., 2006; Karlsson et al., 1999)

As a result, the development of thermophilic techniques to treat phenol-containing wastewater is relatively limited A better understanding of the phenol-degrading microbial community could provide crucial information for better selection of seeding sludge to shorten the start-up time in the mesophilic processes, and facilitate the development of thermophilic processes So far, the phenol-degrading methanogenic microbial consortia was only characterized for biomass samples taken from a laboratory reactor operated at a temperature at 26°C (Fang et al., 2004; Zhang et al., 2005) The

microbial populations responsible for phenol degradation under mesophilic and thermophilic conditions have not been fully identified and characterized

(C) Limited information on full-scale reactor to treat phenol-containing wastewater

Many anaerobic laboratory-scale bioreactors have been successfully used to treat phenol-containing wastewaters However, most of them were fed with synthetic wastewater and had better control of the operational parmeters (e.g., loading rate, recycle rate, temperature and hydraulic retention time [HRT]) These laboratory-scale bioreactors may not provide the same environmental conditions as the full-scale bioreators As a result, the microbial populations found in the laboratory-scale bioreactors or enrichment cultures may not fully represent those in full-scale bioreactors treating wastewaters containing phenol So far, only one full-scale UASB reactor has been constructed and operated to treat the wastewater from phenol production under mesophilic conditions since 1986 (Macarie, 2000) To facilitate the development of full-scale processes, a better understanding of the phenol-degrading microbial community

in a full-scale plant is necessary

(D) Limitation of conventional culture-dependent approaches

The bioreactor where the degradation of organic pollutants takes place traditonally referred to as a “black box” In the past 10 years, microbial populations inside wastewater treatment plants have been extensively studied in order to correlate their identity, abundance and dynamics to the reactor performance In anaerobic wastewater treatment systems for treating terephthalate- or phenol-containing wastewaters, microorganisms

play as the catalysts to degrade the substances However, owing to the limitations associated with the conventional techniques (time-consuming and low culturability of environmental microorganisms) and the difficulty of isolating syntrophic bacteria, only

two mesophilic terephthalate-degrading bacteria (Pelotomaculum terephthalicum and

Pelotomaculum isophthalicum) (Qiu et al., 2004; Qiu et al., 2006) and one

phenol-transforming bacteria (Cryptanaerobacter phenolicus) (Juteau et al., 2005) have been isolated The presence of these isolates P isophthalicum, P terephthalicum and C

phenolicus in the laboratory-, pilot- and full-scale terephthalate- or phenol-degrading

reactors remains unknown It is almost impossible to describe microbial community in the reactors based on the information of limited isolate strains available so far, and the isolates may not even represent as the main population involved in terephthalate or phenol degradation Therefore, the microbial community structures in anaerobic degradation of terephthalate and phenol remain to be further characterized Recently, microbial populations inside wastewater treatment plants have been extensively investigated using 16S rRNA-based culture-independent molecular techniques with an attempt to identify the key players and understand their abundance, dynamics, and

distribution within the “black box” in relation to the reactor performance These

in-depth understandings are important to optimize biological processes

1.3 Objectives

The overall objective of this research is to study the microbial community structures responsible for anaerobic terephthalate and phenol degradation using 16S rRNA gene-based molecular approaches Specific objectives are:

a To characterize the microbial community structure and dynamics in a thermophilic (55°C) anaerobic laboratory-scale hybrid reactor degrading terephthalate-containing wastewater,

b To study the microbial community structure in a terephthalate-degrading anaerobic hybrid bioreactor operated at 46−50°C,

c To identify the important microbial populations in the methanogenic phenol-degrading enrichments under mesophilic (37°C) and thermophilic (55°C) conditions, and

d To investigate the microbial community structure in a mesophilic full-scale phenol-degrading anaerobic fluidized bed reactor

The results will provide evidence that terephthalate can be degraded under 46−50ºC and 55ºC conditions; improve our understanding of syntrophic microorganisms in the terephthalate and phenol degradation systems; identify microorganisms that are important

to the degradation; and provide additional knowledge for future development of methanogenic treatment technologies for terephalate- and phenol-containing wastewaters

1.4 Organization of the thesis

The thesis consists of eight chapters Chapter 1 provides a general introduction, the problem statements and objectives Chapter 2 provides a summary of research publications relevant to this study Chapter 3 describes the experimental materials and methods used Chapters 4 to 7 describe the results and discussion on the microbial community structures in a thermophilic anaerobic hybrid reactor degrading terephthalate

(Chapter 4), a moderate thermophilic anaerobic hybrid reactor degrading terephthalate (Chapter 5), mesophilic and thermophilic phenol-degrading methanogenic consortia (Chapter 6), and a full-scale phenol-degrading bioreactor (Chapter 7) Last, Chapter 8 provides the overall conclusions of this study and recommendations for future works.

