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Formaldehyde degradation in an anaerobic packed-bed bioreactor
ARTICLE IN PRESS
Water Research 38 (2004) 1685–1694
Formaldehyde degradation in an anaerobic packed-bed bioreactor S.V.W.B. Oliveira, E.M. Moraes, M.A.T. Adorno, M.B.A. Varesche, E. Foresti, M. Zaiat* ! ! ! Laboratorio de Processos Biologicos, Departamento de Hidraulica e Saneamento, Escola de Engenharia de Sao * Carlos (EESC), Universidade de Sao * Paulo (USP), Av. Trabalhador Sao-carlense * 400, Sao * Carlos, SP 13566-590, Brazil Received 12 February 2003; received in revised form 12 November 2003; accepted 20 January 2004
Abstract The development of appropriate technologies for the treatment of formaldehyde discharged into the environment is important to minimize its impact. Aerobic systems have been employed, although alternative anaerobic treatments have also been widely studied, mainly due to their low energy consumption and sludge production. However, toxic substances can lead to disturbances in anaerobic reactors. Some research has already been developed on formaldehyde anaerobic biological treatment, but no consensus has yet been reached about its behavior nor has the most efficient system been identified. Aiming at finding supporting evidence for this issue, therefore, this study investigated the degradation and toxicity of formaldehyde in a Horizontal-Flow Anaerobic Immobilized Sludge Reactor. Formaldehyde concentrations of 26.2–1158.6 mg HCHO/L were applied in the reactor, resulting in formaldehyde and chemical oxygen demand removal efficiencies of 99.7% and 92%, respectively. Volatile fatty acids with up to five carbons, found during the degradation of formaldehyde, are believed to indicate that the degradation followed routes unlike those suggested in the literature, which reports the formation of intermediates such as methanol and formic acid. The Monod kinetic model adhered to the experimental data well, with apparent kinetic parameters estimated as rapp max ¼ 2:79 103 mg HCHO/mg SSV h and Ksapp ¼ 242:8 mg HCHO/L. r 2004 Elsevier Ltd. All rights reserved. Keywords: Formaldehyde; Wastewater; Anaerobic treatment; Toxicity; Packed-bed reactor
1. Introduction Formaldehyde is used industrially as a component of some resins and glues in chemical and petrochemical plants, textile processing, paper manufacturing and wood processing, or as an active ingredient in disinfectants and preservatives. These industries can generate wastewaters containing up to 10 g/L of formaldehyde [1]. According to the ranking of environmental impacts generated by 45 chemical products proposed by Edwards et al. [2], formaldehyde ranks in first place. *Corresponding author. Tel.: +55-16-2739546; fax: +55-16273-9550. E-mail address: [email protected] (M. Zaiat).
Many studies have demonstrated the toxicity and the carcinogenicity of this substance [3–5]. As a disinfectant, a 0.5% formaldehyde solution (B5.4 g/L) destroys all species of microorganisms in a period of 6–12 h [6]. The literature contains little information about the anaerobic degradation and toxicity of formaldehyde. The pathway of anaerobic degradation of formaldehyde and the microorganisms involved in this process are not yet well defined. According to Gonz!alez-Gil [7], it is not clear if formaldehyde is converted directly into methane or if intermediate products act as substrates for methanogenic microorganisms. Similarly, there is no consensus about the concentration of formaldehyde that causes inhibition of the anaerobic biomass activity or about the most appropriate
0043-1354/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2004.01.013
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Nomenclature Symbols e bed porosity (dimensionless) CF formaldehyde concentration at a given L=D position (M/L3) CFef effluent formaldehyde concentration (M/L3) CFin influent formaldehyde concentration (M/L3) CX biomass concentration (M/L3) D diameter of the reactor (L) KSapp half-saturation kinetic constant—apparent (M/L3) L length of the reactor (L) Q liquid flow rate (L3/T) app rmax maximum specific formaldehyde conversion rate—apparent (T1)
system to treat formaldehyde-containing wastewaters, the intermediate products of degradation, the microorganisms responsible for each degradation pathway and the importance of co-substrates. Speece [8] stated that the full potential of the anaerobic biomass’s adaptation to many toxicants could be achieved if common sense and patience were used in exposing the biomass initially to relatively low concentrations and gradually increasing the concentrations up to a maximum value. The author also affirmed that formaldehyde is a good example of a compound that is highly toxic in high concentrations, but rapidly biodegradable in lower concentrations. Many reactors have been applied for the evaluation of the anaerobic degradation of formaldehyde, among them are batch reactors [7,9–16], continuous stirred tank reactors [17,18], anaerobic filters [9,17], up-flow anaerobic sludge blanket (UASB) reactors [15]; chemostats [12] and expanded granular sludge bed (EGSB) reactors [1,16]. In those studies, the wastewaters contained formaldehyde concentrations varying from 30 to 3000 mg/L, with or without co-substrate, and the results of inhibition of the microbial activity were quite different. The feeding strategy and the way the formaldehyde concentrations were increased also varied. However, Gonz!alez-Gil [7] found that the available literature on the subject was difficult to interpret as well as insufficient for design purposes. Most of the reports found in the literature indicate that high cellular retention times are indispensable to obtain the best performance of the reactor. Continuousflow reactors were also reported to achieve better results than batch reactors. The purpose of this study was to evaluate the performance of an anaerobic fixed-bed reactor having polyurethane foam as support material for biomass immobilization in the treatment of a formaldehyde-
robs Robs vs
specific formaldehyde conversion rate (T1) formaldehyde conversion rate (M/T L3) liquid superficial velocity (L/T)
Abbreviations COD chemical oxygen demand HAIB horizontal-flow anaerobic immobilized biomass (reactor) HDT hydraulic detention time FLR formaldehyde-loading rate UASB up-flow anaerobic sludge blanket (reactor) VSS volatile suspended solids
based substrate. Additionally, the research was meant to permit the estimation of the kinetic parameters of the biochemical degradation of such a compound.
2. Material and methods The horizontal-flow anaerobic immobilized biomass (HAIB) reactor was comprised of a 1000-mm long (L), 50.4-mm diameter (D) glass cylinder with a total volume of approximately 1995 mL and an L=D ratio of 20 (Fig. 1). The bed porosity (e) was assumed to be 0.4, thus resulting in the liquid volume of 798 mL. Intermediate sampling ports were located at L=D of 4, 8, 12 and 16. The reactor was installed in a temperature-controlled chamber and fed by means of a peristaltic pump. Polyurethane foam cubes (5-mm in size with an apparent density of 23 kg/m3) were used as biomass immobilization support. A synthetic substrate with increasing concentrations of formaldehyde was used in the experiments. Salts, metals and vitamins were supplied according to the medium formulated by Angelidaki et al. [19]. The synthetic wastewater was prepared with formaldehyde concentrations of 26.2, 85.3, 175.9, 394.0, 597.7, 808.0, 989.2 and 1158.6 mg HCHO/L. The substrate was prepared from a solution containing 38% of formaldehyde and 12% of methanol, with a density of 1.081 g/mL. Before starting the experiments, the anaerobic reactor was fed continuously for 1 year with a phenol-based synthetic substrate (50–1200 mg/L), with a hydraulic detention time (HDT) of 12 h at 3071 C [20]. The phenol removal efficiency was approximately 95% during that period. The experiments with formaldehyde-based substrate were performed with a progressive increment of the formaldehyde concentration from 26.2 to 1158.6 mg HCHO/L. The experiment lasted for 151
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Heating Fan
Automatic Control Hydraulic Seal Cubes of polyurethane foam
Reactor
D = 5.0 cm (in) Influent
Effluent
Pump Samplers
L/D=4
L = 100 cm L/D=8 L/D=12
L/D=16
Sample Collecting
Substrate
Fig. 1. Scheme of the bench-scale HAIB reactor.
days, during which the reactor was maintained at a constant temperature of 35 C71 C and operated with an HDT of 12.070.5 h, based on the liquid volume in the reactor. The reactor was operated under each influent formaldehyde concentration until the steady-state regimen was attained, after which the formaldehyde concentration was increased. Spatial formaldehyde profiles were recorded for each influent concentration, based on samples taken along the reactor’s length at L=D of 4, 8, 12 and 16. Two profiles were taken at 2-day intervals for each influent concentration. These profiles were recorded to estimate the apparent kinetic parameters for anaerobic formaldehyde degradation and to evaluate the possible reactions for formaldehyde conversion. In addition to the initial concentrations, which ranged from 26.2 to 1158.6 mg HCHO/L, another profile was obtained for an influent formaldehyde concentration of 1416.8 mg HCHO/L solely for purposes of kinetic studies. Analyses of chemical oxygen demand (COD), alkalinity and solids were performed according to the Standard Methods for the Examination of Water and Wastewater [21]. Modifications proposed by Ripley et al. [22] were applied to determine the alkalinity. The colorimetric method proposed by Bailey and Rankin [23] was used to determine the formaldehyde concentration. The biogas composition was analyzed by gas chromatography (Gow-Mac chromatograph with thermal conductivity detector and Porapak Q column—2 m 1/ 4 in—80 to 100 mesh). The injector, oven and detector temperatures were 50 C, 50 C and 80 C, respectively. Hydrogen was used as the carrier gas.
