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Kinetics of model high molecular weight organic compounds biodegradation in soil aquifer treatment
w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 4 4 1 9 e4 4 2 7
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Kinetics of model high molecular weight organic compounds biodegradation in soil aquifer treatment Peter Fox, Roshan Makam* Department of Civil and Environmental Engineering, Arizona State University, PO Box 5306, Tempe, AZ 85287-5306, USA
article info
abstract
Article history:
Soil Aquifer Treatment (SAT) is a process where treated wastewater is purified during
Received 6 December 2010
transport through unsaturated and saturated zones. Easily biodegradable compounds are
Received in revised form
rapidly removed in the unsaturated zone and the residual organic carbon is comprised of
14 May 2011
primarily high molecular weight compounds. This research focuses on flow in the satu-
Accepted 22 May 2011
rated zone where flow conditions are predictable and high molecular weight compounds
Available online 15 June 2011
are degraded. Flow through the saturated zone was investigated with 4 reactors packed with 2 different particle sizes and operated at 4 different flow rates. The objective was to
Keywords:
evaluate the kinetics of transformation for high molecular weight organics during SAT.
Soil aquifer treatment
Dextran was used as a model compound to eliminate the complexity associated with
High molecular weight organics
studying a mixture of high molecular weight organics. The hydrolysis products of dextran
Hydrolysis
are easily degradable sugars. Batch experiments with media taken from the reactors were
Dextran
used to determine the distribution of microbial activity in the reactors. Zero-order kinetics
Kinetics
were observed for the removal of dextran in batch experiments which is consistent with
Saturated
hydrolysis of high molecular weight organics where extracellular enzymes limit the substrate utilization rate. Biomass and microbial activity measurements demonstrated that the biomass was independent of position in the reactors. A Monod based substrate/ biomass growth kinetic model predicted the performance of dextran removal in the reactors. The rate limiting step appears to be hydrolysis and the overall rate was not affected by surface area even though greater biomass accumulation occurred as the surface area decreased. ª 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
Water Reuse has become important in arid areas throughout the world including regions such as the southwestern United States and the Mideast. Soil aquifer treatment (SAT) is a technology where natural systems have been employed to treat wastewater for indirect potable reuse (Bouwer and Rice, 1984; Bouwer, 1985; Amy et al., 1993; Drewes and Jekel, 1998; Wilson et al., 1995, Wild and Reinhard, 1999). SAT involves
the water quality benefits derived during percolation through vadose zone sediments and subsequent ground water transport. The organic matter in treated wastewater is a complex mixture of simple carbohydrates, amino acids, alcohols, volatile fatty acids mixed with polymers and heteropolymers including proteins (1/3 of COD), polysaccharides (1/5 of COD) and lipids (1/3 of COD) along with Natural Organic Matter (NOM) and Soluble Microbial Products (SMPs) (Raunkjaer et al., 1994). In the unsaturated zone, easily biodegradable low
* Corresponding author. Biotechnology Department, PES Institute of Technology, 82 East End ’B’ Main Road, Jayanagar 9th Block, Bangalore, Karnataka 560069, India. Tel.: þ91 80 26633721. E-mail addresses: [email protected] (P. Fox), [email protected] (R. Makam). 0043-1354/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2011.05.023
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molecular weight organics are biodegraded while residual high molecular weight organics are biodegraded over longer time periods in the saturated zone (Fox et al., 2001; Amy et al., 2006). The removal of dissolved organic carbon may be modeled as a first order reaction where the organic components are divided into fractions with different biodegradation rates. Dissolved organic carbon (DOC) is often used as a surrogate to monitor the removal of organic compounds without consideration for the characteristics of individual organic compounds. During saturated flow, the majority of organic carbon transformations are associated with high molecular weight organics. The high molecular weight fraction cannot be assimilated directly by microorganisms (Levine et al., 1985). Bacteria assimilate high molecular weight compounds via hydrolysis by extracellular enzymes. These extracellular enzymes are either bound to the cell surface (ecto-enzymes) (Chrost, 1991) or released (exoenzymes) (Vetter and Deming, 1999; Wetzel, 1991) into the medium so as to hydrolyze high molecular weight compounds. Cadoret et al. (2002) has demonstrated the utilization of low and high molecular weight substrates as a consequence of extracellular enzymes in whole and dispersed activated sludges (e.g.: azocasein MW ¼ 26,000, amulose azure 32,000 < MW < 86,000). Slow hydrolysis of organic matter by exoenzymes has been demonstrated by Eliosov and Argaman (1995) and Guellil et al. (2001). When substrate utilization kinetics for high molecular weight DOC are dominated by hydrolysis of complex substrates, zero-order kinetics with respect to substrate concentration may be observed. This research focuses on understanding the rate limiting step for the biodegradation of DOC in the saturated zone during SAT. These transformations are vital for the sustainability of SAT and they support microbial activity for long sub-surface travels times allowing for potential co-metabolic reactions of trace anthropogenic compounds (Nalinakumari et al., 2010; Wild and Reinhard, 1999). The objective of this research is to evaluate the kinetics of biodegradation of a model high molecular weight compound during flow through a saturated media. A model compound was chosen to avoid the complexities associated with a mixture of many compounds and provide insight into the mechanisms of removal for a high molecular weight compound. To accomplish this, two different particle size sands were used to provide two different surface areas for microbial attachment and the flow rates were varied. The experimental design maintained aerobic conditions to eliminate complexities from varying redox conditions.
2.
Methods
2.1.
Dextran as a model compound
Dextran was chosen as the model compound because it is a high molecular weight compound (MW ¼ 10,000), mimics polysaccharide soluble microbial products and is readily biodegradable. The hydrolysis products of dextran are sugars that easily biodegradable. Analysis of carbohydrates can be used for monitoring dextran and its hydrolysis products. When combined with dissolved organic carbon measurements, a mass balance on dextran can be done.
2.2.
Reactors
The experimental apparatus was designed to simulate saturated flow in a sand aquifer. The experimental apparatus consisted of 4 cylindrical reactors packed with with two different particle sizes of sand. The reactors were 0.915 m tall with a 0.076 m inner diameter and were constructed of Plexiglass. Two different clean silica sand sieve sizes were used as packing material (Border Products, Arizona). One size was US standard sieve 16 x 30 with a geometric mean diameter of 0.6 mm and the second size was US standard sieve 40 x 60 with a geometric mean diameter of 0.353 mm. The sands were washed with de-ionized water to remove any residual fines and dried before packing. The sands were packed in the reactors to achieve an average dry packing density of 1.4 g/ cm3. Reactors 1 and 4 were packed with 0.6 mm silica sand and Reactors 2 and 3 were packed with 0.353 mm silica sand. The reactors were operated in an upflow mode. The 4 reactors were seeded by feeding filtered nitrified/ denitrified effluent from the Mesa Northwest Reclamation Plant, Arizona for a period of 50 days. After seeding, the reactors were operated with synthetic feed containing dextran (average M.W. ¼ 10,000) (Sigma, St. Louis, Missouri, USA) as the substrate. The synthetic feed had a nominal concentration of 6.8 mg DOC/L added to dechlorinated drinking water. The background DOC of the dechlorinated tap water used to formulate the influent feed was found to be at 1.26 þ 0.4 mg DOC/L. During previous studies using the same tap water in soil columns, less than 0.2 mg/L of the DOC was removed over a 30 day retention time (Nalinakumari et al., 2010). During this study, the retention times were much lower and therefore the natural organic matter in the tap water should not significantly influence the results. Based on the stoichiometry, the tap water contained sufficient nutrients and there was no need add supplemental nutrients. Weekly monitoring of turbidity, UVA254 and dissolved organic carbon was done. The flow rates chosen for simulating sub-surface transport provided a Reynolds Number less than 1 and the Peclet Number ranged from 0.2 to 6. Ground water recharge sites have saturated flow conditions with Reynolds Numbers less than 1 and for most aquifer materials the Peclet Numbers range from 0.2 to 36. The reactors were operated in two phases with different flow rates to evaluate a range of conditions that occur during saturated flow in SAT systems. During Phase 1, Reactors 1 and 2 were operated at 0.5 L/day and Reactors 3 and 4 were operated at 4 L/day. All 4 reactors were operated a minimum of 150 days under saturated aerobic conditions to achieve steady state. Once Phase I was completed, Phase II was initiated. During Phase II, the flow rate to Reactors 1 and 2 was increased 4 fold to 2 L/day and the flow rate to Reactors 3 and 4 was decreased by a factor of 4 to 1 L/day. All 4 reactors were run for at least 50 days under saturated aerobic conditions during Phase II. Table 1 shows the operating conditions for the four reactors during Phases 1 and 2. In this experimental design, the empty bed contact in Reactors 1 and 2 was the always identical and the empty bed contact time in Reactors 3 and 4 was always the same. Table 1 also shows the mean effluent concentrations under these conditions along with the student’s t-test results for comparing the reactors.