Chapter 2

Literature Review

2.1 Anaerobic treatment of terephthalate- and phenol-containing wastewaters

During the past 20 years, anaerobic digestion processes have become an attractive biological approach to treat toxic and poorly biodegradable wastewaters (e.g., petrochemical wastewaters) (Lettinga, 1995) In these processes, organic compounds are degraded and converted into methane and carbon dioxide in the absence of oxygen and other electron acceptors These anaerobic treatments offer a number of advantages over conventional aerobic processes, including low sludge production, high efficiency, simple and flexible, good energy recovery, low cost, high organic loading rate, low space requirement, stable sludge, and low nutrient requirement Nonetheless, these treatment processes are still limited by the long start-up time and recovery time required

So far, different types of anaerobic processes have been developed to treat various

industrial wastewaters They include DSFF, AC, UASB, DAF, IC, EGSB, UAF, continuous stirred tank reactor (CSTR) and anaerobic fluidized bed (AFB) reactors Recent reviews have reported that at least 1330 to 1600 full-scale anaerobic reactors had been constructed and operating around the world (Kleerebezem & Macarie, 2003; Macarie, 2000) Among them, at least 80 to 91 full-scale systems are used for treating wastewaters from petrochemical and related industries (Table 2.1)

Table 2.1 Number of full-scale anaerobic bioreactors constructed for treating petrochemical wastewaters

Fixed bed Type of

Kleerebezem

& Macarie,

2003

2.1.1 PTA wastewater

2.1.1.1 PTA wastewater production

PTA (purified terephthalate, or 1,4-benzenedicarboxylic acid) is used as a raw material in the production of polyester films, textile fibers and PET bottles It is one of the top 50 chemicals manufactured in the world by quantity With an increasing rate at 5 to 10% annually, the amount produced was 9.3 million tons in 1993 (Savostianoff & Didier, 1993), 12.5 million tons in 1996 (Fligg, 1996), and was estimated to be 35 million tons in

2006 (Razo-Flores et al., 2006) Annual PTA production in Asia (e.g., South Korea,

Taiwan, China, Indonesia, Japan, Thailand and Malaysia) represented 66% of the amount produced worldwide (North America, 22% and Europe, 11%)

To be used for the production of commercial fibers and bottles, high-quality terephthalate

is produced based on a well-established chemical process developed by the American Amoco group (Franck & Stadelhofer, 1988) The process consists of oxidation and purification steps (Park & Sheehan, 1996) In the oxidation step, crude terephthalate

(CTA) is produced through wet oxidation of para-xylene with liquid air and acetate as

solvent at a temperature between 175 and 230°C and a pressure between15 and 35 bars CTA usually contains a number of by-products such as benzoate, carboxybenzaldehyde

and p-toluate, and thus needs to be purified through hydrogenation to obtain PTA at

250°C During these two steps, high-strength wastewater containing various organics is discharged Typically, for each ton of PTA producted, 2.5−4.5 m3 of wastewater at a concentration of 20−40 kg COD/m3 is generated (Bushway & Gilman, 1986;

Kleerebezem et al., 1999c; Shelley, 1991; Vanduffel, 1993) The wastewater contains

terephthalate, acetate, benzoate and p-toluate in a decreasing order as the main components (Macarie et al., 1992) As terephthalate, benzoate and p-toluate are not toxic to microorganisms at the concentrations encountered in PTA wastewater (Fajardo et

al., 1997; Kleerebezem et al., 1997), biological processes are suitable for treating this

PTA wastewater

2.1.1.2 Treatment of PTA wastewater

Since the 70s, aerobic biological treatment processes have been frequently applied to the treatment of PTA wastewater (Lau, 1977) These processes include a modified three-stage extended aeration activated sludge system, deep-aeration methods, and a

modified sequential batch reactor process (Lain et al., 1999) However, these aerobic

treatment systems have several disadvantages including long HRT, high demand for oxygen/energy, additional nutrients (nitrogen and phosphorus) requirements, large foot-print, high chemical dosage, and excessive production of waste sludge Due to these disadvantages, Amoco has extended their efforts to develop the anaerobic biological treatment systems as alternatives since the late 1980s

2.1.1.3 Anaerobic treatment under mesophilic conditions

Table 2.2 summarizes some of the full-scale anaerobic bioreactors constructed for the

treatment of PTA wastewater between 1989 and early 2003 (consolidated from Macarie et

al., 2000; Kleerebezem and Macarie, 2003) All the anaerobic processes were mostly

operated under mesophilic methanogenetic conditions The DSFF system was the first anaerobic process successfully developed by US Amoco Company in 1989 (Suzanne,

1991) With a start-up period longer than one year, the reactor was able to achieve a removal efficiency of 80% for COD, and 85% for TOC and organic compounds that can

be identified in PTA wastewater After 1989, various types of full-scale anaerobic reactors have been constructed and demonstrated to treat PTA wastewater with a COD removal efficiency of 75−80% In addition, many pilot- or laboratory-scale bioreactors such as UASB, hybrid, AFB and two-stage systems have been used to treat PTA wastewater However, observations show that the anaerobic process require a long start-up time, ranging 1−6 months in laboratory studies (Cheng et al., 1997a;

