Effects of microcarrier pore characteristics on methanogenic fluidized bed performance

War. Res. Vol. 26, No. 8, pp. I 119-1125, 1992 Printed in Great Britain. All rights reserved

0043-1354/92$5.00+ 0.00 Copyright © 1992Pt~gamonPress Ltd

EFFECTS OF MICROCARRIER PORE CHARACTERISTICS ON METHANOGENIC FLUIDIZED BED PERFORMANCE CAR]- J. Y ~ , YEN HSU and WEN K. SHI~H*@ Environmental Research Laboratory, Department of Systems, University of Pennsylvania, Philadelphia, PA 19104-6315, U.S.A. (First received July 1991; accepted in revised form December 1991)

Al~traet--The cell retention capacities of three porous microcarriers with diversified pore characteristics and Ottawa silica sand were studied in methanogenic fluidized bed reactors with acetic acid as the sole substrate. Batch kinetic experiments on substrate utilization at different initial bulk-liquid substrate concentrations were also performed. The experimental data reveal that, under similar startup conditions, porous microcarriers are capable of reducing the startup times by more than 50% as compared to sand. Furthermore, under pseudo-steadystate conditions at an organic loading of 6 g total organic carbon (TOC)/I-day, porous microcarriers are capable of retaining three times more immobilized cells as compared to sand. More than 90% of total reactor cell mass is immobilized on porous microcarriers as opposed to 80% on sand. As a result, porous microcarriers are conducive for better proliferation of slow-growing methanogenic bacterial consortia. The experimental data clearly indicate that surface area, total pore volume and mean pore diameter should be used concornitantly to obtain better insight into the cell retention capacity of a given porous microcarrier. Batch kinetic data on substrate utilization reveal that mass transfer limitations arc absent in methanogcnic fluidized bed reactors at bulk-liquid TOC concentrations > 10mg/l. The observed maximum substrate utilization rates, which are independent of initial bulk-liquid TOC concentrations ranging from 200 to 1000 mg/1, are low for porous microcarriers as compared to sand (0.5 vs 2.25 day-t). These data confirm the results of the microscopic examinations performed which indicate that porous microcarriers attract Methanothrix type bacterial consortia whereas Ottawa silica sand attracts a mixture of Methanothrix and Methanosarcina. Key words--microcarrier, pore characteristics, fluidized bed, batch kinetics, attached biomass

INTRODUCTION Anaerobic waste treatment technologies offer many advantages including low biomass yield, low nutrient requirements, and biogas production (Speece, 1983). However, there are inherent difficulties and instability in the startup and operation of anaerobic processes, because of the slow growth rates and sequential metabolic pathways of the bacterial consortia involved (Parkin and Owen, 1986; Speece, 1983; Vochten et al., 1988). It is essential that the bacterial consortia be fully established and retained in an anaerobic process, otherwise the process itself will be highly susceptible to extramural perturbations (Camilleri, 1988; Chen, et al., 1986). Among many alternatives the provision of growth support media in an anaerobic process for retaining the bacterial consortia is an effective means that circumvents many operational problems that would otherwise occur (Huysman et al., 1983; Peels et ai., 1984; Switzenbaum et al., 1988). It is evident from the literature that a porous surface or a rough surface is conducive for better development of immobilized cell colonies (Bott *Author to whom all correspondence should be addressed. wR 26/S--H

and Miller, 1983; Bryers, 1987; Characklis, 1990; Switzenbaum et al., 1988; Szewzyk and Schink, 1988). A rough surface and/or internal pore space may provide a more hydrodynamically quiescent environment thereby reducing the detachment of immobilized cells by hydraulic shearing forces (Bryers, 1987; Characklis, 1990; Young and Yang 1989). Murray and van der Berg (1981) found that smooth surfaces such as glass or PVC are more likely to suffer cell loss from hydraulic shearing forces than rough textured materials such as needle-punched polyester. Chang and Rittmann (1988) showed that initial rates of cell loss were different between a rough surfaced granular activated carbon (GAC) carrier and a smooth surfaced G A C carrier. Further evidence indicates that distribution of pore sizes of the porous growth support media is critical to obtaining the optimal immobilized cell densities (Huysman et aL, 1983; Messing and Oppermann 1979; Wang and Wang, 1989; Yee, 1990). Messing and Oppermann (1979) experimentally determined that, to obtain high immobiliz.ed cell densities, at least 70% of the pores should be within the range of the smallest major dimension to five times the largest major dimension of a bacterial cell. Huysman et al. (1983) studied the biogas production rate in downflow anaerobic fixed