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Table 1 e Effluent DOC Concentrations during Phase I and Phase II. Reactor 3
Particle Size (m 10 ) 3
3
1
2
3
4
0.600
0.353
0.353
0.600
Phase I
Flow rate (m /day 10 ) Empty Bed Contact Time (days) Reynolds Number (103) Peclet Number Mean Effluent Concentration (mg/L)
0.500 8.340 0.762 0.762 1.980
0.500 8.340 0.448 0.448 1.740
4.000 1.040 3.584 3.584 2.060
4.000 1.040 6.091 6.091 2.070
Phase II
Flow rate (m3/day 103) Empty Bed Contact Time (days) Reynolds Number (103) Peclet Number Mean Effluent Concentration (mg/L)
2.000 2.080 3.046 3.046 1.950
2.000 2.080 1.792 1.792 1.860
1.000 4.170 0.896 0.896 1.940
1.000 4.170 1.523 1.523 1.930
Student’s t-test
p-value (based on particle size) p-value (based on phase flow rate)
0.183 0.320
0.500 0.172
0.500 0.172
0.183 0.320
2.3.
Batch kinetic tests
A modified biodegradable dissolved organic carbon (BDOC) reactor method was used to measure the kinetics of dextran biodegradation using native sand originally acclimated to tertiary effluent. The native sand used was from the Agua Fria River Basin (passed through a 2 mm screen) at a planned recharge site in Arizona, USA. In addition batch tests were completed with the clean silica sands used in the four reactors. The BDOC sand reactors were 500 ml Erlenmeyer Flasks containing 100 g of biologically active sand in each of the reactors. The biologically active sand was seeded initially with the same Mesa Tertiary Effluent used to seed the reactors. The BDOC reactors were seeded with 300 ml aliquots of effluent for three sequential batch tests where each test was 5 days. The reactors were then acclimated with 300 ml of dextran at a target concentration of 5 mg-C/L. After each 5 day reaction period, the solution was decanted and the acclimated sand in the reactors was washed with 100 ml of solution containing 0.15 M Sodium Chloride with 1 mM Magnesium Chloride. The washing procedure was repeated 3 times before new feed was added to initiate a new batch experiment. The reactors were then incubated with 300 ml of dextran at a nominal concentration of 9.66 mg-C/L to simulate the synthetic feed used in the column studies. The initial concentration in the batch tests was 40% higher than the concentration used in the column studies to provide kinetic information over a larger concentration range. Initial samples were taken to measure the concentration of DOC at the beginning of each experiment after mixing with the sand and biomass. The DOC and UVA254 were monitored every day for a period of 5 days consistent with other BDOC tests (Cha et al., 2004). Also, 5 g of sand was removed daily from the batch reactors and the sand was analyzed for biomass using organic nitrogen and carbohydrate analysis. The results were normalized to the quantity of sand and biomass after each sampling event to account for the removal of the sand and biomass. The data obtained from DOC measurements were used for analyzing the kinetics of dextran utilization for each reactor. The data from organic nitrogen analysis was then used to calculate a yield coefficient. By normalizing the substrate utilization rate data to the biomass content, a specific substrate utilization ratio was
calculated. This value was then compared to values determined for the reactors.
2.4.
Biomass characrerization in the columns
Immediately after Phase II was completed, the sand from the column reactors was extruded and divided into 11 to 12 sections to provide a profile of the sand as a function of reactor length. Each section was approximately 0.076 m in thickness and the sand was analyzed for biomass composition by determining the organic nitrogen, carbohydrate and volatile suspended solid attached to the sand.
2.5.
Analytical methods
2.5.1.
UVA254
The ultraviolet absorbance (UVA) at 254 nm was routinely measured using a Model 8452 A Hewlett Packard Diode Array Spectrophotometer. A 1 cm pathlength was used with a quartz cuvette.
2.5.2.
Dissolved organic carbon
The dissolved organic carbon (DOC) was routinely monitored using a Shimadzu TOC 5000 A Total Organic Carbon Analyzer in accordance with Method 5310 in Standard Methods for Examination of Water and Wastewater (Andrew et al., 2007). The minimum detection level was 0.5 mg/L.
2.5.3.
Biomass extraction and Quantification
The extraction of attached biomass from column reactor media or BDOC reactor media was done using the following procedure. Approximately 5 g of wet media was weighed and transferred into a 10 ml volumetric flask and 3 ml of 20% (w/v) of tricholoroacetic acid (TCA) was added. The flasks were placed in a VWR Scientific Aqua Sonic Model 150 T ultrasonic cleaner and sonicated for 10 min. After sonication, the solution was decanted from the flask and transferred to a second 10 ml volumetric flask. The sand remaining in the flask was rinsed once with 10 ml of water and the rinse water was transferred to the second volumetric flask. The sonication and transfer steps were repeated two more times to complete the separation of attached biomass. The extracts were analyzed
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for organic nitrogen and carbohydrate to characterize the biomass. The sand used in each analysis was then dried and weighed.
2.5.4.
Organic nitrogen analysis
The extracted biomass was quantified for organic nitrogen using a Hach Total Nitrogen kit with the Persulfate Digestion Method in accordance with Method 4500 in Standard Methods for Examination of Water and Wastewater (Andrew et al., 2007) and a Hach DR/4000 Spectrophotometer.
2.5.5.
Carbohydrate analysis
The extracted biomass was also quantified for carbohydrate content. A 1 ml sample was mixed with 1 ml of 5% (w/v) of aqueous phenol solution. Then 5 ml of concentrated sulfuric acid was added (Herbert et al., 1971). A calibration standard with 0e100 mg glucose was used. The carbohydrate was measured at 488 nm using a Model 8452 A Hewlett Packard Diode Array Spectrophotometer. A 1 cm pathlength was used with a quartz cuvette.
2.5.6.
Indirect dextran measurements
Dextran was measured indirectly using both DOC and carbohydrate analyses. The carbohydrate analysis measured both simple and complex carbohydrates and can provided a direct measurement of dextran before biodegradation. There was less than þ3% difference between dextran measurements using carbohydrate analysis as compared to dextran measurements using DOC analysis. The correlation between DOC and carbohydrates was found to be valid throughout the studies even though the carbohydrate analysis will measure sugars in biomass such as riboses. This correlation also demonstrated that measuring carbohydrate on biomass extracted from the sand media was primarily due to biomass and the influence of dextran was negligible.
2.5.7.
Data analysis
And the specific rate of substrate utilization is q ¼ qmax
S Ks þ S
(4)
Where qmax is the maximum specific substrate utilization rate (g-C/g-biomass-t) Assuming hydrolysis is the rate limiting step, Ks is very small since there are not many extracellular enzymes and the concentration of extracellular enzymes is constant (Guellil et al., 2001). The hydrolysis products of dextran are sugars that should be rapidly degraded at a rate faster than the hydrolysis rate. The specific growth rate (Eq. (1)) and specific rate of substrate utilization (Eq. (4)) becomes (5) m ¼ mmax q ¼ qmax ¼ k
(6)
where k ¼ zero-order substrate utilization rate constant (g-C/ g-biomass-day) Considering a fixed-bed and assumingplug flow, steady state conditions with zero-order kinetics with respect to substrate utilization and first order kinetics with respect to biomass growth, we get using the following definitions in the model: L ¼ Length of reactor (m), U velocity in direction of flow (m/day), S ¼ Substrate concentration (g-C/m3), X Biomass concentration (g-biomass/m3), Y Yield Coefficient (g-C/ g-biomass), k zero-order substrate utilization rate constant (g-C/g-biomass-day), b decay coefficient (day1), z axial direction (m), Xf Biofilm density (g/m3), Lf Biofilm thickness (m), q ¼ residence time (day), a specific surface area (m2/m3). Where the biomass concentration may be expressed in terms of the biofilm density and thickness as X ¼ Xf Lf a
(7)
Biofilm kinetics may be analyzed using a classic biofilm model (Fig. 1) where external mass transfer resistance and diffusion through the biofilm are considered. However, if
2.5.7.1. Monod substrate utilization/biomass growth kinetic model. A model was developed for substrate utilization and
Y
biomass growth in the column reactors studied. Monod in 1942 gave an empirical model (Eq. (1)) for microbial growth kinetics introducing the concept of a growth limiting substrate.
dX dS
YX=S dS ¼ YX=S q m¼ X dt
Lf
Bulk Liquid
Diffusion Layer
(1)
where m ¼ specific growth rate (day1), mmax ¼ maximum specific growth rate (day1), S ¼ substrate concentration (g-C/m3), Ks ¼ substrate saturation constant i.e., substrate concentration at half mmax (g-C/m3) In addition to this, Monod also related the Yield Coefficient Yx/s (Eq. (2)) to the specific biomass growth rate and the specific rate of substrate utilization q (Eq. (3)) YX=S ¼
Z
Biofilm
S Ks þ S
Xf
Sand Particle
m ¼ mmax
X
S
L
(2)
(3)
Fig. 1 e Biofilm Model e Flow is In the Z-Direction while Diffusion is in the X-Direction. As Dextran Approaches the Biofilm Extracellular Enzymes Can Hydrolyze the Dextran.