Volatile fatty acid concentrations were determined using a gas chromatograph HP 6869 with flame ionization detector and HP Innovax column— 30 m 0.25 mm (internal diameter) 0.25 mm (film thickness). The injector temperature was 250 C, with a split ratio of 1:20, and the detector temperature was 300 C. The oven temperature was 100 C for 3 min and 180 C for 5 min at 5 C/min. Hydrogen was used as the carrier gas. Formic acid was analyzed through high-pressure liquid chromatography (Shimadzu LC-10 AD VP with UV detector—210 nm—and Aminex 874—300 7.8 mm column). The mobile phase was N2SO4 solution at 0.005 M (0.6 mL/min) and the oven temperature was 35 C. The amount of biomass attached to the polyurethane foam was determined after the solids were detached from the supports in hand-agitated flasks containing glass balls. Biomass microbiological observations were conducted using an Olympus BH2 microscope. The biomass was examined by phase-contrast microscopy before starting the experiments and when the reactor was fed with formaldehyde concentrations of 26.2 and 1158.6 mg/L. Fluorescence was verified using a UV light source attached to a microscope.
3. Results and discussion 3.1. Performance of the packed-bed reactor Table 1 shows the results of formaldehyde degradation and COD removal achieved during 151 days of HAIB reactor operation under the step increase of influent formaldehyde concentrations. Fig. 2 presents
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Table 1 Influent (in) and effluent (ef) COD and formaldehyde concentration (CF ) observed during 151 days of HAIB reactor operation CFin (mg HCHO/L)
CODin (mg/L)
CFef (mg HCHO/L)
CODef (mg/L)
26.273.1 85.375.6 175.9716.5 394.0710.4 597.772.4 808.0723.4 989.2720.8 1158.6729.8
51.675.1 159.4712.4 315.877.0 608.9712.3 930.0717.0 1238.579.5 1494.1740.4 1798.5717.2
3.070.7 2.870.1 2.770.3 3.470.6 3.470.4 3.770.2 3.870.3 3.870.3
13.774.3 21.277.2 22.673.3 42.0710.4 48.972.3 90.8715.2 99.7720.8 99.2717.4
The average values are based on a minimum of four observation data.
Removal efficiency (%)ppp
mg/L HCHO Influent
26.2
85.3
175.9
20
40
60
394.0 597.7 808.0 989.2 1158.6
100 95 90 85 80 75 70 65 60 55 50 0
80 t (day)
100
120
140
160
Fig. 2. Temporal variation of COD (’), and formaldehyde () removal efficiencies.
the formaldehyde and COD removal efficiencies recorded throughout the experiment. The startup period was very short, since operating instability was only observed up to the 20th day of operation when the reactor was fed with formaldehyde concentration of 26.2 mg/L. This rapid acclimatization period may be attributed to the previous reactor operation with phenol-based substrate [20] and to the high biomass retention provided by the polyurethane foam. The average biomass concentration attached to the polyurethane foam, expressed as volatile suspended solids (VSS), was found to be 44.877.3 mg VSS/mL of foam, while the total biomass concentration was 26.9 g VSS/L, based on the total volume of the reactor, or 67.2 g VSS/L, based on the liquid volume. These data, which were obtained from samplings taken along the experimental time, indicate that the biomass concentration was practically constant throughout the experiments owing to the balance between biomass growth and washout. The biomass washout was estimated as 93 mg VSS/day, resulting in an overall biomass washout of 14 g of VSS along all the experimental time, corresponding to approximately 26% of the total biomass in the reactor (B54 g).
The anaerobic biodegradation of formaldehyde was found to be suitable for all the influent concentrations assayed. The effluent formaldehyde concentration presented slight variations as the influent concentration was increased and formaldehyde removal efficiencies of more than 95% were reached, except in the initial phase. Moreover, no operating instabilities occurred with the increase of the influent formaldehyde concentration. The influent COD increased slightly as the formaldehyde influent concentration was increased; even so, the mean COD removal efficiency of 92% was reached. The ratio between COD and formaldehyde concentration in the influent (CODin/CFin) was 1.770.2, but this ratio was found to vary for samples taken from the effluent stream, reaching the mean value of 2679 for influent concentrations of over 808.0 mg HCHO/L. Intermediate products or VSS could have been responsible for this variation, but low values of volatile fatty acids concentrations and VSS were detected in effluent samples, indicating that other by-products, undetected in this research, contributed to the effluent COD. Although no experimental tests were conducted, the removal of formaldehyde by adsorption in the reactor was considered negligible. This assumption was based on the studies of Qu and Bhattacharya [12] and Omil et al. [14], who reported only 10–11% of abiotic formaldehyde removal in anaerobic bioreactors. Fig. 3 presents the formaldehyde-loading rate (FLR) applied to the reactor as a function of the formaldehyde removal rate. A linear function fitted the experimental data well, indicating that the maximum degradation capacity of the system was not exceeded. The maximum FLR applied was 2316 mg HCHO/ L day or 0.086 g HCHO/g VSS day. A value of 0.05 g HCHO/g VSS day was applied by Sharma et al. [18], while Qu and Bhattacharya [12] reached a value of 0.164 g HCHO/g VSS day. The highest FLR value (0.6 g HCHO/g VSS day) was applied by Vidal et al. [15] because the biomass concentration in the reactor was low (5 g VSS/L). A formaldehyde influent concentration of 8 g/L should be applied in the HAIB reactor
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1158.6 mg HCHO/L. The presence of this volatile acid in the effluent motivated the search for volatile acids at intermediate sampling ports along the reactor’s length. Therefore, samples were taken at L=D of 4 and 8 in some experiments. Low concentrations of propionic (max. 7.9 mg/L), isobutyric (max. 12.1 mg/L), n-butyric (max. 2.4 mg/L), and isovaleric (max. 5.2 mg/L) acids were detected in experiments with initial formaldehyde concentrations of 808.0, 989.2 and 1158.6 mg HCHO/L. The maximum concentrations of volatile acids were detected at L=D of 8 when the reactor was fed with 1158.6 mg HCHO/L. Relatively high acetic acid concentrations were detected at L=D of 4 and 8 for influent formaldehyde concentrations exceeding 85.3 mg HCHO/L, with a minimum of 18.3 mg/L and a maximum of 231.4 mg/L. Acetic acid concentrations of 231.4 and 218.2 mg/L were
mg/L HCHO Influent 26.2
2500 2250 2000 1750
CH4 and CO2 (%)
Rate of HCHO removal (mg/L.day) pp
operated with a 12-h HDT to reach the specific FLR applied by Vidal et al. [15]. Table 2 compares the results obtained in this work with those of other studies in the literature. The pH values were 7.670.4 for influent samples and 7.970.4 for the effluent ones. The bicarbonate alkalinity showed stable values for influent (942770 mg CaCO3/L) and effluent (936797 mg CaCO3/L) samples, indicating the stability of the anaerobic process. The biogas was composed of approximately 80% of methane and 20% of carbon dioxide (Fig. 4). No variation was found in the relation CH4 to CO2 along the experimental time, except during the first 20 days of operation, when the biomass was in the acclimatization period. Low concentrations of about 20 mg/L of acetic acid were detected in the effluent samples when the reactor was fed with formaldehyde solution with 989.2 and
1500 1250 1000 750 500 250 0 0
250
500
750
1000
1250 1500
1750 2000
1689
0
2250 2500
85.3
175.9
40
60
394.0
597.7
808.0 989.2
1158.6
100 90 80 70 60 50 40 30 20 10 0 20
80
100
120
140
160
t (day)
HCHO Loading rate (mg/L.day)
Fig. 3. Rate of formaldehyde removal as a function of the FLR.
Fig. 4. Temporal variation of CH4 (m), and CO2 () concentration in the biogas.
Table 2 Some results from the literature obtained in continuous systems treating formaldehyde-containing wastewater Reactor
CFin (mg HCHO/L)
HDT (day)
Anaerobic filter Continuous stirring tank Continuous stirring tank with immobilized biomass
100–400 — —
1 10 10
Chemostat EGSB EGSB UASB UASB HAIB
100–1110 333 200/400/600 50–2000 95–950 26.2–1158.6
14 1.25 — 0.62 0.62 0.5
Limiting dose Temperature (mg HCHO/L) ( C) 400 125 375
1110 — — 1000 380 No limiting dose was observed
Formaldehyde removal efficiency (%)
Reference
35 35 35
— 85–88 95–98
(a) (b) (b)
35 — — 37 37 35
99.9 >93 High 98 (of COD) 95 >95
(c) (d) (e) (f) (f) This work
(a) Parkin et al. [9]; (b) Sharma et al. [18]; (c) Qu and Bhattacharya [12]; (d) Zoutberg and de Been [1]; (e) Gonzalez-Gil et al. [16]; and (f) Vidal et al. [15].