Kleerebezem et al., 1997; Kleerebezem et al., 1999c; Macarie et al., 1992) to more than 1 year in full-scale reactors (Razo-Flores et al., 2006)

The UASB system has been used to treat a variety of industrial wastewater (Macarie & Guyot, 1992) Estimatedly, more than 1000 full-scale UASB reactors were in use

worldwide (Sekiguchi et al., 2001a) In this system, influent moves upward through a

thick blanket of anaerobic sludge granules (0.5−2.5 mm in diameter) that consist of high concentrations of immobilized microorganisms with excellent settling properties (Lettinga, 1995) Mixing in the reactor is achieved mainly by hydraulic flow and then

by the gas bubbles generated Granulation in UASB reactors is reported to be more favorable under high upflow liquid velocity, neutral pH, and short HRT with polymer and cations addition, suitable seeding biomass, and fast degraded substrate (Liu & Tay, 2002;

Liu & Tay, 2004; Tiwari et al., 2006) This process has been successfully demonstrated

to treat terephthalate-containing wastewater (Table 2.2; Wu et al., 2001)

Furthermore, hybrid system or a combination of fixed-film reactor and UASB reactor has been developed to treat PTA wastewater In this process, carrier media (plastic ring or polyurethane particles) are placed in the upper region of the reactor (zone of fixed-biofilm process) and the inoculum is seeded at the base of the reactor (UASB form)

As active anaerobic sludge is retained by sludge granulation or biofilm formation, the hybrid system can function effectively with a higher terephthalate removal rate than in

UASB reactors (Kleerebezem et al., 1999c)

PTA wastewater can also be treated using AFB system (Cheng et al., 1997b) In this

process, granular activated carbon (GAC) is commonly used as the carrier to provide sufficient surface area for microbial adhesion and adsorption of organic compounds In GAC-AFB processes, sludge granules are formed by different microbial cells in close physical association Comparing to the conventional activated sludge, granular sludge

in GAC-AFB processes has a better settling property Cheng et al (1997b) observed

that during the first three months of operation, COD removal was approximate 60% from

the degradation of acetate, benzoate and terephthalate but not p-toluate After 600 days, the COD removal was increased to 85% as the p-toluate could also be degraded

It has been reported that terephthalate degradation can be strongly inhibited in the presence of other readily degradable substrates (i.e., acetate and benzoate) in PTA

wastewater (Cheng et al., 1997a; Kleerebezem et al., 1997; Kleerebezem et al., 1999a; Kleerebezem et al., 1999b; Macarie & Guyot, 1992) To address this issue, a two-stage anaerobic reactor was used to treat PTA wastewater (Kleerebezem et al., 1997;

Kleerebezem et al., 2005) In this system, the readily degradable substrates (i.e., acetate

and benzoate) were removed in the first reactor, and the slowly degradable terephthalate were degraded effectively in the second reactor No degradation of terephthalate was observed in the first stage when acetate and benzoate were present during the operation

up to 300 days (Kleerebezem et al., 2005)

Table 2.2 Full-scale anaerobic bioreactors treating PTA wastewaters*

Year Company and location Type of reactor

Reactor volume (m 3 )

Organic loading (kg COD/m 3 d)

Organic loading (ton COD/d)

COD removal (%) Constructor

2.1.1.4 Anaerobic treatment under thermophilic conditions

Thermophilic anaerobic processes (normally operated at a temperature between 50 and 60°C) have been used in recent years for the treatment of industrial wastes and wastewaters discharged at high temperatures These processes are capable of accommodating a very high loading rate (80−100 kg COD/m3 • day) with good removal

efficiency (van Lier, 1996; van Lier et al., 1997) As the production of PTA is carried

out under high pressure and temperature, the temperature of the discharged PTA wastewater can be as high as 56°C Thus, thermophilic anaerobic treatment processes are potentially attractive for PTA wastewater Unfortunately, there is no full-scale anaerobic thermophilic bioreactors being used to treat PTA wastewater, and the biodegradability of those organic compounds present in PTA wastewater at 55°C is poorly known

Only two case studies have been reported to treat PTA under thermophilic conditions The first study employed a thermophilic anaerobic process for the degradation of

terephthalate as the sole substrate (Kleerebezem et al., 1999c) However, this study

failed to obtain a thermophilic terephthalate-degrading microbial community using both UASB bioreactors and batch cultures up to a period of 130 days The authors concluded that the seed sludge probably did not contain thermophilic terephthalate-degrading organisms, and the operational conditions were inappropriate for the cultivation of such

organisms (Kleerebezem et al., 1999c) The second study employed a laboratory-scale hybrid reactor to treat synthetic PTA wastewater up to 800 days (Thierry et al., 1999) It

was observed that all the compounds (i.e., terephthalate, phthalate, benzoate, trimellitate

and acetate) except p-toluate present in the PTA wastewater could be readily degraded at

55°C The maximum loading rate applicable to the system was 16 kg COD/m3 • day,

which is compatible with a full scale thermophilic treatment system (Thierry et al., 1999)

This result suggested the earlier observations reported by Kleerebezem et al (1999c) was probably due to the absence of thermophilic terephthalate-degrading microorganisms in the inoculum