1119

CARL J. Y ~ et aL

1120

Ca~ pIIMP Feed GAS METER F--J

TIMER ~ ~nt

r~ers

Sand

Fig. 1. Fluidized bed reactor flow scheme (not to scale). film reactors and observed a strong positive influence by media porosity on cell colonization. Recently, W a n g a n d W a n g (1989) mathematically calculated the theoretical m a x i m u m cell retention capacities of microcarriers with different pore sizes and found that a m e a n pore diameter within a range of 2-5 times the m e a n cell diameter would yield the m a x i m u m immobilized cell densities. This paper reports and discusses the effects o f microcarrier pore characteristics on the performance of methanogenic fluidized bed reactors, with acetic acid as the sole substrate. Three porous microcarriers and n o n - p o r o u s silica sand were evaluated and compared for their cell retention capacities. The pseudosteady-state performance data of methanogenic fluidized bed reactors operated at an organic loading of 6 g total organic carbon (TOC)/I-day were used for comparison purposes. Furthermore, batch kinetic experiments o n substrate utilization in methanogenic fluidized bed reactors were also performed at different initial substrate concentrations.

The feed was pumped into the reactor through the top cap using a timer-controlled peristaltic pump on an hourly cycle. The effluent was collected by gravity through a loop connected to a port on the reactor wall. The recycle flow was drawn from the enlarged top section using a peristaltic pump and fed into the conical bottom section for fluidization of microcarriers. A wet tip gas meter was used for providing continuous readings on the volume of biogas produced. A "T"-shaped connector sealed with a septa was installed on the gas line for obtaining biogas samples for analysis of biogas composition. Reactors were located in a heated enclosure with its ambient temperature maintained at 34-36°C.

APPROACH

Table 1. Microcarrier characteristics*t RI R2 R3 R4 Specific gravity 2.21 2.36 2.27 2.65 Dry bulk density (g/ml) 0.44 0.31 0.46 1.53 Surface area (mZ/g) 46 1.3 0.2 0.004 Pore volume (ml/g) 1.19 1.47 0.66 - Volume fraction (ml/g) 0.04-0.1/~m 0.23 0.01 --0. I-I .0 p m 0.29 0 . 0 4 0.02 - -

Reactor design Four identical fluidized bed reactors were fabricated (Fig. I). A 7.5 cm i.d., 30 cm long acrylic tube was connected to a 2.5 cm i.d., 120 era long acrylic tube by a truncated Nalgene Imhoff cone to form the body of the reactor. The enlarged top section was used as a gas-solid separator and was made airtight using an acrylic cap sealed with a rubber "o"-ring. A conical section filled with garnet sand was attached to the bottom of the reactor and used as a flow distributor. Two sampling ports were installed on the reactor wall to obtain both liquid and media samples. The feed line (Tygon tubing) was changed weeHy and the recycle line (Norprene tubing) was changed every 3 weeks. The liquid volume in the reactor was 21.

Microcarriers Three porous microcarriers with diversified pore characteristics and the Ottawa silica sand were evaluated. They were sieved to obtain a narrow size distribution between ,*25 and 610/zm. Table 1 summarizes the characteristics of microcarriers evaluated.

Feed composition and bacterial inoculum Table 2 lists the composition of the feed tested. The feed TOC concentration was approx. 5000 rag/1. The feed was prepared 3 times per week by diluting the stock solutions

1.0-10.0 #m

0.28

0.88

0.10

--

10.0-50.0 # m

O. 12

0.54

0.47

--

Mean pore diameter ~m) 0.14 6.5 30.9 -*Rh calcined diatomaceous earth and day; R2: flux calcined diatomaceous earth; R3: calcined diatomaceous earth and day, R4: Ottawa silica sand. tMicrocarrier size: 425-610pm.