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detected for influent formaldehyde concentrations of 1158.6 mg HCHO/L (at L=D of 8) and 989.2 mg HCHO/ L (at L=D of 4), respectively.
suggests the oxidation of formaldehyde into formic acid and its reduction to methanol as
3.2. Formaldehyde conversion pathway
HCHO þ H2 -CH3 OH;
The presence of volatile acids along the reactor’s length possibly indicated that the formaldehyde conversion pathway in this system was different from that presented by Gonzalez-Gil et al. [16], according to whom the formaldehyde was transformed into formic acid and methanol. Analyses were also performed at intermediate sampling ports to detect the presence of formic acid and methanol. Formic acid was detected at L=D of 4 in the concentration of 95.8 mg/L for the influent formaldehyde concentration of 1158.6 mg HCHO/L, while methanol was not detected. Under this condition, the formic acid was consumed from L=D of 4 to L=D of 8, while the acetic acid was produced in the same segment. The intermediate compounds (with 2–5 carbons) detected along the reactor’s length and in the effluent stream (acetic acid) strongly indicated that biological or chemical synthesis was occurring, since formaldehyde was the sole carbon source (with 12% of methanol). Formaldehyde is a substance of immense chemical reactivity [24], so chemical synthesis appears to be the most likely explanation for these findings. Due to its high reactivity, formaldehyde can form polymers in aqueous solution [6]. The reactions are rapid in the absence of methanol, which is added to formaldehyde solutions to prevent polymerization. In aqueous solution, formaldehyde is almost completely hydrated to methylene glycol, which may polymerize to form a series of polyoxymethylene glycol [25]. In the HAIB reactor, the methanol (12%) in the synthetic substrate could have been consumed rapidly in the anaerobic reactor, thus allowing the formaldehyde to become polymerized. Methanol is a compound that degrades easily in anaerobic environments, mainly at such low concentrations. This hypothesis may explain the detection of compounds as volatile fatty acids, along the reactor’s length, which are intermediate in the anaerobic degradation of the formed polymers. Another possibility is aldol condensation, which occurs in the presence of weak bases, forming glycolic aldehyde and carbohydrates [24]. Karrer [26] reported on the production of carbohydrates from simple or multiple aldol condensation of formaldehyde, such as aldohexose. Therefore, the aldol condensation of formaldehyde, with the formation of six-carbon carbohydrates, may also explain the detection of intermediate products with up to five carbons. Therefore, the mechanism for formaldehyde degradation proposed by Gonzalez-Gil et al. [16] could not be confirmed in this work. The proposed mechanism
2HCHO þ H2 O-CH3 OH þ HCOOH ðTotalÞ:
HCHO þ H2 O-H2 þ HCOOH;
This possible biodegradation mechanism is similar to the ‘‘Cannizzaro Reaction’’ (Stanislao Cannizzaro, 1826–1910), in which two aldehyde groups are transformed into the corresponding hydroxyl functions, existing separately or in combination as an ester [27,28]. Formaldehyde, one aldehyde without a-hydrogens atoms, undergoes the ‘‘Cannizzaro Reaction’’ to yield methanol and formic acid [27] as NaOH
2HCHO þ H2 O ! CH3 OH þ HCOOH: 3.3. Kinetics of formaldehyde degradation As discussed previously, temporal profiles of formaldehyde concentrations were taken for each operating condition, with different influent formaldehyde concentrations, in order to estimate the apparent kinetic parameters for anaerobic formaldehyde degradation. The spatial profiles revealed that the application of an HDT of 4.8 h (L=D ¼ 8) would suffice to achieve the maximum performance when the reactor was fed with formaldehyde concentrations ranging from 26.2 to 175.9 mg HCHO/L since, at L=D of 8 (HDT of 4.8 h), the formaldehyde concentration was similar to that observed in the effluent stream. For influent formaldehyde concentrations ranging from 394.0 to 1158.6 mg HCHO/L, application of a 7-h HDT would be adequate to provide the same performance achieved with the HDT of 12 h. This analysis was based on the consideration that the HAIB reactor behaves as a plugflow reactor [29] and that the packed bed is homogeneous, thus preventing channeling and dead zones. Efficient formaldehyde degradation was therefore achieved by applying a HDT equal to or less than 4.8 h (L=D ¼ 8) for each initial concentration assayed (26.2–1158.6 mg HCOH/L). Under this operating condition, the formaldehyde removal efficiency ranged from 88% to 99%, with effluent formaldehyde concentrations of 2.7–19.4 mg HCHO/L. This positive result may be attributed to the hydrodynamic behavior of the HAIB reactor, which provides acclimatization for the microorganisms on the primary substrate and intermediate products in different segments along the reactor’s length [30]. In this flow pattern, not all microorganisms are subjected to possible toxic compounds. In addition to high cellular concentration, biomass immobilization may be another factor that explains the
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Robs ¼
evs dCF : D dðL=DÞ
Q : eA
ð2Þ
In Eq. (2), A is the total cross-sectional area of the reactor and Q is the liquid flow rate. Initial formaldehyde conversion rates (Robs ) were obtained for each initial formaldehyde concentration (CFin ) using the Microcal Origin 5.0s software. A function was adjusted to each spatial profile and values of ½dCF =dðL=DÞ were obtained by deriving the function at L=D equal to zero. Thus, the initial reaction rate was obtained from the mass balance (expression 1) for each influent formaldehyde concentration. Specific rates were then estimated by robs ¼
Robs : CX
ð3Þ
In this equation, CX is the concentration of biomass in the reactor, based on the liquid volume, which was considered constant and equal to 67.200 mg VSS/L. The Monod-type kinetic model was adjusted to the experimental data, using the Levenberg–Marquardt non-linear regression method (Microcal Origin 5.0s), as follows: robs ¼ rapp max
CF : KSapp þ CF
2.50E-03 2.00E-03 1.50E-03 1.00E-03 5.00E-04 0.00E+00 0
ð1Þ
In Eq. (1), Robs is the overall formaldehyde conversion rate; e; the bed porosity; vs ; the liquid superficial velocity; D; the diameter of the reactor; L; the length of the reactor and CF the formaldehyde concentration at a given L=D position. The liquid superficial velocity was kept constant throughout the experiment at 8.36 cm/h and was calculated as a function of the total cross-sectional area of the reactor (A) and the liquid flow rate (Q) as vs ¼
3.00E-03 robs (mg HCHO/mg VSS.L)ppp
positive results obtained for formaldehyde degradation. The liquid and solid-phase mass transfer resistances provide a ‘‘protected environment’’ inside the biofilm, preventing severe inhibitory effects. Moreover, there is some evidence that microorganisms organized in biofilms are inherently more resistant [31]. The kinetic parameters were estimated from the spatial profiles through the mass balance in the heterogeneous reactor, which was considered as a plug-flow reactor [29]
ð4Þ
The maximum specific conversion rate (rapp max ) and the half saturation constant (KSapp ) are apparent kinetic parameters, since they are dependent on both the mass transfer coefficients and the intrinsic kinetic parameters. The Monod kinetic model adhered to the experimental data well (Fig. 5) and the apparent kinetic app parameters rapp were estimated as 2.79 max and KS 3 (70.37) 10 mg HCHO/mg VSS h and 242.8
1691
250
500
750
1000
1250
1500
CF (mg HCHO/L)
Fig. 5. Monod kinetic model (—) adjusted to the experimental data ().
(7114.1) mg HCHO/L, respectively, with correlation coefficient of 0.8854. It should be noted that no inhibition was observed in the range of formaldehyde concentrations studied here. The kinetic behavior does not predict substrate or product inhibition.