2.1.2 Phenolic wastewater

2.1.2.1 Phenolic wastewater production

The terms “phenols”, “total phenols” or “phenolics” used for wastewaters or contaminated sites denote simple phenol or a mixture of phenolic compounds, such as

phenol, cresol isomers (ortho-, meta- and para-), resorcinol, hydriquinone and dimethyl

phenol Phenols are often found in wastewaters generated from coal gasification, coke ovens, and petroleum-related manufacturing processes (e.g., phenolic resin, adhesives, fiberglass and herbicide) (Kirk-Othmer, 1978) The concentration of phenol and phenolic compounds in the wastewater varies from 1−1.7 x 104 mg/L, and the COD

contributed by phenol and phenolic compounds ranges from 40 to 80% of the total COD

in the wastewater (Veeresh et al., 2005) In coal conversion processes and coal ovens,

the wastewaters generally consist of phenol (60%) and cresol isomers (30%) (Nakhla & Suidan, 1995)

Phenol can be produced from partial oxidation of beneze, a process known as the

“cumene process” or “Raschig process” It can also be found in the byproducts of coal oxidation Phenol is mostly used in the production of phenolic resins, followed by the manufacturing of nylon and other synthetic fibers Phenolic resins are used in the plywood adhesive, construction, automotive and appliance industries With sickeningly sweet and tarry odor, phenol is known to be toxic, carcinogenic, mutagenic and

teratogenic (Autenrieth et al., 1991) In natural environments, phenol can affect aquatic

life when the concentration exceeds 1 mg/L, and is regarded as a high priority environmental pollutant in most countries Thus, stringent effluent discharge limits of less

than 0.5 mg/L are typically imposed (Chang et al., 1995; Tay et al., 2001)

2.1.2.2 Treatment of phenol-containing wastewater

Although phenol is highly toxic and a strong growth inhibitor for microorganisms, biological treatment processes are able to treat phenol at concentrations between 50 and

500 mg/L (Patterson, 1975) During the 1980s, anaerobic biological treatment of phenolic wastewaters was widely practiced Since 1990, anaerobic treatment has become more attractive to treat phenol-containing wastewaters

2.1.2.3 Anaerobic treatment under mesophilic conditions

A number of laboratory-scale anaerobic bioreactors including GAC-anaerobic filter (AF)

(Khan et al., 1981; Suidan et al., 1981; Suidan et al., 1983a), GAC-expanded bed (Wang

et al., 1986) and GAC-AFB (Kim et al., 1986; Lao, 2002) reactors have been used to

treat phenolic wastewaters GAC-AF and GAC-AFB processes have been further applied to treat the phenol-containing wastewaters from coal gasification (Nakhla &

Suidan, 1995; Suidan et al., 1983b), but their performances are sometimes limited by

issues related to media plugging, gas/liquid separation with GAC-AF, high recycle ratio and high operating cost In the last 10 years, UASB process has been widely used for treating phenol-containing wastewaters Veeresh and co-worker (2005) reviewed the treatment of phenol using UASB process and highlighted the operational problems related to reactor start-up, shock loading, and unknown operating parameters (Table 2.3)

In the start-up period of UASB processes, easily degradable substrates such as sucrose, glucose and volatile fatty acids (VFAs) are commonly used as co-substrates to serve as an energy source for the growth of microorganisms With the use of a co-substrate, the acclimation time for start-up has been reported to be between 45 and 300 days It is observed that phenol could be efficiently removed at a concentration between 280 and

1260 mg/L in UASB processes Veeresh and co-workers (2005) further indicated that some of the operational problems such as the relatively long acclimation period, small granule size and decrease in phenol removal efficiency at higher loading rates, sensitivity

to temperature, and long recovery periods could be overcome by dilution through recirculation of effluents So far, many laboratory-scale bioreactors have been demonstrated to treat phenol-containing wastewaters, but only one full-scale UASB

reactor has been constructed to treat the wastewater generated from phenol manufacturing processes since 1986 (Table 2.3) (Macarie, 2000)

Table 2.3 Performance of UASB reactors treating phenol-containing wastewaters under mesophilic and methanogenic conditions*

Scale Temp

(ºC)

Reactor volume (L)

Co-substrate for activity enhancement (start-up time, day)

HRT (d)

Influent (mg/L)

Organic loading rate (kg COD/m 3 d)

Removal efficiency phenol (%)

Lab 37 2.8 Sucrose

(134)

0.5

(55)

0.5- 0.6

2.1.2.4 Anaerobic treatment under thermophilic conditions

Fang and co-workers (2006) successfully operated a UASB reactor to treat synthetic phenol-containing wastewater for 224 days under thermophilic conditions (55°C) Over 99% of phenol at an initial concentration of 630 mg/L was effectively degraded at an HRT of 40 hr During the start-up period, the reactor was fed with sucrose as the co-substrate The HRT was subsequently decreased stepwise from 60 to 48, 40 and