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hydrolysis by extracellular enzymes is the rate limiting step, diffusion into the biofilm is only important for the products of hydrolysis. For dextran, the hydrolysis products are rapidly degraded and diffusion into the biofilm was assumed to not be rate limiting. A Steady State Substrate Mass Balance may be written assuming external mass transfer to the biofilm is negligible.
0¼V
dS ¼ dt
! dS U kXf Lf a V dz
DS=Dt ¼ ks whereDS ¼ mass substrate utilized over the time Dt
(13)
The yield of biomass per mass of substrate utilized substrate (YX/S) is obtained using equation 14 where DXX is the biomass growth over the time Dt. YX=S ¼ DXX=DS
(8)
Steady State Biomass Mass Balance 0 ¼ aV
The rate of substrate (S) utilization is obtained using Eq. (13).
dXf Lf ¼ ðYk bÞXf Lf aV dt
(9)
(14)
where YX/S yield of biomass on substrate (mg-X/mg-C) The specific substrate utilization rate (kbatch) for the batch experiment is obtained using Eq. (15). kbatch ¼ ðks =XXÞ ð300ml=1000ml=LÞ
(15)
Recognizing that the substrate utilization rate-k Xf is the rate of substrate utilization, then Eq. (9) reduces to Eq. (10) where J is the flux into the biofilm in gC/m2ed.
where kbatch specific substrate utilization rate (mg-C/mgbiomass-day) The specific growth rate is then obtained according to Eq. (16).
JY ¼ bXf Lf
m ¼ YX=S kbatch
(10)
which is the classic result for a steady state biofilm where growth equals decay. Solving Eq. (8) for X where X XfLfa yields X¼
ðS0 SÞU Lk
(11)
Since hydrolysis limits substrate utilization to zero-order kinetics, the model predicts biomass content becomes independent of position.
2.6.
(16) 1
where m specific growth rate (day ) For each particle size of soil, the kinetic parameters are tabulated in Table 2. The specific substrate utilization rate constants for column experiments (kcolumn) are then obtained according to Eq. 17 and listed in Table 3. kcolumn ¼ kbatch particle surface area 5=residence time (17) The predicted biomass from the model is then obtained using Eq. (18) and is listed in Table 3.
Batch experiments data analysis
Predicted Biomass ¼ X V Batch experiments were analyzed for substrate utilization and microbial growth kinetics. Analysis was done to determine the zero-order substrate utilization rate (ks). The specific substrate utilization rate (kbatch) was determined by normalizing to biomass attached to the sand which was measured in terms of organic nitrogen per gram of sand. The quantity of attached biomass (XX) in mg was calculated using organic nitrogen data assuming an empirical cell composition of C5H7O2N according to Eq. (12). In Eq. (12), Org-N is the measured organic nitrogen in mg-N/g-sand. XX ¼ ðOrg N=g sandÞ 100 g sand 113 mg C5 H7 O2 N=14 mgN
(12)
where XX ¼ amount of biomass formed (mg-X)
(18)
where V volume of reactor (L), X concentration of biomass according to Eq. (11) (mg-X/m3)
3.
Results and discussion
3.1.
Column/Reactor experiments with dextran
The influent and effluent DOC concentrations were monitored as a function of time for the four reactors and the average concentrations were used to evaluate the reactors for the two phases of operation. Effluent DOC was used as a surrogate to understand the removal of dextran in the soil columns since these values correlated with carbohydrate analysis as
Table 2 e Kinetic parameters from Batch Kinetic Experiments. Reactor
1 2 3 4
Particle Size (m 103)
Substrate rate constant ks (mg-C/L/day)
Final Biomass-N per gram of sand DN (mg-N/g-sand)
Amount of Biomass formed X (mg-X)
Amount of Substrate utilized S (mg-C)
Yield of Biomass on Substrate YX/S (mg-X/mg-C)
Specific substrate utilization rate kbatch (mg-C/mg-X/day)
Specific Growth Rate m (day1)
0.600 0.353 0.353 0.600
0.4426 0.8856 0.8856 0.4426
0.023 0.041 0.041 0.023
18.564 33.093 33.093 18.564
0.664 1.328 1.328 0.664
27.958 24.920 24.920 27.958
0.0072 0.0080 0.0080 0.0072
0.2 0.2 0.2 0.2
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Table 3 e Biomass according to substrate utilization/microbial growth model and experimentation Reactor
1 2 3 4
Experimental Experimental Predicted Predicted Particle Flux Flow rate Particle Biofilm Size (m 103) (m3/day 103) surface area (mg-C/m2/day) Thickness (mm) (m2 106) 0.600 0.353 0.353 0.600
0.500 0.500 4.000 4.000
1.133 0.392 0.392 1.133
1.550 0.910 7.290 12.390
mentioned above. Phase I was run for a period of 150 days such that the DOC concentration became constant with time reflecting the development of an acclimated population and pseudo-steady state conditions. The reactors were operated under saturated and aerobic conditions. The feed dissolved oxygen (DO) was maintained near equilibrium with atmospheric oxygen with an approximate concentration of 6 mg/L. Aerobic conditions in the reactors were maintained as 0.7 mg of oxygen was needed per mg of dextran according to stoichiometry (Eq. (19)) and the average feed dextran concentration was 6.7 mg/L resulting in an effluent dissolved oxygen concentration greater than 2 mg/L. þ 0:042C6 H10 O5 þ 0:02HCO 3 þ 0:02NH4 þ 0:15O2 /0:02C5 H7 O2 N
þ 0:25CO2 þ 0:188H2 O (19) Routine monitoring of DO, Turbidity, UV254 and DOC was performed on the reactors. The mean feed concentration which includes the background DOC of tap water was 6.7 þ 0.5 mg DOC/L and the average effluent concentrations ranged from 1.70 to 2.07 mg DOC/L for Phase 1 (Table 1). During Phase 2, the effluent concentrations in all four reactors were very similar and ranged only from 1.86 to 1.95 mg DOC/L (Table 1). Also, listed in Table 1 are the Reynolds Number and Peclet Number corresponding to the mean effluent concentrations during Phase 1 and Phase 2. A Student’s t-test was performed on the mean effluent concentrations in the reactors based on particle size and flow rate for both the Phases
Fig. 2 e BDOC Kinetic Experiment with different sands.
0.004 0.001 0.010 0.028
Column Predicted Experiment Rate Biomass Biomass Constant (Eq (18)) (mg-X) kcolumn (mg-X) (mg-C/mg-X/L/day) 0.0176 0.0068 0.0541 0.1406
508.48 1363.38 1287.20 501.12
393.110 1185.680 1196.140 498.060
and was found to have no statistical difference between the various mean effluent concentrations.
3.1.1.
Batch kinetic experiments with dextran as substrate
Batch experiments with the modified BDOC reactors were completed using Agua Fria sand and two clean silica sands sieved to the same geometric diameter as used in the column reactors which was 0.353 mm and 0.6 mm. Prior to running the kinetic experiments the BDOC reactors were acclimated with Mesa Tertiary Effluent as described above. Acclimatization was determined complete when the final DOC concentration after each test was the same as the previous test. After acclimation, the kinetic experiments were carried out with a nominal initial dextran concentration 40% higher than the influent to the reactors. The results are summarized in Fig. 2. Fig. 2 shows that the substrate concentration decreases linearly as a function of time which is consistent with zeroorder kinetics for the different types of sand studied. i.e., Agua Fria sand, 0.353 mm silica sand and 0.6 mm silica sand. The UVA254 in the effluent increased from zero to 0.4 cm1 for 0.353 mm silica sand and from zero to 0.18 cm1 for 0.6 mm silica sand during the batch tests. The increase in UVA254 is consistent with the presence of aromatic compounds as the microorganisms were producing or desorbing soluble microbial products. The larger increase with the 0.353 mm silica sand is consistent with the larger surface area and biomass content. The increase in UVA254 was not enough to significantly impact the DOC concentration. The organic nitrogen data used to evaluate the growth of biomass
Fig. 3 e Organic Nitrogen data during the kinetic experiment with different sands.