3.4. Microbiological observations Table 3 summarizes the findings obtained from the microscopic observation throughout the experiment under different influent formaldehyde concentrations. In the inoculum from the HAIB reactor treating phenol, Bolan˜os et al. [20] observed the prevalence of methanogenic Archaea cells, which are morphologically similar to Methanosaeta and fluorescent rods. Straight rods, rods with rounded ends and rods with thin extremities were also frequent and were associated with Domain Bacteria. Among the methanogenic archaea, the organisms similar to Methanosaeta dominated for influent formaldehyde concentrations equal or lower than 394.0 mg HCHO/L. For the higher concentrations, the populations of Methanosaeta sp. and Methanosarcina sp. were found to be in equilibrium. On the other hand, the presence of fluorescent rods that could be associated to hydrognotrophic organisms was very rare. It is well known that Methanosaeta sp. grows chemoorganotrophically in a medium containing vitamins and acetate as the only organic compounds. Some strains require yeast extract and methane is formed exclusively from the methyl group of acetate. Formate, H2–CO2, methanol and methylamines cannot be used for growth and methane formation. In this work, the acetic acid concentration ranged from 18.3 to 231.4 mg/L and the medium [19] contained vitamins and yeast extract (0.5 mg/L). Janssen [32] isolated cultures of Methanosaeta spp. and good growth occurred when acetate was added in concentrations ranging from 590 to 2950 mg/L, with
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Table 3 Microbial morphologies in the inoculum and in samples of each phase Morphology
Inoculum
Influent formaldehyde concentration (mg HCHO/L) 175.9
394.0
597.7
808.0
989.2
1158.6
Domain Bacteria Straight rod Rod with extremities thin Rod with rounded ends Curved rod 1 Curved rod 2 Cocci Thin-filament Filament
++ ++ ++ + + + +
++ ++ ++ + + + +
++ ++ ++ + + +
++ ++ ++ + + +
++ ++ +++ + +++ + +
+++ +++ ++ + +++ + +
+++ +++ ++ + ++ + +
Domain Archaea Fluorescent rod Methanosaeta sp. Methanosarcina sp.
++ +++
+ +++ +
+ +++ ++
+ +++ +++
+ +++ +++
+ +++ +++
+ +++ +++
Cysts
+
+++, Predominant; ++, frequent; +, rare; , absent.
approximately 1 mol of methane formed per mol of acetate utilized. The culture vessels contained 50 mL of medium and a headspace of 75 mL of N2 plus CO2 (4:1 v/v) at 101 kPa. In the HAIB reactor, fed with formaldehyde or phenol [20], the configuration of the reactor, the type of support material and the concentration of acetate probably favored the growth of such Archaea cells. In the HAIB reactor treating formaldehyde, both Methanosaeta and Methanosarcina were found to prevail with influent formaldehyde concentrations higher than 394.0 mg HCHO/L. Organisms similar to Methanosaeta probably prevailed in the preferential use of acetate [33], while Methanosarcina preferentially used the methanol. The influent methanol concentration ranged from 124.4 to 365.9 mg/L for formaldehyde concentrations ranging from 597.7 to 1158.6 mg HCHO/L. These cells, which are acetotrophic methanogens, compete with each other for acetate. All Methanosarcina species use methanol and methylamines as catabolic substrates. Most species can also use H2–CO2 and acetate. The other strains do not use H2–CO2 as the sole energy substrate. However, if cells are grown in the presence of both H2–CO2 and acetate, the H2–CO2 is used first, followed shortly thereafter by the acetate. Acetate is degraded by the aceticlastic reaction, with the methyl group reduced to CH4 and the carboxyl group oxidized to CO2 [34]. Gonz!alez-Gil et al. [16], however, verified the prevalence of Methanosarcina in EGSB reactor treating formaldehyde. The sludge used by these researchers was adapted to methanol and they found that a portion of
the formaldehyde was quickly transformed into methanol. The presence of methanol probably favored the growth of Methanosarcina [34]. Other anaerobic microorganisms (straight rod, curved rod, rod with thin extremities, rod with rounded ends, cocci, thin-filament, filament) participated in the degradation of the formaldehyde of their polymeric products and of the long chain organic acids.
4. Conclusions The results obtained in this study of formaldehyde degradation in a HAIB reactor led to the following conclusions: The HAIB reactor was suitable for the treatment of formaldehyde-containing wastewater, presenting operating stability throughout the experiment. The average formaldehyde and COD removal efficiencies were 92% and 95%, respectively. Efficient formaldehyde degradation was achieved by applying an HDT equal to or less than 4.8 h for influent formaldehyde concentrations ranging from 26.2 to 1158.6 mg HCOH/L. The polyurethane foam favored the biomass retention in the reactor, with the average biomass reaching the concentration of 26.88 g VSS/L, which allowed for specific formaldehyde loads of over 0.086 g HCHO/ g VSS day to be attained for a formaldehyde load of 2.316 g HCHO/L day. The presence of organic acids with 2–5 carbons along the reactor’s length and in the effluent stream indicated
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the probable polymerization of formaldehyde in the absence of methanol, which may have been rapidly consumed in the anaerobic reactor. Aldol condensation of formaldehyde, with the formation of six-carbon carbohydrates, may also explain the detection of intermediate products with up to five carbons. The probable polymerization of formaldehyde, the use of immobilized cells on inert and macroporous support, high biomass concentrations and a hydrodynamic flow pattern similar to plug flow were believed to be the main factors responsible for the excellent performance of the HAIB reactor in the degradation of formaldehyde. Microscopic exams revealed a biomass containing multiple morphologies. This diversification likely contributed to the assimilation of the formaldehyde and to the intermediate products of degradation. Methanosaeta-like organisms probably used acetate preferentially, while Methanosarcina fed preferentially on methanol. The fast adaptation of the biomass to formaldehyde was attributed to its acclimatization to phenol in a previous experiment. The Monod kinetic model provided a suitable representation of the anaerobic degradation of formal3 dehyde with rapp (73.7 104) mg max ¼ 2:79 10 app HCHO/mg VSS h and KS ¼ 242:8 (7114.1) mg HCHO/L. No inhibition of cellular activity occurred with formaldehyde concentrations of up to 1416.8 mg HCHO/L.
Acknowledgements This study was supported by the Funda@*ao de Amparo a" Pesquisa do Estado de S*ao Paulo (FAPESP). The authors acknowledge the grants received from Funda@*ao Coordena@*ao de Aperfei@oamento de Pessoal de N!ıvel Superior (CAPES) and Conselho Nacional de ! Desenvolvimento Cient!ıfico e Tecnologico (CNPq).
References [1] Zoutberg GR, de Been P. The Biobeds EGSB (expanded granular sludge bed) system covers shortcomings of the up flow anaerobic sludge blanket reactor in the chemical industry. Water Sci Technol 1997;35:183–8. [2] Edwards FG, Egemen E, Brennan R, Nirmalakhandan N. Ranking of toxics release inventory chemicals using a level III fugacity model and toxicity. Water Sci Technol 1999;39:83–90. [3] Grafstrom RC, Curren RD, Harris CC. Genotoxicity of formaldehyde in cultured human bronchial fibroblasts. Science 1985;228:89–91. [4] Casteel SW, Vernon RJ, Bailey EM. Formaldehyde: toxicology and hazards. Vet Hum Toxicol 1987;29:31–3.
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[5] Conaway CC, Whysner J, Verna LK, Williams GM. Formaldehyde mechanistic data and risk assessment: endogenous protection from DNA adduct formation. Pharmacol Therapeut 1996;71:29–55. [6] Mingoia Q. Qu!ımica farmac#eutica. S*ao Paulo: Editora Universidade de S*ao Paulo—EDUSP; 1967. 787pp. [7] Gonz!alez-Gil G. Conversion of methanotrophic substrates in anaerobic reactors, metals, mass transfer and toxicity. PhD theses, Wageningen University, The Netherlands, 2000. 157pp. [8] Speece RE. Anaerobic biotechnology for industrial wastewaters. Nashville, Tennessee: Archae Press; 1996. 393pp. [9] Parkin GF, Speece RE, Yang CH, Kocher WM. Response of methane fermentation systems to industrial toxicants. J WPCF 1983;55:44–53. [10] Hickey RF, Vanderwielen J, Switzenbaum MS. The effects of organic toxicants on methane production and hydrogen gas levels during the anaerobic digestion of waste activated sludge. Water Res 1987;21:1417–27. [11] Todini O, Hulshoff Pol L. Anaerobic degradation of benzaldehyde in methanogenic granular sludge: the influence of additional substrates. Appl Microbiol Biotechnol 1992;38:417–20. [12] Qu M, Bhattacharya SK. Toxicity and biodegradation of formaldehyde in anaerobic methanogenic culture. Biotechnol Bioeng 1997;55:727–36. [13] Lu Z, Hegemann W. Anaerobic toxicity and biodegradation of formaldehyde in batch cultures. Water Res 1998;32:209–15. [14] Omil F, M!endez D, Vidal G, M!endez R, Lema JM. Biodegradation of formaldehyde under anaerobic conditions. Enzyme Microb Tech 1999;24:255–62. [15] Vidal G, Jiang ZP, Omil F, Thalasso, F, M!endez R, Lema JM. Continuous anaerobic treatment of wastewaters containing formaldehyde and urea. Bioresour Technol 1999;70:283–91. [16] Gonz!alez-Gil G, Kleerebezem R, van Aelst A, Zoutberg GR, Versprille AI, Lettinga G. Toxicity effects of formaldehyde on methanol degrading sludge and its anaerobic conversion in biobed expanded granular sludge bed (EGSB) reactors. Water Sci Technol 1999;40: 195–202. [17] Parkin GF, Speece RE. Attached versus suspended growth anaerobic reactors: response to toxic substrates. Water Sci Technol 1983;15:261–89. [18] Sharma S, Ramakrishna C, Desai JD, Bhatt NM. Anaerobic biodegradation of a petrochemical waste-water using biomass support particles. Appl Microbiol Biotechnol 1994;40:768–71. [19] Angelidaki I, Petersen SP, Ahring BK. Effects of lipids on thermophilic anaerobic digestion and reduction of lipid inhibition upon addition of bentonite. Appl Microbiol Biotechnol 1990;33:469–72. [20] Bolan˜os ML, Varesche MBA, Zaiat M, Foresti E. Phenol degradation in horizontal-flow anaerobic immobilized biomass (HAIB) reactor under mesophilic conditions. Water Sci Technol 2001;44:167–74. [21] Standard Methods for Examination of Water and Wastewater, 20th ed. Washington, DC, USA: American Public Health Association/American Water Works Association/ Water Environment Federation; 1998.