28 hr Removal efficiency, which was over 96% at the HRT of 40 hr, became unstable (<77%) at an HRT of 28 hr The maximum specific methanogenic activity (SMA) of the thermophilic phenol-degrading sludge was 0.09 g-CH4-COD/g-volatile suspended solids (VSS) • day The SMA was substantially lower than 0.19 and 0.24 g-CH4-COD/g-VSS •

day, respectively, observed with anaerobic reactors operated at 26°C (Fang et al., 2004) and 37°C (Fang et al., 1996)

compounds) becomes exergonic Therefore, degradation of substances through syntrophic association is dependent on the presence of the H2- or acetate-consuming microorganism such as methanogen

Syntrophic association is also an important mechanism taking place in the anaerobic degradation of terephthalate and phenol Positive ∆Go' is required to initially convert

terephthalate and phenol to acetate and hydrogen (Table 2.4) (Kleerebezem et al., 1999a;

Schink, 1997) As the initial conversions of terephthalate and phenol are thermodynamically unfavourable, the hydrogen or acetate consuming reactions (e.g., methanogensis) have to be coupled to make the overall reactions energetically possible

Table 2.4 Syntrophic degradation of organic matters and the standard Gibbs free-energy

Fermentative reaction ∆G o ' (kJ/reaction)

2.2.2 Proposed terephthalate degradation pathway

Anaerobic degradation of terephthalate was first reported under denitrifying condition, and decarboxylation steps were suggested to be involved in the initial degradation of terephthalate to benzoyl-CoA (Nozawa & Maruyama, 1988) It is proposed that under methanogenic condition, terephthalate could be degraded to acetate and hydrogen by

fermentative bacteria, and then acetotrophic and hydrogenotrophic methanogens could convert acetate and hydrogen to the final products, methane and carbon dioxide

(Kleerebezen et al., 1999a, b) This syntrophic degradation was demonstrated by

incubating the methanogenic cultures with terephthalate in the presence of a

methanogensis inhibitor, bromoethanesulfonate (BES) (Kleerebezen et al., 1999a) As a

result, significant concentrations of methanogenic substrates (e.g., acetate and hydrogen) were accumulated in the cultures It is further proposed that terephthalate was initially converted to a transient intermediate benzoate via decarboxylation (Fig 2.1), based on the observation that a methanogenic terephthalate-degrading consortium was strongly

inhibited by the presence of benzoate and acetate (Kleerebezem et al., 1999a, b) The

corresponding values for maximum specific growth rate (µsmax) and biomass yields (Yxtots) were reported to be higher for growth on benzoate than on terephthalate Due to the difficulty of isolating the syntrophic bacteria, the proposed degradation remains to be verified To date, anaerobic degradation of terephthalate was only conducted under mesophilic conditions but not thermophilic conditions

2.2.3 Proposed phenol degradation pathway

Phenol degradation by anaerobic consortia under methanogenic condition was first

reported by Wang et al., (1986) and Young & Rivera (1985) two decades ago Since

then, many studies have suggested that phenol is initially transformed to benzoate via

reductive carboxylation (Charest et al., 1999; Gallert et al., 1991; Karlsson et al., 1999;

Londry & Fedorak, 1991) and subsequently through the benzoyl-CoA pathway to acetate and hydrogen (Heider & Fuchs, 1997) Karlsson and co-workers (1999) suggested that the initial conversion of phenol to benzoate under mesophilic condition can lead to a decrease in H2 levels, providing that a suitable condition is there for the degradation of phenol to acetate (Fig 2.2) In the degradation of phenol under thermophilic conditions, benzoate was not observed as an intermediate in the BES-amended cultures, suggesting a high turnover rate of benzoate or a different degradation pathway of phenol under

thermophilic conditions (Karlsson et al., 1999).

(B) C6H5OH + 4H2O → 3.5CH4 + 2.5CO2

Figure 2.2 The phenol degradation pathway, based on phenol transformation to benzoate and

acetate in BES-amended cultures (A) (Karlsson et al., 1999) Overall phenol degradation equation

under methanogenic condition (B)

(A)

Besides the most recognized degradation pathway (i.e., through benzoate), it has also been reported that phenol can be degraded via caproate to acetate and hydrogen (Evans,

1977; Fang et al., 2006) Phenol is reduced through cyclohexanone, caproate and

adipate, VFAs, and finally to methane in the presence of nitrate (Evans, 1977)

However, Fang et al (2006) showed that the phenol-degrading sludge was able to convert

caproate into methane, but was not able to degrade cyclohexanone These results indicated that the degradation pathway did not agree completely with that reported by Evans (1977) Thus, the degradation pathway of phenol via caproate needs to be further confirmed

2.3 Microbial communities for terephthalate and phenol degradation

2.3.1 Microorganisms involved in terephthalate degradation

2.3.1.1 Culture-dependent studies

Two mesophilic terephthalate-degrading bacteria, Pelotomaculum terephthalicum and