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Biomass (C5H7O2N) (mg-X/g-sand)
Reactor Biomass 0.40 Reactor Reactor Reactor Reactor
0.35 0.30
3 4 2 1
0.25 0.20 0.15 0.10 0.05 0.00
0
2
4 6 8 Reactor Section (Bottom-Top)
10
12
Fig. 4 e Reactor Biomass versus Reactor Sections.
on the sand is shown in Fig. 3. The gradual increase with reaction time for the different types of sand is consistent with microbial growth. From the kinetic experiments with the BDOC reactors, the rate of DOC biodegradation kinetics was observed to be zero-order with respect to substrate concentration. The rate for the smaller particle size was found to be approximately twice that of the larger particle size. The smaller particle size had approximately three times the surface area of the larger particle size. Assuming the microbial population was primarily attached, the surface area appeared to directly influence the removal rate. The BDOC reactor experiments support a zero-order relationship for substrate utilization which might be expected when hydrolysis of high molecular weight compounds is the rate limiting step. The specific substrate utilization rate, yield of biomass on substrate and the specific growth rate were calculated according to the Batch Experiments Data Analysis and are listed in Table 2. The rate constants for the column experiments were then obtained in accordance with Eq. (17).
3.1.2.
Biomass data from columns
Once, the column experiments were completed the sand in the reactors were cut into 11 or 12 sections and were analyzed for biomass using Org-N, carbohydrate and volatile suspended solids. Fig. 4 shows the biomass profiles for the 4 reactors based on the Org-N measurements. The Organic-N profiles,
Fig. 5 e Activity Kinetics for Reactor 1.
Fig. 6 e Activity Kinetics for Reactor 2.
which represent a measure of the protein content of the cells was relatively constant along the length of the reactors. The organic-N could also represent extracellular polymers, nucleic acids and the organic-N does not distinguish between active cells and cellular debris. We see from Fig. 4 that the biomass growth was similar for reactors 1 and 4 and for reactors 2 and 3. This indicates that the biomass growth was independent of the applied substrate flux and was primarily affected by the surface area and hence the particle size. Table 3 lists the quantity of biomass according to the biomass growth model (Eq. (18)) and from the experimental investigation. We see from Table 3 that the biomass is proportional to the surface area in the reactors. The biomass increases by a factor of 3 for a 3-fold increase in individual particle surface area when comparing the reactors with 0.6 mm sand to reactors with 0.353 mm sand. Table 3 also shows that the biofilm thickness is less than one micron. Therefore, diffusion in the biofilm can be assumed and this verifies the assumption made in model development. A fully-penetrated biofilm with the traditional Monod model for substrate utilization/biomass growth agrees with experimental results.
3.1.3.
Activity kinetics
The sand samples extruded as sections from the reactors were analyzed for microbial activity using the modified BDOC
Fig. 7 e Activity Kinetics for Reactor 3.
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Fig. 8 e Activity Kinetics for Reactor 4.
kinetic test. Sand extruded from every 4th section of each of the reactors was tested to determine the removal of dextran over a period of 5 days. The activity test data for sand samples taken from reactors 1 through 4 is presented in Figs. 5e8, respectively. The kinetics were found to be zero-order with respect to substrate removal for samples from all the reactors. For each section of the reactors, the rate was independent of the distance from the influent to the effluent. Hence, the overall rate for a reactor could be calculated based on the results for the average of sections from a reactor. The individual rate for reactor 1 was found to be equal to 0.1734 mg-C/L-day, for reactor 2 it was 0.2849 mg-C/L-day, for reactor 3 it was 0.3989 mg-C/L-day and for reactor 4 it was 0.3257 mg-C/L-day. The overall rates for the column reactors were calculated using the ratio of total mass of sand in the columns divided by the mass used for batch testing. These values were found to be equal to 10.404 mg-C/L-day for reactor 1, 17.094 mg-C/L/day for reactor 2, 23.934 mg-C/L/day for reactor 3 and 19.542 mg-C/ L/day for reactor 4. Reactors 1 and 2 always had the same hydraulic retention time and Reactors 3 and 4 always had the same hydraulic retention time. We see that the reactors with smaller particle size had higher rates than that of reactors with larger particle diameter for the same flow rates (i.e. Reactor 2 Reactor 1 and Reactor 3 Reactor 4). The smaller difference between Reactors 3 and 4 could be from an accumulation of biomass during Phase 1 when the loading rate was 8 times the loading rate in Phase II. Similar data was observed when the BDOC reactors were used to evaluate kinetics using a single dextran feed concentration. This is interesting since the BDOC reactors were acclimated in a batch system while the sand from the reactors was acclimated in a column, yet the effects of surface area were similar.
4.
Summary
Dextran (average MW 10,000 Da) was observed to biodegrade with zero-order substrate utilization kinetics during batch kinetic experiments. This is consistent with the expected kinetics when hydrolysis is the rate limiting step. During continuous flow column experiments, the biomass distribution did not vary significantly based on activity measurements
and organic nitrogen analyses. Four column experiments were completed with two different particle sizes (hence surface area contact) and 4 different flow rates. The removal of substrate was independent of flow rate and particle size. However, the surface area had a positive relationship with biomass accumulation. The ratio of organic-N (hence biomass) was a factor of 3 higher for 0.353 mm particle size when compared with 0.6 mm particle size in one set of paired columns (Columns 1 and 2) and a factor of 2.4 higher for 0.353 mm particle size when compared with 0.6 mm particle size in the second set of paired columns (Columns 3 and 4). The surface area for the columns with a particle size of 0.353 mm was 3 times greater than the columns with 0.6 mm particle size. The experimental results demonstrated that biological removal of a biodegradable high molecular weight compound was robust during flow over a porous media. The Monod based substrate/growth kinetic model does predict the removal of a single type of biodegradable high molecular weight and the biofilm thickness was insufficient to cause diffusion limitations. The rate limiting step appears to be hydrolysis. Hydrolysis may be the rate limiting step for mixtures of high molecular weight compounds that are present in actual systems. The mixtures contain compounds with different biodegradabilities and their hydrolysis products might not be easily biodegradable resulting in higher order kinetics.
references
Amy, G., Wilson, L.G., Conroy, A., Chahbandour, J., Ahai, W., Siddiqui, M., 1993. Fate of chlorination byproducts and nitrogen species during effluent recharge and soil aquifer treatment. Water and Environmental Research 65, 726e734. Amy, G.L., Drewes, J., Westerhoff, P., 2006. Organic matter in soilaquifer treatment systems. Journal of Environmental Engineering 132 (11), 1447e1458. Andrew, D.E., Rice, E.W., Baird, R.B., 2007. In: Standard Methods for the Examination of Water and Wastewater, first ed. American Public Health Association. Bouwer, H., 1985. Renovation of Wastewater with Rapidinfiltration Land Treatment Systems. In: Asano, T. (Ed.), Artificial Recharge of Groundwater. Butterworth, Boston, pp. 249e282. Bouwer, H., Rice, R.C., 1984. Renovation of wastewater at the 23rd avenue rapid infiltration project. Journal of Water Pollution Control Federation 36, 76e83. Cadoret, Aurore, Conrad, Arnaud, Block, Jean-Claude, 2002. Availability of low and high molecular weight substrates to extracellular enzymes in whole and dispersed activated sludges. Enzyme and Microbial Technology 31, 179e186. Cha, W., Choi, H.C., Fox, P., 2004. Abiotic and biotic removal mechanisms for organic carbon during soil aquifer treatment. Water Environment Research 76, 756e804. Chrost, R.J., 1991. Environmental control of the synthesis and activity of aquatic microbial ecto-enzymes. In: Chrost, R.J. (Ed.), Microbial Enzymes in Aquatic Environments. Springer, New York, pp. 29e59. Drewes, J.E., Jekel, M., 1998. Behavior of DOC and AOX using advanced treated wastewater for groundwater recharge. Water Research 32, 3125e3133. Eliosov, B., Argaman, Y., 1995. Hydrolysis of particulate organics in activated sludge systems. Water Research 29, 155e163.