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[22] Ripley LE, Boyle WC, Converse JC. Improved alkalimetric monitoring for anaerobic digestion of high-strength wastes. J WPCF 1986;58:406–11. [23] Bailey BW, Rankin JM. New spectrophotometric method for determination of formaldehyde. Anal Chem 1971; 43:782–4. [24] Johnson AW, Buchanan JG, Elmore DT, Harvey WE, Walker J. Aldehydes and ketones—formaldehyde (CAP. VIII). In: Rodd EH, editor. Chemistry of carbon compounds, vol. IA. New York: Elsevier Publishing Company; 1951. p. 472–5. [25] Winkelman JGM, Voorwinde OK, Ottens M, Beenackers AACM, Jansen LPBM. Kinetics and chemical equilibrium of the hydration of formaldehyde. Chem Eng Sci 2002;57:4067–76. [26] Karrer P. Lehrbuch der organischen chemie, 11th ed. Stuttgart: Georg Thieme; 1950. 1082pp. [27] Geissman TA. The canizzaro reaction. In: Adans R, editor. Organic reactions, vol II. New York: Wiley; 1944. p. 94–113.
[28] Pavlov B, T!erentiev A. Chimie organique. Moscou: ! Editions Mir; 1967. 640pp. [29] Nardi IR, Zaiat M, Foresti E. Influence of the tracer characteristics on hydrodynamic models of packed-bed bioreactors. Bioprocess Eng 1999;21:469–76. [30] Zaiat M, Vieira LGT, Foresti E. Spatial and temporal variations of monitoring performance parameters in horizontal-flow anaerobic immobilized sludge (HAIS) reactor treating synthetic substrate. Water Res 1997;31:1760–6. [31] Jenkinson HF, Lappin-Scott HM. Biofilms adhere to stay. Trends Microbiol 2001;9:9–10. [32] Janssen PH. Selective enrichment and purification of cultures of Methanosaeta spp. J Microbiol Meth 2003; 52:239–44. [33] Zehnder AJB. Genus III. Methanotrix. In: Holt JG-inchief, editor. Bergeys’s manual of systematic bacteriology. Baltimore: Williams & Wilkins; 1984. p. 2207–9. [34] Madigan MT, Martinko JM, Parker J. Brock biology of microorganisms, 8th ed. Englewood Cliffs, NJ: Prentice Hall; 1997. 986pp.
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Formaldehyde degradation in an anaerobic packed-bed bioreactor S.V.W.B. Oliveira, E.M. Moraes, M.A.T. Adorno, M.B.A. Varesche, E. Foresti, M. Zaiat* ! ! ! Laboratorio de Processos Biologicos, Departamento de Hidraulica e Saneamento, Escola de Engenharia de Sao * Carlos (EESC), Universidade de Sao * Paulo (USP), Av. Trabalhador Sao-carlense * 400, Sao * Carlos, SP 13566-590, Brazil Received 12 February 2003; received in revised form 12 November 2003; accepted 20 January 2004
Abstract The development of appropriate technologies for the treatment of formaldehyde discharged into the environment is important to minimize its impact. Aerobic systems have been employed, although alternative anaerobic treatments have also been widely studied, mainly due to their low energy consumption and sludge production. However, toxic substances can lead to disturbances in anaerobic reactors. Some research has already been developed on formaldehyde anaerobic biological treatment, but no consensus has yet been reached about its behavior nor has the most efficient system been identified. Aiming at finding supporting evidence for this issue, therefore, this study investigated the degradation and toxicity of formaldehyde in a Horizontal-Flow Anaerobic Immobilized Sludge Reactor. Formaldehyde concentrations of 26.2–1158.6 mg HCHO/L were applied in the reactor, resulting in formaldehyde and chemical oxygen demand removal efficiencies of 99.7% and 92%, respectively. Volatile fatty acids with up to five carbons, found during the degradation of formaldehyde, are believed to indicate that the degradation followed routes unlike those suggested in the literature, which reports the formation of intermediates such as methanol and formic acid. The Monod kinetic model adhered to the experimental data well, with apparent kinetic parameters estimated as rapp max ¼ 2:79 103 mg HCHO/mg SSV h and Ksapp ¼ 242:8 mg HCHO/L. r 2004 Elsevier Ltd. All rights reserved. Keywords: Formaldehyde; Wastewater; Anaerobic treatment; Toxicity; Packed-bed reactor
1. Introduction Formaldehyde is used industrially as a component of some resins and glues in chemical and petrochemical plants, textile processing, paper manufacturing and wood processing, or as an active ingredient in disinfectants and preservatives. These industries can generate wastewaters containing up to 10 g/L of formaldehyde [1]. According to the ranking of environmental impacts generated by 45 chemical products proposed by Edwards et al. [2], formaldehyde ranks in first place. *Corresponding author. Tel.: +55-16-2739546; fax: +55-16273-9550. E-mail address: [email protected] (M. Zaiat).
Many studies have demonstrated the toxicity and the carcinogenicity of this substance [3–5]. As a disinfectant, a 0.5% formaldehyde solution (B5.4 g/L) destroys all species of microorganisms in a period of 6–12 h [6]. The literature contains little information about the anaerobic degradation and toxicity of formaldehyde. The pathway of anaerobic degradation of formaldehyde and the microorganisms involved in this process are not yet well defined. According to Gonz!alez-Gil [7], it is not clear if formaldehyde is converted directly into methane or if intermediate products act as substrates for methanogenic microorganisms. Similarly, there is no consensus about the concentration of formaldehyde that causes inhibition of the anaerobic biomass activity or about the most appropriate
0043-1354/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2004.01.013
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Nomenclature Symbols e bed porosity (dimensionless) CF formaldehyde concentration at a given L=D position (M/L3) CFef effluent formaldehyde concentration (M/L3) CFin influent formaldehyde concentration (M/L3) CX biomass concentration (M/L3) D diameter of the reactor (L) KSapp half-saturation kinetic constant—apparent (M/L3) L length of the reactor (L) Q liquid flow rate (L3/T) app rmax maximum specific formaldehyde conversion rate—apparent (T1)
system to treat formaldehyde-containing wastewaters, the intermediate products of degradation, the microorganisms responsible for each degradation pathway and the importance of co-substrates. Speece [8] stated that the full potential of the anaerobic biomass’s adaptation to many toxicants could be achieved if common sense and patience were used in exposing the biomass initially to relatively low concentrations and gradually increasing the concentrations up to a maximum value. The author also affirmed that formaldehyde is a good example of a compound that is highly toxic in high concentrations, but rapidly biodegradable in lower concentrations. Many reactors have been applied for the evaluation of the anaerobic degradation of formaldehyde, among them are batch reactors [7,9–16], continuous stirred tank reactors [17,18], anaerobic filters [9,17], up-flow anaerobic sludge blanket (UASB) reactors [15]; chemostats [12] and expanded granular sludge bed (EGSB) reactors [1,16]. In those studies, the wastewaters contained formaldehyde concentrations varying from 30 to 3000 mg/L, with or without co-substrate, and the results of inhibition of the microbial activity were quite different. The feeding strategy and the way the formaldehyde concentrations were increased also varied. However, Gonz!alez-Gil [7] found that the available literature on the subject was difficult to interpret as well as insufficient for design purposes. Most of the reports found in the literature indicate that high cellular retention times are indispensable to obtain the best performance of the reactor. Continuousflow reactors were also reported to achieve better results than batch reactors. The purpose of this study was to evaluate the performance of an anaerobic fixed-bed reactor having polyurethane foam as support material for biomass immobilization in the treatment of a formaldehyde-
robs Robs vs
specific formaldehyde conversion rate (T1) formaldehyde conversion rate (M/T L3) liquid superficial velocity (L/T)
Abbreviations COD chemical oxygen demand HAIB horizontal-flow anaerobic immobilized biomass (reactor) HDT hydraulic detention time FLR formaldehyde-loading rate UASB up-flow anaerobic sludge blanket (reactor) VSS volatile suspended solids
based substrate. Additionally, the research was meant to permit the estimation of the kinetic parameters of the biochemical degradation of such a compound.