Pelotomaculum isophthalicum have been isolated (Qiu et al., 2004, 2006) These two

isolates are from the Desulfotomaculum subcluster Ih, which contains species that do not show the abilities of dissimilatory sulfate and sulfite reduction (Imachi et al., 2006)

mesophilic (37°C) phthalate isomer-degrading bacterium isolated from a terephthalate-degrading enrichment The growth of strain JTT is extremely slow - up to

3 months are needed to completely degrade 1 mM terephthalate The bacterial strain can grow on crotonate, hydroquinone and 2, 5-dihydroxybenzoate in pure culture Thecells are 0.8−1.0 µm wide and 2.0−3.0 µm long, occurring singly or in pairs The spores

in sphere are located at the center of a cell The culture formed tiny light brown-colored and lens-shaped colonies (0.1−0.15 mm in diameter) on agar medium containing 10 mM

and 0.02% yeast extract after 1 month of incubation In co-culture with Methanospirillum

terephthalate, but not ortho-phthalate) and a number of low-molecular weight aromatic

compounds, such as benzoate, hydroquinone, 2-hydroxybenzoate, 3-hydroxybenzoate,

2,5-dihydroxybenzoate, 3-phenyl-propionate

phthalate isomer-degrading bacterium isolated/purified from an isophthalate-degrading enrichment that was originally derived from a UASB granular sludge treating the wastewater from the manufacturing of terephthalate and isophthalate Isolation of strain

JIT is reported to be difficult since no substrates were able to support the growth of this strain in pure culture, and the growth was extremely slow in the media containg

isophthalate in the presence of Methanospirillum hungatei cells A defined “pure

co-culture” of strain JIT was obtained by co-cultured with M hungatei with

3-hydroxybenzoate (5 mM) and yeast extract (0.02%) as the carbon substrate The cells are 0.8−1.0 µm wide and 2.0−3.0 µm long, occurring singly or in pairs The spores are spherical and at central of the cell Tiny colonies, light brown in color and lens-shaped are formed in the presence of methanogens on agar medium after 3 months of incubation

In co-culture with M hungatei, strain JIT could degrade three phthalate isomers (i.e.,

isophthalate, terephthalate and ortho-phthalate), benzoate and 3-hydroxybenzoate No

substrates were found to support the growth of strain JIT axenically

microscopy observation revealed that bamboo-shaped cells (Methanosaeta-like), fat rods (Methanobrevibacter- or Methanobacterium-like) and short rods were the three

predominant morphotypes in the consortium 16S rRNA gene clone library and phylogenetic analysis showed that 78.5% of the bacterial clones belonged to the

Deltaproteobacteria, and the remaining assigned to the green non-sulfur bacteria (7.5%), Synergistes (0.9%) and unidentified divisions (13.1%) In the predominat Deltaproteobacteria, a novel clone cluster (consisted of 8 operational taxonomy units

[OTUs], 66.8% of total bacterial clones) was firstly observed in the terephthalate-degrading sludge, it was subsequently referred as group TA in

Deltaproteobacteria

Fluorescence in situ hybridization (FISH) analysis with 16S rRNA-targeted specific

oligonucleotide probes in thin-section of granules was used to verify the presence of this

yet-to-be cultured Deltaproteobacteria group TA and the spatial distribution of microorganisms within anaerobic terephthalate-degrading granular sludge (Wu et al.,

2001) Unlike easily degraded substrates (i.e., glucose, hydrolyzed protein, sucrose,

brewery wastes, and a mixture of sucrose/acetate/propionate) at high concentration (Fang

et al., 1994; Guiot et al., 1992; MacLeod et al., 1990; Sekiguchi et al., 1999), no layered

microstructure was observed with the terephthalate-degrading granules Bacterial and archaeal cells were randomly distributed in the terephthalate-degrading granules with

approximate equal abundance (Wu et al., 2001) Since the degradation of terephalate

takes place slowly on the surface of granules, it can also penetrate into the granules and

be degraded at the inner part of granules As a result, non-layered structures were

observed in the terephthalate-degrading granules (Wu et al., 2001) Furthermore, the rod-shape Deltaproteobacteria group TA cells (87% of the total bacterial cells) were the predominat bacterial populations throughout the granules (Wu et al., 2001) For the

domain Archaea, members in Methanosaeta, Methanospirillum and Methanobacteriaceae were present at a significant level in the terephthalate-degrading

granules (Wu et al., 2001)

2.3.2 Microorganisms involved in phenol degradation

2.3.2.1 Culture-dependent study

A phenol-transforming bacteria, Cryptanaerobacter phenolicus strain LR7.2T has been

isolated from a mixture of swamp water, sewage sludge, swine waste and soil (Juteau et

10-days incubation on agar, the strain formed colonies (1 mm in diameter) with diffuse margins and brownish-color The cells were 1 µm wide and 2 µm long Strain LR7.2Tcould transform phenol or 4-hydroxybenzoate into benzoate to generate energy for growth This supports the possibility that phenol is initially carboxylated to benzoate and

other fatty acids No sporulation was observed with strain LR7.2T, but its ability to form spores is possible as pasteurization was used during the enrichment process for isolation Syntrophic degradation of phenol was not evaluated for strain LR7.2T in co-culture with methanogens