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Fox, P., Narayanaswamy, K., Genz, A., Drewes, J.E., 2001. Water quality transformations during soil aquifer treatment at the nesa Northwest water reclamation plant USA. Water Science and Technology 43 (10), 343e350. Guellil, A., Boualam, M., Quiquampoix, H., Ginsestet, P., Audic, J.M., Block, J.C., 2001. Hydrolysis of wastewater colloidal organic matter by extracellular enzymes extracted from activated sludge flocs. Water Science and Technology 43 (6), 33e40. Herbert, D., Phipps, P.J., Strange, R.E., 1971. Carbohydrate analysis. vol. 5B. In: Norris, J.R., Ribbons, D.W. (Eds.), Methods in Microbiology. Academic Press, New York, pp. 265e301. Levine, A.D., Tchobanoglous, G., Asano, T., 1985. Characterization of the size distribution of contaminants in wastewater: treatment and reuse implications. Journal of Water Pollution Control Federation 57, 805e816. Nalinakumari, B., Cha, W., Fox, P., 2010. Effects of primary substrate concentration on N-nitrosodimethylamine (NDMA) during simulated aquifer recharge. ASCE Journal of Environmental Engineering 136 (4), 373e380.
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Raunkjaer K, Hvitved-Jacobsen T., Nielsen P.H. 1994. Measurement of Pools of Protein, Carbohydrate and Lipid in Domestic Wastewater. 28(2), 251e262. Vetter, Y.A., Deming, J.W., 1999. Growth rates of marine bacterial isolates on particulate organic substrates solubilized by freely released extracellular enzymes. Microbial Ecology 37, 86e94. Wetzel, R.G., 1991. Extracellular enzymatic interactions: storage, redistribution and inter specific communication. In: Chrost, R.J. (Ed.), Microbial Enzymes in Aquatic Environments. Springer, New York, pp. 6e28. Wild, D., Reinhard, M., 1999. Biodegradation residual of 4-Octylphenoxyacetic acid in laboratory columns under groundwater recharge conditions. Environ. Sci. Technol. 33 (24), 4422e4426. Wilson, L.G., Amy, G.L., Gerba, C.P., Gordon, H., Johnson, B., Miller, J., 1995. Water quality changes during soil aquifer treatment of tertiary effluent. Water and Environmental Research 67, 371e376.
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Kinetics of model high molecular weight organic compounds biodegradation in soil aquifer treatment Peter Fox, Roshan Makam* Department of Civil and Environmental Engineering, Arizona State University, PO Box 5306, Tempe, AZ 85287-5306, USA
article info
abstract
Article history:
Soil Aquifer Treatment (SAT) is a process where treated wastewater is purified during
Received 6 December 2010
transport through unsaturated and saturated zones. Easily biodegradable compounds are
Received in revised form
rapidly removed in the unsaturated zone and the residual organic carbon is comprised of
14 May 2011
primarily high molecular weight compounds. This research focuses on flow in the satu-
Accepted 22 May 2011
rated zone where flow conditions are predictable and high molecular weight compounds
Available online 15 June 2011
are degraded. Flow through the saturated zone was investigated with 4 reactors packed with 2 different particle sizes and operated at 4 different flow rates. The objective was to
Keywords:
evaluate the kinetics of transformation for high molecular weight organics during SAT.
Soil aquifer treatment
Dextran was used as a model compound to eliminate the complexity associated with
High molecular weight organics
studying a mixture of high molecular weight organics. The hydrolysis products of dextran
Hydrolysis
are easily degradable sugars. Batch experiments with media taken from the reactors were
Dextran
used to determine the distribution of microbial activity in the reactors. Zero-order kinetics
Kinetics
were observed for the removal of dextran in batch experiments which is consistent with
Saturated
hydrolysis of high molecular weight organics where extracellular enzymes limit the substrate utilization rate. Biomass and microbial activity measurements demonstrated that the biomass was independent of position in the reactors. A Monod based substrate/ biomass growth kinetic model predicted the performance of dextran removal in the reactors. The rate limiting step appears to be hydrolysis and the overall rate was not affected by surface area even though greater biomass accumulation occurred as the surface area decreased. ª 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
Water Reuse has become important in arid areas throughout the world including regions such as the southwestern United States and the Mideast. Soil aquifer treatment (SAT) is a technology where natural systems have been employed to treat wastewater for indirect potable reuse (Bouwer and Rice, 1984; Bouwer, 1985; Amy et al., 1993; Drewes and Jekel, 1998; Wilson et al., 1995, Wild and Reinhard, 1999). SAT involves
the water quality benefits derived during percolation through vadose zone sediments and subsequent ground water transport. The organic matter in treated wastewater is a complex mixture of simple carbohydrates, amino acids, alcohols, volatile fatty acids mixed with polymers and heteropolymers including proteins (1/3 of COD), polysaccharides (1/5 of COD) and lipids (1/3 of COD) along with Natural Organic Matter (NOM) and Soluble Microbial Products (SMPs) (Raunkjaer et al., 1994). In the unsaturated zone, easily biodegradable low
* Corresponding author. Biotechnology Department, PES Institute of Technology, 82 East End ’B’ Main Road, Jayanagar 9th Block, Bangalore, Karnataka 560069, India. Tel.: þ91 80 26633721. E-mail addresses: [email protected] (P. Fox), [email protected] (R. Makam). 0043-1354/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2011.05.023
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molecular weight organics are biodegraded while residual high molecular weight organics are biodegraded over longer time periods in the saturated zone (Fox et al., 2001; Amy et al., 2006). The removal of dissolved organic carbon may be modeled as a first order reaction where the organic components are divided into fractions with different biodegradation rates. Dissolved organic carbon (DOC) is often used as a surrogate to monitor the removal of organic compounds without consideration for the characteristics of individual organic compounds. During saturated flow, the majority of organic carbon transformations are associated with high molecular weight organics. The high molecular weight fraction cannot be assimilated directly by microorganisms (Levine et al., 1985). Bacteria assimilate high molecular weight compounds via hydrolysis by extracellular enzymes. These extracellular enzymes are either bound to the cell surface (ecto-enzymes) (Chrost, 1991) or released (exoenzymes) (Vetter and Deming, 1999; Wetzel, 1991) into the medium so as to hydrolyze high molecular weight compounds. Cadoret et al. (2002) has demonstrated the utilization of low and high molecular weight substrates as a consequence of extracellular enzymes in whole and dispersed activated sludges (e.g.: azocasein MW ¼ 26,000, amulose azure 32,000 < MW < 86,000). Slow hydrolysis of organic matter by exoenzymes has been demonstrated by Eliosov and Argaman (1995) and Guellil et al. (2001). When substrate utilization kinetics for high molecular weight DOC are dominated by hydrolysis of complex substrates, zero-order kinetics with respect to substrate concentration may be observed. This research focuses on understanding the rate limiting step for the biodegradation of DOC in the saturated zone during SAT. These transformations are vital for the sustainability of SAT and they support microbial activity for long sub-surface travels times allowing for potential co-metabolic reactions of trace anthropogenic compounds (Nalinakumari et al., 2010; Wild and Reinhard, 1999). The objective of this research is to evaluate the kinetics of biodegradation of a model high molecular weight compound during flow through a saturated media. A model compound was chosen to avoid the complexities associated with a mixture of many compounds and provide insight into the mechanisms of removal for a high molecular weight compound. To accomplish this, two different particle size sands were used to provide two different surface areas for microbial attachment and the flow rates were varied. The experimental design maintained aerobic conditions to eliminate complexities from varying redox conditions.
2.
Methods
2.1.
Dextran as a model compound
Dextran was chosen as the model compound because it is a high molecular weight compound (MW ¼ 10,000), mimics polysaccharide soluble microbial products and is readily biodegradable. The hydrolysis products of dextran are sugars that easily biodegradable. Analysis of carbohydrates can be used for monitoring dextran and its hydrolysis products. When combined with dissolved organic carbon measurements, a mass balance on dextran can be done.
2.2.