2. Material and methods The horizontal-flow anaerobic immobilized biomass (HAIB) reactor was comprised of a 1000-mm long (L), 50.4-mm diameter (D) glass cylinder with a total volume of approximately 1995 mL and an L=D ratio of 20 (Fig. 1). The bed porosity (e) was assumed to be 0.4, thus resulting in the liquid volume of 798 mL. Intermediate sampling ports were located at L=D of 4, 8, 12 and 16. The reactor was installed in a temperature-controlled chamber and fed by means of a peristaltic pump. Polyurethane foam cubes (5-mm in size with an apparent density of 23 kg/m3) were used as biomass immobilization support. A synthetic substrate with increasing concentrations of formaldehyde was used in the experiments. Salts, metals and vitamins were supplied according to the medium formulated by Angelidaki et al. [19]. The synthetic wastewater was prepared with formaldehyde concentrations of 26.2, 85.3, 175.9, 394.0, 597.7, 808.0, 989.2 and 1158.6 mg HCHO/L. The substrate was prepared from a solution containing 38% of formaldehyde and 12% of methanol, with a density of 1.081 g/mL. Before starting the experiments, the anaerobic reactor was fed continuously for 1 year with a phenol-based synthetic substrate (50–1200 mg/L), with a hydraulic detention time (HDT) of 12 h at 3071 C [20]. The phenol removal efficiency was approximately 95% during that period. The experiments with formaldehyde-based substrate were performed with a progressive increment of the formaldehyde concentration from 26.2 to 1158.6 mg HCHO/L. The experiment lasted for 151
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Heating Fan
Automatic Control Hydraulic Seal Cubes of polyurethane foam
Reactor
D = 5.0 cm (in) Influent
Effluent
Pump Samplers
L/D=4
L = 100 cm L/D=8 L/D=12
L/D=16
Sample Collecting
Substrate
Fig. 1. Scheme of the bench-scale HAIB reactor.
days, during which the reactor was maintained at a constant temperature of 35 C71 C and operated with an HDT of 12.070.5 h, based on the liquid volume in the reactor. The reactor was operated under each influent formaldehyde concentration until the steady-state regimen was attained, after which the formaldehyde concentration was increased. Spatial formaldehyde profiles were recorded for each influent concentration, based on samples taken along the reactor’s length at L=D of 4, 8, 12 and 16. Two profiles were taken at 2-day intervals for each influent concentration. These profiles were recorded to estimate the apparent kinetic parameters for anaerobic formaldehyde degradation and to evaluate the possible reactions for formaldehyde conversion. In addition to the initial concentrations, which ranged from 26.2 to 1158.6 mg HCHO/L, another profile was obtained for an influent formaldehyde concentration of 1416.8 mg HCHO/L solely for purposes of kinetic studies. Analyses of chemical oxygen demand (COD), alkalinity and solids were performed according to the Standard Methods for the Examination of Water and Wastewater [21]. Modifications proposed by Ripley et al. [22] were applied to determine the alkalinity. The colorimetric method proposed by Bailey and Rankin [23] was used to determine the formaldehyde concentration. The biogas composition was analyzed by gas chromatography (Gow-Mac chromatograph with thermal conductivity detector and Porapak Q column—2 m 1/ 4 in—80 to 100 mesh). The injector, oven and detector temperatures were 50 C, 50 C and 80 C, respectively. Hydrogen was used as the carrier gas.
Volatile fatty acid concentrations were determined using a gas chromatograph HP 6869 with flame ionization detector and HP Innovax column— 30 m 0.25 mm (internal diameter) 0.25 mm (film thickness). The injector temperature was 250 C, with a split ratio of 1:20, and the detector temperature was 300 C. The oven temperature was 100 C for 3 min and 180 C for 5 min at 5 C/min. Hydrogen was used as the carrier gas. Formic acid was analyzed through high-pressure liquid chromatography (Shimadzu LC-10 AD VP with UV detector—210 nm—and Aminex 874—300 7.8 mm column). The mobile phase was N2SO4 solution at 0.005 M (0.6 mL/min) and the oven temperature was 35 C. The amount of biomass attached to the polyurethane foam was determined after the solids were detached from the supports in hand-agitated flasks containing glass balls. Biomass microbiological observations were conducted using an Olympus BH2 microscope. The biomass was examined by phase-contrast microscopy before starting the experiments and when the reactor was fed with formaldehyde concentrations of 26.2 and 1158.6 mg/L. Fluorescence was verified using a UV light source attached to a microscope.
3. Results and discussion 3.1. Performance of the packed-bed reactor Table 1 shows the results of formaldehyde degradation and COD removal achieved during 151 days of HAIB reactor operation under the step increase of influent formaldehyde concentrations. Fig. 2 presents
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Table 1 Influent (in) and effluent (ef) COD and formaldehyde concentration (CF ) observed during 151 days of HAIB reactor operation CFin (mg HCHO/L)
CODin (mg/L)
CFef (mg HCHO/L)
CODef (mg/L)
26.273.1 85.375.6 175.9716.5 394.0710.4 597.772.4 808.0723.4 989.2720.8 1158.6729.8
51.675.1 159.4712.4 315.877.0 608.9712.3 930.0717.0 1238.579.5 1494.1740.4 1798.5717.2
3.070.7 2.870.1 2.770.3 3.470.6 3.470.4 3.770.2 3.870.3 3.870.3
13.774.3 21.277.2 22.673.3 42.0710.4 48.972.3 90.8715.2 99.7720.8 99.2717.4
The average values are based on a minimum of four observation data.
Removal efficiency (%)ppp
mg/L HCHO Influent
26.2
85.3
175.9
20
40
60
394.0 597.7 808.0 989.2 1158.6
100 95 90 85 80 75 70 65 60 55 50 0
80 t (day)
100
120
140
160
Fig. 2. Temporal variation of COD (’), and formaldehyde () removal efficiencies.
the formaldehyde and COD removal efficiencies recorded throughout the experiment. The startup period was very short, since operating instability was only observed up to the 20th day of operation when the reactor was fed with formaldehyde concentration of 26.2 mg/L. This rapid acclimatization period may be attributed to the previous reactor operation with phenol-based substrate [20] and to the high biomass retention provided by the polyurethane foam. The average biomass concentration attached to the polyurethane foam, expressed as volatile suspended solids (VSS), was found to be 44.877.3 mg VSS/mL of foam, while the total biomass concentration was 26.9 g VSS/L, based on the total volume of the reactor, or 67.2 g VSS/L, based on the liquid volume. These data, which were obtained from samplings taken along the experimental time, indicate that the biomass concentration was practically constant throughout the experiments owing to the balance between biomass growth and washout. The biomass washout was estimated as 93 mg VSS/day, resulting in an overall biomass washout of 14 g of VSS along all the experimental time, corresponding to approximately 26% of the total biomass in the reactor (B54 g).