2.3.2.1 Culture-independent studies

Using electron microscopy, filamentous Methanothrix spp were observed to be the

predominant methanogen on the surface and interior of a granule together with members

of Syntrophus-, Methanospirillum-, and Methanobrevibacter-like microorganisms in the mesophilic phenol-degrading UASB reactors (Chang et al., 1995; Fang et al., 1996) Using rRNA-based molecular tools, Fang and co-workers (Fang et al., 2004; Zhang et al.,

2005) studied the microbial populations in the phenol-degrading UASB reactors operated

at ambient temperature (26°C) The cloning results suggested that phenol was first

converted to benzoate possibly by Desulfotomaculum spp and Clostridium spp., and then

to acetate and H2/CO2 by Syntrophus spp The intermediate by-products (acetate and

H2/CO2) were further converted to methane and CO2 by members of Methanosaetaceae,

Methanomicrobiales and Methanobacteriaceae FISH analysis revealed that

methanogens (74%) were more abundant than bacteria (26%) populations in this UASB system Like anaerobic degradation of terephthalate, the initial conversion of phenol has been identified as the rate-limiting step As a result, non-layered structure of phenol-degrading granules was observed in the UASB reactors operated at 26°C (Fang et

Fang and co-workers (2006) also enriched a phenol-degrading methanogenic consortium under thermophilic conditions (55°C) Among those 54 bacterial clones screened, no dominant microbial populations could be identified However, they observed that the microbial populations under thermophilic conditions were different from those observed

in the bioreactor operated under ambient temperature The microbial communities involving in thermophilic phenol degradation remain to be characterized

Chapter 3

Materials and Methods

3.1 Terephthalate- and phenol-degrading microbial consortia

3.1.1 Thermophilic anaerobic terephthalate-degrading reactor

A 1.2-liter laboratory-scale hybrid reactor, packed with 69 polypropylene Pall rings (Flexiring®, Koch Inc.) (total volume = 400 mL), was used to enrich anaerobic microbial consortia degrading terephthalate under thermophilic conditions (Fig 3.1) The reactor was inoculated with seed sludge that previously showed thermophilic

terephthalate-degrading activity (Thierry et al., 1999) Prior to the inoculation, the seed

sludge consisted of non-granulated particles smaller than 0.23 mm (>90% of total suspended solids) and was stored at room temperature without feeding for 316 days The reactor was initially fed with synthetic PTA wastewater (pH 6.7) containing

terephthalate (1.32 g/L), benzoate (0.5 g/L), p-toluate (0.5 g/L), trimellitate (0.24 g/L),

phthalate (0.12 g/L), acetate (0.5 g/L), NH4Cl (0.4 g/L), K2HPO4 (0.1 g/L), FeSO4.7 H2O

(0.04 g/L) and trace metals (El Mamouni et al., 1995) After 118 days of operation, the

synthetic feed was replaced with terephthalate as the sole carbon source at a concentration of 3.41 g/L (COD concentration = 4.93 g/L) to further enrich the terephthalate-degrading microbial consortia The reactor temperature was controlled at 55°C by recirculating heated water through its double-jacket column Throughout the study, the HRT and volumetric organic loading rate (Bv) were maintained at 1 day and 5

kg COD/m3 • day, respectively Complete mixing was provided to prevent the development of substrate gradient or stratified microbial communities in the reactor column Reactor performance was evaluated by monitoring the removal efficiency of

terephthalate, benzoate, p-toluate, trimellitate phthalate, and acetate as well as total and

soluble COD Biomass samples were collected from the sludge bed on Days 103, 172,

200 and 259, and immediately stored in anaerobic serum bottles upon collection Due to sampling difficulty, the biomass from the packing material was only taken after the shutdown of the reactor on Day 272 The sludge samples were used for microbiological analyses

Figure 3.1 Sketch of the laboratory-scale thermophilic anaerobic terephthalate-

degrading hybrid reactor

3.1.2 Anaerobic terephthalate-degrading reactor operated at 46−50°C

A 1-liter laboratory-scale hybrid bioreactor with 78 packing materials (sera siporax, Sera Germany) was used to enrich the anaerobic microbial consortia that degrades terephthalate under 46−50°C conditions (Fig 3.2) The seeding sludge was obtained

from the mesophilic UASB reactors treating dimethylterephthalate (Temex and Petrocel, Mexico) The reactor temperature was controlled by recirculating heated water through

a double-jacket column The actual temperatures inside the reactor were 46 ± 0.5°C for sludge bed and 50 ± 0.5°C for the upper medium part Terephthalate was used as the sole carbon and energy source Reactor operation was divided into six stages in terms

of terephthalate-loading During stage 2 and 6, the overall terephthalate loading per day was gradually increased from 0.7 to 2.1 g terephthalate/L • day (1.02 to 5.22 kg COD/m3 • day) Reactor performance was determined based on the removal efficiency of COD and terephthalate Biomass was collected from both packing materials and sludge bed for further microbial analyses

Figure 3.2 Sketch of the laboratory-scale anaerobic terephthalate-degrading hybrid reactor operated at 46−50°C, and the packing materials on Day 346