Reactors
The experimental apparatus was designed to simulate saturated flow in a sand aquifer. The experimental apparatus consisted of 4 cylindrical reactors packed with with two different particle sizes of sand. The reactors were 0.915 m tall with a 0.076 m inner diameter and were constructed of Plexiglass. Two different clean silica sand sieve sizes were used as packing material (Border Products, Arizona). One size was US standard sieve 16 x 30 with a geometric mean diameter of 0.6 mm and the second size was US standard sieve 40 x 60 with a geometric mean diameter of 0.353 mm. The sands were washed with de-ionized water to remove any residual fines and dried before packing. The sands were packed in the reactors to achieve an average dry packing density of 1.4 g/ cm3. Reactors 1 and 4 were packed with 0.6 mm silica sand and Reactors 2 and 3 were packed with 0.353 mm silica sand. The reactors were operated in an upflow mode. The 4 reactors were seeded by feeding filtered nitrified/ denitrified effluent from the Mesa Northwest Reclamation Plant, Arizona for a period of 50 days. After seeding, the reactors were operated with synthetic feed containing dextran (average M.W. ¼ 10,000) (Sigma, St. Louis, Missouri, USA) as the substrate. The synthetic feed had a nominal concentration of 6.8 mg DOC/L added to dechlorinated drinking water. The background DOC of the dechlorinated tap water used to formulate the influent feed was found to be at 1.26 þ 0.4 mg DOC/L. During previous studies using the same tap water in soil columns, less than 0.2 mg/L of the DOC was removed over a 30 day retention time (Nalinakumari et al., 2010). During this study, the retention times were much lower and therefore the natural organic matter in the tap water should not significantly influence the results. Based on the stoichiometry, the tap water contained sufficient nutrients and there was no need add supplemental nutrients. Weekly monitoring of turbidity, UVA254 and dissolved organic carbon was done. The flow rates chosen for simulating sub-surface transport provided a Reynolds Number less than 1 and the Peclet Number ranged from 0.2 to 6. Ground water recharge sites have saturated flow conditions with Reynolds Numbers less than 1 and for most aquifer materials the Peclet Numbers range from 0.2 to 36. The reactors were operated in two phases with different flow rates to evaluate a range of conditions that occur during saturated flow in SAT systems. During Phase 1, Reactors 1 and 2 were operated at 0.5 L/day and Reactors 3 and 4 were operated at 4 L/day. All 4 reactors were operated a minimum of 150 days under saturated aerobic conditions to achieve steady state. Once Phase I was completed, Phase II was initiated. During Phase II, the flow rate to Reactors 1 and 2 was increased 4 fold to 2 L/day and the flow rate to Reactors 3 and 4 was decreased by a factor of 4 to 1 L/day. All 4 reactors were run for at least 50 days under saturated aerobic conditions during Phase II. Table 1 shows the operating conditions for the four reactors during Phases 1 and 2. In this experimental design, the empty bed contact in Reactors 1 and 2 was the always identical and the empty bed contact time in Reactors 3 and 4 was always the same. Table 1 also shows the mean effluent concentrations under these conditions along with the student’s t-test results for comparing the reactors.
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Table 1 e Effluent DOC Concentrations during Phase I and Phase II. Reactor 3
Particle Size (m 10 ) 3
3
1
2
3
4
0.600
0.353
0.353
0.600
Phase I
Flow rate (m /day 10 ) Empty Bed Contact Time (days) Reynolds Number (103) Peclet Number Mean Effluent Concentration (mg/L)
0.500 8.340 0.762 0.762 1.980
0.500 8.340 0.448 0.448 1.740
4.000 1.040 3.584 3.584 2.060
4.000 1.040 6.091 6.091 2.070
Phase II
Flow rate (m3/day 103) Empty Bed Contact Time (days) Reynolds Number (103) Peclet Number Mean Effluent Concentration (mg/L)
2.000 2.080 3.046 3.046 1.950
2.000 2.080 1.792 1.792 1.860
1.000 4.170 0.896 0.896 1.940
1.000 4.170 1.523 1.523 1.930
Student’s t-test
p-value (based on particle size) p-value (based on phase flow rate)
0.183 0.320
0.500 0.172
0.500 0.172
0.183 0.320
2.3.
Batch kinetic tests
A modified biodegradable dissolved organic carbon (BDOC) reactor method was used to measure the kinetics of dextran biodegradation using native sand originally acclimated to tertiary effluent. The native sand used was from the Agua Fria River Basin (passed through a 2 mm screen) at a planned recharge site in Arizona, USA. In addition batch tests were completed with the clean silica sands used in the four reactors. The BDOC sand reactors were 500 ml Erlenmeyer Flasks containing 100 g of biologically active sand in each of the reactors. The biologically active sand was seeded initially with the same Mesa Tertiary Effluent used to seed the reactors. The BDOC reactors were seeded with 300 ml aliquots of effluent for three sequential batch tests where each test was 5 days. The reactors were then acclimated with 300 ml of dextran at a target concentration of 5 mg-C/L. After each 5 day reaction period, the solution was decanted and the acclimated sand in the reactors was washed with 100 ml of solution containing 0.15 M Sodium Chloride with 1 mM Magnesium Chloride. The washing procedure was repeated 3 times before new feed was added to initiate a new batch experiment. The reactors were then incubated with 300 ml of dextran at a nominal concentration of 9.66 mg-C/L to simulate the synthetic feed used in the column studies. The initial concentration in the batch tests was 40% higher than the concentration used in the column studies to provide kinetic information over a larger concentration range. Initial samples were taken to measure the concentration of DOC at the beginning of each experiment after mixing with the sand and biomass. The DOC and UVA254 were monitored every day for a period of 5 days consistent with other BDOC tests (Cha et al., 2004). Also, 5 g of sand was removed daily from the batch reactors and the sand was analyzed for biomass using organic nitrogen and carbohydrate analysis. The results were normalized to the quantity of sand and biomass after each sampling event to account for the removal of the sand and biomass. The data obtained from DOC measurements were used for analyzing the kinetics of dextran utilization for each reactor. The data from organic nitrogen analysis was then used to calculate a yield coefficient. By normalizing the substrate utilization rate data to the biomass content, a specific substrate utilization ratio was
calculated. This value was then compared to values determined for the reactors.
2.4.
Biomass characrerization in the columns
Immediately after Phase II was completed, the sand from the column reactors was extruded and divided into 11 to 12 sections to provide a profile of the sand as a function of reactor length. Each section was approximately 0.076 m in thickness and the sand was analyzed for biomass composition by determining the organic nitrogen, carbohydrate and volatile suspended solid attached to the sand.
2.5.
Analytical methods
2.5.1.
UVA254
The ultraviolet absorbance (UVA) at 254 nm was routinely measured using a Model 8452 A Hewlett Packard Diode Array Spectrophotometer. A 1 cm pathlength was used with a quartz cuvette.
2.5.2.
Dissolved organic carbon
The dissolved organic carbon (DOC) was routinely monitored using a Shimadzu TOC 5000 A Total Organic Carbon Analyzer in accordance with Method 5310 in Standard Methods for Examination of Water and Wastewater (Andrew et al., 2007). The minimum detection level was 0.5 mg/L.
2.5.3.
Biomass extraction and Quantification
The extraction of attached biomass from column reactor media or BDOC reactor media was done using the following procedure. Approximately 5 g of wet media was weighed and transferred into a 10 ml volumetric flask and 3 ml of 20% (w/v) of tricholoroacetic acid (TCA) was added. The flasks were placed in a VWR Scientific Aqua Sonic Model 150 T ultrasonic cleaner and sonicated for 10 min. After sonication, the solution was decanted from the flask and transferred to a second 10 ml volumetric flask. The sand remaining in the flask was rinsed once with 10 ml of water and the rinse water was transferred to the second volumetric flask. The sonication and transfer steps were repeated two more times to complete the separation of attached biomass. The extracts were analyzed
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for organic nitrogen and carbohydrate to characterize the biomass. The sand used in each analysis was then dried and weighed.
2.5.4.
Organic nitrogen analysis
The extracted biomass was quantified for organic nitrogen using a Hach Total Nitrogen kit with the Persulfate Digestion Method in accordance with Method 4500 in Standard Methods for Examination of Water and Wastewater (Andrew et al., 2007) and a Hach DR/4000 Spectrophotometer.
2.5.5.
Carbohydrate analysis
The extracted biomass was also quantified for carbohydrate content. A 1 ml sample was mixed with 1 ml of 5% (w/v) of aqueous phenol solution. Then 5 ml of concentrated sulfuric acid was added (Herbert et al., 1971). A calibration standard with 0e100 mg glucose was used. The carbohydrate was measured at 488 nm using a Model 8452 A Hewlett Packard Diode Array Spectrophotometer. A 1 cm pathlength was used with a quartz cuvette.
2.5.6.
Indirect dextran measurements
Dextran was measured indirectly using both DOC and carbohydrate analyses. The carbohydrate analysis measured both simple and complex carbohydrates and can provided a direct measurement of dextran before biodegradation. There was less than þ3% difference between dextran measurements using carbohydrate analysis as compared to dextran measurements using DOC analysis. The correlation between DOC and carbohydrates was found to be valid throughout the studies even though the carbohydrate analysis will measure sugars in biomass such as riboses. This correlation also demonstrated that measuring carbohydrate on biomass extracted from the sand media was primarily due to biomass and the influence of dextran was negligible.