The anaerobic biodegradation of formaldehyde was found to be suitable for all the influent concentrations assayed. The effluent formaldehyde concentration presented slight variations as the influent concentration was increased and formaldehyde removal efficiencies of more than 95% were reached, except in the initial phase. Moreover, no operating instabilities occurred with the increase of the influent formaldehyde concentration. The influent COD increased slightly as the formaldehyde influent concentration was increased; even so, the mean COD removal efficiency of 92% was reached. The ratio between COD and formaldehyde concentration in the influent (CODin/CFin) was 1.770.2, but this ratio was found to vary for samples taken from the effluent stream, reaching the mean value of 2679 for influent concentrations of over 808.0 mg HCHO/L. Intermediate products or VSS could have been responsible for this variation, but low values of volatile fatty acids concentrations and VSS were detected in effluent samples, indicating that other by-products, undetected in this research, contributed to the effluent COD. Although no experimental tests were conducted, the removal of formaldehyde by adsorption in the reactor was considered negligible. This assumption was based on the studies of Qu and Bhattacharya [12] and Omil et al. [14], who reported only 10–11% of abiotic formaldehyde removal in anaerobic bioreactors. Fig. 3 presents the formaldehyde-loading rate (FLR) applied to the reactor as a function of the formaldehyde removal rate. A linear function fitted the experimental data well, indicating that the maximum degradation capacity of the system was not exceeded. The maximum FLR applied was 2316 mg HCHO/ L day or 0.086 g HCHO/g VSS day. A value of 0.05 g HCHO/g VSS day was applied by Sharma et al. [18], while Qu and Bhattacharya [12] reached a value of 0.164 g HCHO/g VSS day. The highest FLR value (0.6 g HCHO/g VSS day) was applied by Vidal et al. [15] because the biomass concentration in the reactor was low (5 g VSS/L). A formaldehyde influent concentration of 8 g/L should be applied in the HAIB reactor
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1158.6 mg HCHO/L. The presence of this volatile acid in the effluent motivated the search for volatile acids at intermediate sampling ports along the reactor’s length. Therefore, samples were taken at L=D of 4 and 8 in some experiments. Low concentrations of propionic (max. 7.9 mg/L), isobutyric (max. 12.1 mg/L), n-butyric (max. 2.4 mg/L), and isovaleric (max. 5.2 mg/L) acids were detected in experiments with initial formaldehyde concentrations of 808.0, 989.2 and 1158.6 mg HCHO/L. The maximum concentrations of volatile acids were detected at L=D of 8 when the reactor was fed with 1158.6 mg HCHO/L. Relatively high acetic acid concentrations were detected at L=D of 4 and 8 for influent formaldehyde concentrations exceeding 85.3 mg HCHO/L, with a minimum of 18.3 mg/L and a maximum of 231.4 mg/L. Acetic acid concentrations of 231.4 and 218.2 mg/L were
mg/L HCHO Influent 26.2
2500 2250 2000 1750
CH4 and CO2 (%)
Rate of HCHO removal (mg/L.day) pp
operated with a 12-h HDT to reach the specific FLR applied by Vidal et al. [15]. Table 2 compares the results obtained in this work with those of other studies in the literature. The pH values were 7.670.4 for influent samples and 7.970.4 for the effluent ones. The bicarbonate alkalinity showed stable values for influent (942770 mg CaCO3/L) and effluent (936797 mg CaCO3/L) samples, indicating the stability of the anaerobic process. The biogas was composed of approximately 80% of methane and 20% of carbon dioxide (Fig. 4). No variation was found in the relation CH4 to CO2 along the experimental time, except during the first 20 days of operation, when the biomass was in the acclimatization period. Low concentrations of about 20 mg/L of acetic acid were detected in the effluent samples when the reactor was fed with formaldehyde solution with 989.2 and
1500 1250 1000 750 500 250 0 0
250
500
750
1000
1250 1500
1750 2000
1689
0
2250 2500
85.3
175.9
40
60
394.0
597.7
808.0 989.2
1158.6
100 90 80 70 60 50 40 30 20 10 0 20
80
100
120
140
160
t (day)
HCHO Loading rate (mg/L.day)
Fig. 3. Rate of formaldehyde removal as a function of the FLR.
Fig. 4. Temporal variation of CH4 (m), and CO2 () concentration in the biogas.
Table 2 Some results from the literature obtained in continuous systems treating formaldehyde-containing wastewater Reactor
CFin (mg HCHO/L)
HDT (day)
Anaerobic filter Continuous stirring tank Continuous stirring tank with immobilized biomass
100–400 — —
1 10 10
Chemostat EGSB EGSB UASB UASB HAIB
100–1110 333 200/400/600 50–2000 95–950 26.2–1158.6
14 1.25 — 0.62 0.62 0.5
Limiting dose Temperature (mg HCHO/L) ( C) 400 125 375
1110 — — 1000 380 No limiting dose was observed
Formaldehyde removal efficiency (%)
Reference
35 35 35
— 85–88 95–98
(a) (b) (b)
35 — — 37 37 35
99.9 >93 High 98 (of COD) 95 >95
(c) (d) (e) (f) (f) This work
(a) Parkin et al. [9]; (b) Sharma et al. [18]; (c) Qu and Bhattacharya [12]; (d) Zoutberg and de Been [1]; (e) Gonzalez-Gil et al. [16]; and (f) Vidal et al. [15].
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detected for influent formaldehyde concentrations of 1158.6 mg HCHO/L (at L=D of 8) and 989.2 mg HCHO/ L (at L=D of 4), respectively.
suggests the oxidation of formaldehyde into formic acid and its reduction to methanol as
3.2. Formaldehyde conversion pathway
HCHO þ H2 -CH3 OH;
The presence of volatile acids along the reactor’s length possibly indicated that the formaldehyde conversion pathway in this system was different from that presented by Gonzalez-Gil et al. [16], according to whom the formaldehyde was transformed into formic acid and methanol. Analyses were also performed at intermediate sampling ports to detect the presence of formic acid and methanol. Formic acid was detected at L=D of 4 in the concentration of 95.8 mg/L for the influent formaldehyde concentration of 1158.6 mg HCHO/L, while methanol was not detected. Under this condition, the formic acid was consumed from L=D of 4 to L=D of 8, while the acetic acid was produced in the same segment. The intermediate compounds (with 2–5 carbons) detected along the reactor’s length and in the effluent stream (acetic acid) strongly indicated that biological or chemical synthesis was occurring, since formaldehyde was the sole carbon source (with 12% of methanol). Formaldehyde is a substance of immense chemical reactivity [24], so chemical synthesis appears to be the most likely explanation for these findings. Due to its high reactivity, formaldehyde can form polymers in aqueous solution [6]. The reactions are rapid in the absence of methanol, which is added to formaldehyde solutions to prevent polymerization. In aqueous solution, formaldehyde is almost completely hydrated to methylene glycol, which may polymerize to form a series of polyoxymethylene glycol [25]. In the HAIB reactor, the methanol (12%) in the synthetic substrate could have been consumed rapidly in the anaerobic reactor, thus allowing the formaldehyde to become polymerized. Methanol is a compound that degrades easily in anaerobic environments, mainly at such low concentrations. This hypothesis may explain the detection of compounds as volatile fatty acids, along the reactor’s length, which are intermediate in the anaerobic degradation of the formed polymers. Another possibility is aldol condensation, which occurs in the presence of weak bases, forming glycolic aldehyde and carbohydrates [24]. Karrer [26] reported on the production of carbohydrates from simple or multiple aldol condensation of formaldehyde, such as aldohexose. Therefore, the aldol condensation of formaldehyde, with the formation of six-carbon carbohydrates, may also explain the detection of intermediate products with up to five carbons. Therefore, the mechanism for formaldehyde degradation proposed by Gonzalez-Gil et al. [16] could not be confirmed in this work. The proposed mechanism
2HCHO þ H2 O-CH3 OH þ HCOOH ðTotalÞ:
HCHO þ H2 O-H2 þ HCOOH;
This possible biodegradation mechanism is similar to the ‘‘Cannizzaro Reaction’’ (Stanislao Cannizzaro, 1826–1910), in which two aldehyde groups are transformed into the corresponding hydroxyl functions, existing separately or in combination as an ester [27,28]. Formaldehyde, one aldehyde without a-hydrogens atoms, undergoes the ‘‘Cannizzaro Reaction’’ to yield methanol and formic acid [27] as NaOH
2HCHO þ H2 O ! CH3 OH þ HCOOH: 3.3. Kinetics of formaldehyde degradation As discussed previously, temporal profiles of formaldehyde concentrations were taken for each operating condition, with different influent formaldehyde concentrations, in order to estimate the apparent kinetic parameters for anaerobic formaldehyde degradation. The spatial profiles revealed that the application of an HDT of 4.8 h (L=D ¼ 8) would suffice to achieve the maximum performance when the reactor was fed with formaldehyde concentrations ranging from 26.2 to 175.9 mg HCHO/L since, at L=D of 8 (HDT of 4.8 h), the formaldehyde concentration was similar to that observed in the effluent stream. For influent formaldehyde concentrations ranging from 394.0 to 1158.6 mg HCHO/L, application of a 7-h HDT would be adequate to provide the same performance achieved with the HDT of 12 h. This analysis was based on the consideration that the HAIB reactor behaves as a plugflow reactor [29] and that the packed bed is homogeneous, thus preventing channeling and dead zones. Efficient formaldehyde degradation was therefore achieved by applying a HDT equal to or less than 4.8 h (L=D ¼ 8) for each initial concentration assayed (26.2–1158.6 mg HCOH/L). Under this operating condition, the formaldehyde removal efficiency ranged from 88% to 99%, with effluent formaldehyde concentrations of 2.7–19.4 mg HCHO/L. This positive result may be attributed to the hydrodynamic behavior of the HAIB reactor, which provides acclimatization for the microorganisms on the primary substrate and intermediate products in different segments along the reactor’s length [30]. In this flow pattern, not all microorganisms are subjected to possible toxic compounds. In addition to high cellular concentration, biomass immobilization may be another factor that explains the
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Robs ¼
evs dCF : D dðL=DÞ
Q : eA
ð2Þ
In Eq. (2), A is the total cross-sectional area of the reactor and Q is the liquid flow rate. Initial formaldehyde conversion rates (Robs ) were obtained for each initial formaldehyde concentration (CFin ) using the Microcal Origin 5.0s software. A function was adjusted to each spatial profile and values of ½dCF =dðL=DÞ were obtained by deriving the function at L=D equal to zero. Thus, the initial reaction rate was obtained from the mass balance (expression 1) for each influent formaldehyde concentration. Specific rates were then estimated by robs ¼
Robs : CX
ð3Þ
In this equation, CX is the concentration of biomass in the reactor, based on the liquid volume, which was considered constant and equal to 67.200 mg VSS/L. The Monod-type kinetic model was adjusted to the experimental data, using the Levenberg–Marquardt non-linear regression method (Microcal Origin 5.0s), as follows: robs ¼ rapp max
CF : KSapp þ CF
2.50E-03 2.00E-03 1.50E-03 1.00E-03 5.00E-04 0.00E+00 0
ð1Þ
In Eq. (1), Robs is the overall formaldehyde conversion rate; e; the bed porosity; vs ; the liquid superficial velocity; D; the diameter of the reactor; L; the length of the reactor and CF the formaldehyde concentration at a given L=D position. The liquid superficial velocity was kept constant throughout the experiment at 8.36 cm/h and was calculated as a function of the total cross-sectional area of the reactor (A) and the liquid flow rate (Q) as vs ¼
3.00E-03 robs (mg HCHO/mg VSS.L)ppp
positive results obtained for formaldehyde degradation. The liquid and solid-phase mass transfer resistances provide a ‘‘protected environment’’ inside the biofilm, preventing severe inhibitory effects. Moreover, there is some evidence that microorganisms organized in biofilms are inherently more resistant [31]. The kinetic parameters were estimated from the spatial profiles through the mass balance in the heterogeneous reactor, which was considered as a plug-flow reactor [29]
ð4Þ
The maximum specific conversion rate (rapp max ) and the half saturation constant (KSapp ) are apparent kinetic parameters, since they are dependent on both the mass transfer coefficients and the intrinsic kinetic parameters. The Monod kinetic model adhered to the experimental data well (Fig. 5) and the apparent kinetic app parameters rapp were estimated as 2.79 max and KS 3 (70.37) 10 mg HCHO/mg VSS h and 242.8
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250
500
750
1000
1250
1500
CF (mg HCHO/L)
Fig. 5. Monod kinetic model (—) adjusted to the experimental data ().