3.1.3 Mesophilic and thermophilic phenol-degrading enrichments

The seeding sludge used for the cultivation of phenol-degrading consortia was taken from

an anaerobic digester at a local wastewater treatment plant receiving both industrial (~ 66.6 % of total influent volume) and domestic wastewaters The anaerobic digester was operated at ambient temperature (28−30 °C), and the concentrations of suspended solid and volatile suspended solid (VSS) were approximately 30 g/L and 14 g/L, respectively Prior to inoculation, the sludge was gently washed with 0.1 M Phosphate buffered saline (PBS) solution Five milliliters of seeding sludge were inoculated into 120-mL serum bottles containing 50 mL of anaerobic culture medium pre-sparged with 80% N2−20%

CO2 (v/v) Concentrated phenol solution was added as sole carbon source to a final concentration of 100 mg/L, and incubated at 37°C and 55°C without shaking Once the phenol was completely degraded, the concentration used in subsequent culture transfers (10% inoculum, v/v) was increased stepwise to 250, 500 and finally 750 mg/L Phenol

at a concentration of 750 mg/L was used in subsequent culture transfers The cultures (5 mL) were anaerobically transferred using a syringe and needle This enrichment process was conducted for up to 18 months During incubation, biogas production was monitoring on a weekly basis Afterward, the effective phenol-degrading mesophilic enrichment (MP) (after 10 successive transfers) and thermophilic enrichment (TP) (after

7 successive transfers) were used for batch degradation assays and microbial community analyses

3.1.4 Full-scale phenol-degrading anaerobic sludge sample

The biological GAC sample was obtained from a full-scale AFB reactor treating phenolic resin manufacturing industrial wastewater The reactor was with a total volume of 275

m3 (5 m in diameter and 16 m in height) It was operated with a HRT of 1.5 day and an upflow velocity at 20 m/h under mesophilic conditions The influent wastewater contained 2500−3000 mg/L COD (approximately 60% was contributed by phenol) During the sampling period, the reactor had a COD removal efficiency of about 85% under a loading rate of 3−5 kg COD/m3 • day Sample was collected from one of the sampling port at about 5 m in height from bottom The approximate 2−4 mm plate-like thick GAC particles were collected

Figure 3.3 Full-scale phenol-degrading anaerobic granular sludge sample

3.2 Anaerobic degradation batch test

3.2.1 Anaerobic culture medium

The anaerobic culture medium (pH 7.2−7.4) used in this study contained inorganic nutrients (mg/L) (NH4Cl, 170; CaCl2⋅2H2O, 17; MgCl2⋅6H2O, 125; FeCl3⋅6H2O, 4.1; KCl, 90; MnCl2⋅4H2O, 1.4; CoCl2⋅6H2O, 2.1; H3BO3, 0.4; CuCl2⋅2H2O, 0.19; Na2MoO4⋅2H2O, 0.18; and ZnCl2, 0.15), and micro nutrients (µg/L) (biotin, 4; folic acid, 4; pyridoxine HCl, 20; riboflavin, 10; thiamine, 10; pantothenic acid, 10; nicotinic acid, 10; vitamin B12, 0.2; 4-aminobenzoic acid, 10; and thioctic, 10) This medium was used in the initial enriching culture and the batch degradation test

A bicarbonate-buffered medium was used for further enrichment (Table 3.1) It contained (mg/L): K2HPO4, 400; trace element solution, 10 mL; MgCl2, 100; CaCl2-2H2O, 100; NH4Cl, 1000; resazurin (oxygen indicator), 1; NaHCO3, 1000; cystein-HCl, 500; vitamins solution, 10 mL; Na2S-9H2O, 250 All compounds were heat sterilized (i.e., solutions were boiled for 15 min) and flushed with nitrogen gas, except for the vitamins solution which was sterilized by filtration through a 0.2 µm membrane The medium was anaerobically distributed into 120 mL serum bottles, each containing 50 mL of liquid All the bottles were sealed with butyl rubber stoppers

Table 3.1 Trace elements and vitamins composition in anaerobic medium

3.2.2 Batch substrate degradation

Sludge samples or enrichment cultures were anaerobically transferred (10% inoculum,

v/v) into 120 mL serum bottles containing 50 mL of freshly prepared culture medium and

the test substrates (e.g., terephthalate, phenol and benzoate) under an atmosphere of 80%

N2−20% CO2 (v/v) and dark without shaking During incubation, substrates depletion

and methane production were measured periodically Every 2−3 days, three duplicates

of 0.5 mL liquid samples were withdrawn, and filtered immediately through a 0.45 µm

filter prior to chemical analysis Bio-gas composition (i.e., methane, hydrogen and

carbon dioxide), soluble volatile fatty acids and aromatic compounds (i.e., terephthalate,

phenol and benzoate) were determined as described in below

3.2.3 Chemical analyses

COD was analyzed according to the Standard Method (APHA et al., 1998) Soluble

short-chain volatile fatty acids (i.e acetate, propionate and butyrate) were determined