2.5.7.
Data analysis
And the specific rate of substrate utilization is q ¼ qmax
S Ks þ S
(4)
Where qmax is the maximum specific substrate utilization rate (g-C/g-biomass-t) Assuming hydrolysis is the rate limiting step, Ks is very small since there are not many extracellular enzymes and the concentration of extracellular enzymes is constant (Guellil et al., 2001). The hydrolysis products of dextran are sugars that should be rapidly degraded at a rate faster than the hydrolysis rate. The specific growth rate (Eq. (1)) and specific rate of substrate utilization (Eq. (4)) becomes (5) m ¼ mmax q ¼ qmax ¼ k
(6)
where k ¼ zero-order substrate utilization rate constant (g-C/ g-biomass-day) Considering a fixed-bed and assumingplug flow, steady state conditions with zero-order kinetics with respect to substrate utilization and first order kinetics with respect to biomass growth, we get using the following definitions in the model: L ¼ Length of reactor (m), U velocity in direction of flow (m/day), S ¼ Substrate concentration (g-C/m3), X Biomass concentration (g-biomass/m3), Y Yield Coefficient (g-C/ g-biomass), k zero-order substrate utilization rate constant (g-C/g-biomass-day), b decay coefficient (day1), z axial direction (m), Xf Biofilm density (g/m3), Lf Biofilm thickness (m), q ¼ residence time (day), a specific surface area (m2/m3). Where the biomass concentration may be expressed in terms of the biofilm density and thickness as X ¼ Xf Lf a
(7)
Biofilm kinetics may be analyzed using a classic biofilm model (Fig. 1) where external mass transfer resistance and diffusion through the biofilm are considered. However, if
2.5.7.1. Monod substrate utilization/biomass growth kinetic model. A model was developed for substrate utilization and
Y
biomass growth in the column reactors studied. Monod in 1942 gave an empirical model (Eq. (1)) for microbial growth kinetics introducing the concept of a growth limiting substrate.
dX dS
YX=S dS ¼ YX=S q m¼ X dt
Lf
Bulk Liquid
Diffusion Layer
(1)
where m ¼ specific growth rate (day1), mmax ¼ maximum specific growth rate (day1), S ¼ substrate concentration (g-C/m3), Ks ¼ substrate saturation constant i.e., substrate concentration at half mmax (g-C/m3) In addition to this, Monod also related the Yield Coefficient Yx/s (Eq. (2)) to the specific biomass growth rate and the specific rate of substrate utilization q (Eq. (3)) YX=S ¼
Z
Biofilm
S Ks þ S
Xf
Sand Particle
m ¼ mmax
X
S
L
(2)
(3)
Fig. 1 e Biofilm Model e Flow is In the Z-Direction while Diffusion is in the X-Direction. As Dextran Approaches the Biofilm Extracellular Enzymes Can Hydrolyze the Dextran.
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hydrolysis by extracellular enzymes is the rate limiting step, diffusion into the biofilm is only important for the products of hydrolysis. For dextran, the hydrolysis products are rapidly degraded and diffusion into the biofilm was assumed to not be rate limiting. A Steady State Substrate Mass Balance may be written assuming external mass transfer to the biofilm is negligible.
0¼V
dS ¼ dt
! dS U kXf Lf a V dz
DS=Dt ¼ ks whereDS ¼ mass substrate utilized over the time Dt
(13)
The yield of biomass per mass of substrate utilized substrate (YX/S) is obtained using equation 14 where DXX is the biomass growth over the time Dt. YX=S ¼ DXX=DS
(8)
Steady State Biomass Mass Balance 0 ¼ aV
The rate of substrate (S) utilization is obtained using Eq. (13).
dXf Lf ¼ ðYk bÞXf Lf aV dt
(9)
(14)
where YX/S yield of biomass on substrate (mg-X/mg-C) The specific substrate utilization rate (kbatch) for the batch experiment is obtained using Eq. (15). kbatch ¼ ðks =XXÞ ð300ml=1000ml=LÞ
(15)
Recognizing that the substrate utilization rate-k Xf is the rate of substrate utilization, then Eq. (9) reduces to Eq. (10) where J is the flux into the biofilm in gC/m2ed.
where kbatch specific substrate utilization rate (mg-C/mgbiomass-day) The specific growth rate is then obtained according to Eq. (16).
JY ¼ bXf Lf
m ¼ YX=S kbatch
(10)
which is the classic result for a steady state biofilm where growth equals decay. Solving Eq. (8) for X where X XfLfa yields X¼
ðS0 SÞU Lk
(11)
Since hydrolysis limits substrate utilization to zero-order kinetics, the model predicts biomass content becomes independent of position.
2.6.
(16) 1
where m specific growth rate (day ) For each particle size of soil, the kinetic parameters are tabulated in Table 2. The specific substrate utilization rate constants for column experiments (kcolumn) are then obtained according to Eq. 17 and listed in Table 3. kcolumn ¼ kbatch particle surface area 5=residence time (17) The predicted biomass from the model is then obtained using Eq. (18) and is listed in Table 3.
Batch experiments data analysis
Predicted Biomass ¼ X V Batch experiments were analyzed for substrate utilization and microbial growth kinetics. Analysis was done to determine the zero-order substrate utilization rate (ks). The specific substrate utilization rate (kbatch) was determined by normalizing to biomass attached to the sand which was measured in terms of organic nitrogen per gram of sand. The quantity of attached biomass (XX) in mg was calculated using organic nitrogen data assuming an empirical cell composition of C5H7O2N according to Eq. (12). In Eq. (12), Org-N is the measured organic nitrogen in mg-N/g-sand. XX ¼ ðOrg N=g sandÞ 100 g sand 113 mg C5 H7 O2 N=14 mgN
(12)
where XX ¼ amount of biomass formed (mg-X)
(18)
where V volume of reactor (L), X concentration of biomass according to Eq. (11) (mg-X/m3)
3.
Results and discussion
3.1.
Column/Reactor experiments with dextran
The influent and effluent DOC concentrations were monitored as a function of time for the four reactors and the average concentrations were used to evaluate the reactors for the two phases of operation. Effluent DOC was used as a surrogate to understand the removal of dextran in the soil columns since these values correlated with carbohydrate analysis as
Table 2 e Kinetic parameters from Batch Kinetic Experiments. Reactor
1 2 3 4
Particle Size (m 103)
Substrate rate constant ks (mg-C/L/day)
Final Biomass-N per gram of sand DN (mg-N/g-sand)
Amount of Biomass formed X (mg-X)
Amount of Substrate utilized S (mg-C)
Yield of Biomass on Substrate YX/S (mg-X/mg-C)
Specific substrate utilization rate kbatch (mg-C/mg-X/day)
Specific Growth Rate m (day1)
0.600 0.353 0.353 0.600
0.4426 0.8856 0.8856 0.4426
0.023 0.041 0.041 0.023
18.564 33.093 33.093 18.564
0.664 1.328 1.328 0.664
27.958 24.920 24.920 27.958
0.0072 0.0080 0.0080 0.0072
0.2 0.2 0.2 0.2
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Table 3 e Biomass according to substrate utilization/microbial growth model and experimentation Reactor
1 2 3 4
Experimental Experimental Predicted Predicted Particle Flux Flow rate Particle Biofilm Size (m 103) (m3/day 103) surface area (mg-C/m2/day) Thickness (mm) (m2 106) 0.600 0.353 0.353 0.600
0.500 0.500 4.000 4.000
1.133 0.392 0.392 1.133
1.550 0.910 7.290 12.390
mentioned above. Phase I was run for a period of 150 days such that the DOC concentration became constant with time reflecting the development of an acclimated population and pseudo-steady state conditions. The reactors were operated under saturated and aerobic conditions. The feed dissolved oxygen (DO) was maintained near equilibrium with atmospheric oxygen with an approximate concentration of 6 mg/L. Aerobic conditions in the reactors were maintained as 0.7 mg of oxygen was needed per mg of dextran according to stoichiometry (Eq. (19)) and the average feed dextran concentration was 6.7 mg/L resulting in an effluent dissolved oxygen concentration greater than 2 mg/L. þ 0:042C6 H10 O5 þ 0:02HCO 3 þ 0:02NH4 þ 0:15O2 /0:02C5 H7 O2 N
þ 0:25CO2 þ 0:188H2 O (19) Routine monitoring of DO, Turbidity, UV254 and DOC was performed on the reactors. The mean feed concentration which includes the background DOC of tap water was 6.7 þ 0.5 mg DOC/L and the average effluent concentrations ranged from 1.70 to 2.07 mg DOC/L for Phase 1 (Table 1). During Phase 2, the effluent concentrations in all four reactors were very similar and ranged only from 1.86 to 1.95 mg DOC/L (Table 1). Also, listed in Table 1 are the Reynolds Number and Peclet Number corresponding to the mean effluent concentrations during Phase 1 and Phase 2. A Student’s t-test was performed on the mean effluent concentrations in the reactors based on particle size and flow rate for both the Phases
Fig. 2 e BDOC Kinetic Experiment with different sands.