(7114.1) mg HCHO/L, respectively, with correlation coefficient of 0.8854. It should be noted that no inhibition was observed in the range of formaldehyde concentrations studied here. The kinetic behavior does not predict substrate or product inhibition.
3.4. Microbiological observations Table 3 summarizes the findings obtained from the microscopic observation throughout the experiment under different influent formaldehyde concentrations. In the inoculum from the HAIB reactor treating phenol, Bolan˜os et al. [20] observed the prevalence of methanogenic Archaea cells, which are morphologically similar to Methanosaeta and fluorescent rods. Straight rods, rods with rounded ends and rods with thin extremities were also frequent and were associated with Domain Bacteria. Among the methanogenic archaea, the organisms similar to Methanosaeta dominated for influent formaldehyde concentrations equal or lower than 394.0 mg HCHO/L. For the higher concentrations, the populations of Methanosaeta sp. and Methanosarcina sp. were found to be in equilibrium. On the other hand, the presence of fluorescent rods that could be associated to hydrognotrophic organisms was very rare. It is well known that Methanosaeta sp. grows chemoorganotrophically in a medium containing vitamins and acetate as the only organic compounds. Some strains require yeast extract and methane is formed exclusively from the methyl group of acetate. Formate, H2–CO2, methanol and methylamines cannot be used for growth and methane formation. In this work, the acetic acid concentration ranged from 18.3 to 231.4 mg/L and the medium [19] contained vitamins and yeast extract (0.5 mg/L). Janssen [32] isolated cultures of Methanosaeta spp. and good growth occurred when acetate was added in concentrations ranging from 590 to 2950 mg/L, with
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Table 3 Microbial morphologies in the inoculum and in samples of each phase Morphology
Inoculum
Influent formaldehyde concentration (mg HCHO/L) 175.9
394.0
597.7
808.0
989.2
1158.6
Domain Bacteria Straight rod Rod with extremities thin Rod with rounded ends Curved rod 1 Curved rod 2 Cocci Thin-filament Filament
++ ++ ++ + + + +
++ ++ ++ + + + +
++ ++ ++ + + +
++ ++ ++ + + +
++ ++ +++ + +++ + +
+++ +++ ++ + +++ + +
+++ +++ ++ + ++ + +
Domain Archaea Fluorescent rod Methanosaeta sp. Methanosarcina sp.
++ +++
+ +++ +
+ +++ ++
+ +++ +++
+ +++ +++
+ +++ +++
+ +++ +++
Cysts
+
+++, Predominant; ++, frequent; +, rare; , absent.
approximately 1 mol of methane formed per mol of acetate utilized. The culture vessels contained 50 mL of medium and a headspace of 75 mL of N2 plus CO2 (4:1 v/v) at 101 kPa. In the HAIB reactor, fed with formaldehyde or phenol [20], the configuration of the reactor, the type of support material and the concentration of acetate probably favored the growth of such Archaea cells. In the HAIB reactor treating formaldehyde, both Methanosaeta and Methanosarcina were found to prevail with influent formaldehyde concentrations higher than 394.0 mg HCHO/L. Organisms similar to Methanosaeta probably prevailed in the preferential use of acetate [33], while Methanosarcina preferentially used the methanol. The influent methanol concentration ranged from 124.4 to 365.9 mg/L for formaldehyde concentrations ranging from 597.7 to 1158.6 mg HCHO/L. These cells, which are acetotrophic methanogens, compete with each other for acetate. All Methanosarcina species use methanol and methylamines as catabolic substrates. Most species can also use H2–CO2 and acetate. The other strains do not use H2–CO2 as the sole energy substrate. However, if cells are grown in the presence of both H2–CO2 and acetate, the H2–CO2 is used first, followed shortly thereafter by the acetate. Acetate is degraded by the aceticlastic reaction, with the methyl group reduced to CH4 and the carboxyl group oxidized to CO2 [34]. Gonz!alez-Gil et al. [16], however, verified the prevalence of Methanosarcina in EGSB reactor treating formaldehyde. The sludge used by these researchers was adapted to methanol and they found that a portion of
the formaldehyde was quickly transformed into methanol. The presence of methanol probably favored the growth of Methanosarcina [34]. Other anaerobic microorganisms (straight rod, curved rod, rod with thin extremities, rod with rounded ends, cocci, thin-filament, filament) participated in the degradation of the formaldehyde of their polymeric products and of the long chain organic acids.
4. Conclusions The results obtained in this study of formaldehyde degradation in a HAIB reactor led to the following conclusions: The HAIB reactor was suitable for the treatment of formaldehyde-containing wastewater, presenting operating stability throughout the experiment. The average formaldehyde and COD removal efficiencies were 92% and 95%, respectively. Efficient formaldehyde degradation was achieved by applying an HDT equal to or less than 4.8 h for influent formaldehyde concentrations ranging from 26.2 to 1158.6 mg HCOH/L. The polyurethane foam favored the biomass retention in the reactor, with the average biomass reaching the concentration of 26.88 g VSS/L, which allowed for specific formaldehyde loads of over 0.086 g HCHO/ g VSS day to be attained for a formaldehyde load of 2.316 g HCHO/L day. The presence of organic acids with 2–5 carbons along the reactor’s length and in the effluent stream indicated
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the probable polymerization of formaldehyde in the absence of methanol, which may have been rapidly consumed in the anaerobic reactor. Aldol condensation of formaldehyde, with the formation of six-carbon carbohydrates, may also explain the detection of intermediate products with up to five carbons. The probable polymerization of formaldehyde, the use of immobilized cells on inert and macroporous support, high biomass concentrations and a hydrodynamic flow pattern similar to plug flow were believed to be the main factors responsible for the excellent performance of the HAIB reactor in the degradation of formaldehyde. Microscopic exams revealed a biomass containing multiple morphologies. This diversification likely contributed to the assimilation of the formaldehyde and to the intermediate products of degradation. Methanosaeta-like organisms probably used acetate preferentially, while Methanosarcina fed preferentially on methanol. The fast adaptation of the biomass to formaldehyde was attributed to its acclimatization to phenol in a previous experiment. The Monod kinetic model provided a suitable representation of the anaerobic degradation of formal3 dehyde with rapp (73.7 104) mg max ¼ 2:79 10 app HCHO/mg VSS h and KS ¼ 242:8 (7114.1) mg HCHO/L. No inhibition of cellular activity occurred with formaldehyde concentrations of up to 1416.8 mg HCHO/L.
Acknowledgements This study was supported by the Funda@*ao de Amparo a" Pesquisa do Estado de S*ao Paulo (FAPESP). The authors acknowledge the grants received from Funda@*ao Coordena@*ao de Aperfei@oamento de Pessoal de N!ıvel Superior (CAPES) and Conselho Nacional de ! Desenvolvimento Cient!ıfico e Tecnologico (CNPq).
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