0.004 0.001 0.010 0.028
Column Predicted Experiment Rate Biomass Biomass Constant (Eq (18)) (mg-X) kcolumn (mg-X) (mg-C/mg-X/L/day) 0.0176 0.0068 0.0541 0.1406
508.48 1363.38 1287.20 501.12
393.110 1185.680 1196.140 498.060
and was found to have no statistical difference between the various mean effluent concentrations.
3.1.1.
Batch kinetic experiments with dextran as substrate
Batch experiments with the modified BDOC reactors were completed using Agua Fria sand and two clean silica sands sieved to the same geometric diameter as used in the column reactors which was 0.353 mm and 0.6 mm. Prior to running the kinetic experiments the BDOC reactors were acclimated with Mesa Tertiary Effluent as described above. Acclimatization was determined complete when the final DOC concentration after each test was the same as the previous test. After acclimation, the kinetic experiments were carried out with a nominal initial dextran concentration 40% higher than the influent to the reactors. The results are summarized in Fig. 2. Fig. 2 shows that the substrate concentration decreases linearly as a function of time which is consistent with zeroorder kinetics for the different types of sand studied. i.e., Agua Fria sand, 0.353 mm silica sand and 0.6 mm silica sand. The UVA254 in the effluent increased from zero to 0.4 cm1 for 0.353 mm silica sand and from zero to 0.18 cm1 for 0.6 mm silica sand during the batch tests. The increase in UVA254 is consistent with the presence of aromatic compounds as the microorganisms were producing or desorbing soluble microbial products. The larger increase with the 0.353 mm silica sand is consistent with the larger surface area and biomass content. The increase in UVA254 was not enough to significantly impact the DOC concentration. The organic nitrogen data used to evaluate the growth of biomass
Fig. 3 e Organic Nitrogen data during the kinetic experiment with different sands.
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Biomass (C5H7O2N) (mg-X/g-sand)
Reactor Biomass 0.40 Reactor Reactor Reactor Reactor
0.35 0.30
3 4 2 1
0.25 0.20 0.15 0.10 0.05 0.00
0
2
4 6 8 Reactor Section (Bottom-Top)
10
12
Fig. 4 e Reactor Biomass versus Reactor Sections.
on the sand is shown in Fig. 3. The gradual increase with reaction time for the different types of sand is consistent with microbial growth. From the kinetic experiments with the BDOC reactors, the rate of DOC biodegradation kinetics was observed to be zero-order with respect to substrate concentration. The rate for the smaller particle size was found to be approximately twice that of the larger particle size. The smaller particle size had approximately three times the surface area of the larger particle size. Assuming the microbial population was primarily attached, the surface area appeared to directly influence the removal rate. The BDOC reactor experiments support a zero-order relationship for substrate utilization which might be expected when hydrolysis of high molecular weight compounds is the rate limiting step. The specific substrate utilization rate, yield of biomass on substrate and the specific growth rate were calculated according to the Batch Experiments Data Analysis and are listed in Table 2. The rate constants for the column experiments were then obtained in accordance with Eq. (17).
3.1.2.
Biomass data from columns
Once, the column experiments were completed the sand in the reactors were cut into 11 or 12 sections and were analyzed for biomass using Org-N, carbohydrate and volatile suspended solids. Fig. 4 shows the biomass profiles for the 4 reactors based on the Org-N measurements. The Organic-N profiles,
Fig. 5 e Activity Kinetics for Reactor 1.
Fig. 6 e Activity Kinetics for Reactor 2.
which represent a measure of the protein content of the cells was relatively constant along the length of the reactors. The organic-N could also represent extracellular polymers, nucleic acids and the organic-N does not distinguish between active cells and cellular debris. We see from Fig. 4 that the biomass growth was similar for reactors 1 and 4 and for reactors 2 and 3. This indicates that the biomass growth was independent of the applied substrate flux and was primarily affected by the surface area and hence the particle size. Table 3 lists the quantity of biomass according to the biomass growth model (Eq. (18)) and from the experimental investigation. We see from Table 3 that the biomass is proportional to the surface area in the reactors. The biomass increases by a factor of 3 for a 3-fold increase in individual particle surface area when comparing the reactors with 0.6 mm sand to reactors with 0.353 mm sand. Table 3 also shows that the biofilm thickness is less than one micron. Therefore, diffusion in the biofilm can be assumed and this verifies the assumption made in model development. A fully-penetrated biofilm with the traditional Monod model for substrate utilization/biomass growth agrees with experimental results.
3.1.3.
Activity kinetics
The sand samples extruded as sections from the reactors were analyzed for microbial activity using the modified BDOC
Fig. 7 e Activity Kinetics for Reactor 3.
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w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 4 4 1 9 e4 4 2 7
Fig. 8 e Activity Kinetics for Reactor 4.
kinetic test. Sand extruded from every 4th section of each of the reactors was tested to determine the removal of dextran over a period of 5 days. The activity test data for sand samples taken from reactors 1 through 4 is presented in Figs. 5e8, respectively. The kinetics were found to be zero-order with respect to substrate removal for samples from all the reactors. For each section of the reactors, the rate was independent of the distance from the influent to the effluent. Hence, the overall rate for a reactor could be calculated based on the results for the average of sections from a reactor. The individual rate for reactor 1 was found to be equal to 0.1734 mg-C/L-day, for reactor 2 it was 0.2849 mg-C/L-day, for reactor 3 it was 0.3989 mg-C/L-day and for reactor 4 it was 0.3257 mg-C/L-day. The overall rates for the column reactors were calculated using the ratio of total mass of sand in the columns divided by the mass used for batch testing. These values were found to be equal to 10.404 mg-C/L-day for reactor 1, 17.094 mg-C/L/day for reactor 2, 23.934 mg-C/L/day for reactor 3 and 19.542 mg-C/ L/day for reactor 4. Reactors 1 and 2 always had the same hydraulic retention time and Reactors 3 and 4 always had the same hydraulic retention time. We see that the reactors with smaller particle size had higher rates than that of reactors with larger particle diameter for the same flow rates (i.e. Reactor 2 Reactor 1 and Reactor 3 Reactor 4). The smaller difference between Reactors 3 and 4 could be from an accumulation of biomass during Phase 1 when the loading rate was 8 times the loading rate in Phase II. Similar data was observed when the BDOC reactors were used to evaluate kinetics using a single dextran feed concentration. This is interesting since the BDOC reactors were acclimated in a batch system while the sand from the reactors was acclimated in a column, yet the effects of surface area were similar.
4.
Summary
Dextran (average MW 10,000 Da) was observed to biodegrade with zero-order substrate utilization kinetics during batch kinetic experiments. This is consistent with the expected kinetics when hydrolysis is the rate limiting step. During continuous flow column experiments, the biomass distribution did not vary significantly based on activity measurements
and organic nitrogen analyses. Four column experiments were completed with two different particle sizes (hence surface area contact) and 4 different flow rates. The removal of substrate was independent of flow rate and particle size. However, the surface area had a positive relationship with biomass accumulation. The ratio of organic-N (hence biomass) was a factor of 3 higher for 0.353 mm particle size when compared with 0.6 mm particle size in one set of paired columns (Columns 1 and 2) and a factor of 2.4 higher for 0.353 mm particle size when compared with 0.6 mm particle size in the second set of paired columns (Columns 3 and 4). The surface area for the columns with a particle size of 0.353 mm was 3 times greater than the columns with 0.6 mm particle size. The experimental results demonstrated that biological removal of a biodegradable high molecular weight compound was robust during flow over a porous media. The Monod based substrate/growth kinetic model does predict the removal of a single type of biodegradable high molecular weight and the biofilm thickness was insufficient to cause diffusion limitations. The rate limiting step appears to be hydrolysis. Hydrolysis may be the rate limiting step for mixtures of high molecular weight compounds that are present in actual systems. The mixtures contain compounds with different biodegradabilities and their hydrolysis products might not be easily biodegradable resulting in higher order kinetics.
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