Micropore effects on methanogenic fluidized beds Table 2. Feed composition Constitutent Conoentration(mg/I) CH3COOH 13,125 Yeast extract 25 NH4CI 1200 MgSO( • 7H20 400 KCI 400 Na2S •9H20 307 CaC12• 2H20 55 (NH4)2HPO, 80 FeCI2•4H20 32.5 CoC12•6H20 10 KI 10 MnCI2' 4H20 0.8 CuCI2• 2H20 0.5 ZnCI2 0.5 AICI3•6H:O 0.5 NaMoO4 "2H20 0.5 H3BO3 0.5 NiCI2. 6H20 0.5 Cysteine I0 NaHCO3 4500 with the distilled water and refrigerating to prevent growth. Reagent grade chemicals were used with the exception of NaHCO 3 which was technical grade. A mixture of the acetate enriched bacterial culture and the mixed liquor collected from an existing anaerobic fluidized bed reactor being fed with lactose was used as the bacterial inoeulum for reactor startup. No further acclimation of the bacterial inoculum with acetic acid was provided.

Experimental design (1) Reactor startup. 335 ml microcarriers, 265 ml bacterial inoculum at 1330mg VSS/I (VSS: volatile suspended solids), and 1400ml feed at 840rag TOC/lwere added to each reactor. The reactors were put on total recycle for 4 days and then switched to semi-continuous feeding, with the feed TOC at 5000 mg/l and the organic loading at I g TOC/I-day. The media bed expansion in each reactor was controlled at 30% by maintaining the recycle flow rate at 510 ml/min. The increase in the organic loading was administered by increasing the feed rate to the reactor. Each increase in the organic loading was limited to 10-20'/. of the previous loading level and implemented only when stable reactor performance was attained. This startup strategy was continued to allow further buildup of immobilized cells until the symptoms of overloading such as low effluent pH and high CO2 percentage in the biogas appeared. Then the organic loading was immediately reduced to 6 g TOC/l-day. (2) Pseudo-steady-state experiments. The reactors were operated at an organic loading of 6 g TOC/l-day until stable reactor performance and constant reactor immobilized cell mass were attained. Then daily measurements on the following parameters were performed for approx. I month to insure that representative data were obtained: feed and effluent TOC, feed and effluent acetic acid, feed and effluent total alkalinity, biogas volume and composition and effluent SS/VSS. In addition, six measurements on reactor immobilized cell mass were also performed during this period. Reactor pH and temperature were monitored continuously and adjusted as required. (3) Substrate utilization kinetic experiments. Upon the completion of the pseudo-steady-state experiments, the batch kinetics of substrate utilization in methanogenic fluidized bed reactors were examined, with initial TOC concentrations ranged from 200 to 1000mg/l. The reactors were operated under semi-continuous feeding conditions (organic loading: 6 g TOC/l-day) for a week between batch kinetic experiments. This was designed to insure that the residual mixed liquor TOC concentration in the reactor prior to a specific batch kinetic experiment was negligible. At the beginning of a batch kinetic experiment, the feed

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was discontinued and a portion of reactor mixed liquor removed. Then the feed was added to replenish the liquid withdrawn and to attain a specific initial mixed liquor TOC concentration. The first mixed liquor sample was taken 8-10 min after the addition of feed to insure that complete mixed conditions had been attained. A number of mixed liquor samples were taken at the predetermined intervals and analyzed for TOC, acetic acid and SS/VSS. In addition, biogas volume and composition were also analyzed at these intervals. The reactor immobilized cell mass was measured at the beginning and end of the experiment.

Analytical techniques The analytical work was performed in accordance with

Standard Methods (APHA et aL, 1985). A RosemontXertex DC-80 TOC analyzer was employed for TOC measurements. A Hath Carle 100 gas chromatograph (GC) with a thermal conductivity detector (TCD) was employed for biogas composition determinations. A Varian 3400 GC with a flame ionization detector (FID) was employed for acetic acid analysis. The sample was directly injected into a J&W megabore (0.53ram x 10m) FFAP column with hydrogen as the cartier gas. Effluent VSS was used as a measurement of free cell mass in the mixed liquor. The immobilized cell mass was measured as follows. The expanded bed material was collected through a sampling port (5 -10ml), and dried at 103°C for 24h in a ceramic evaporating dish. The dried sample was then muffled at 550°C for 1 h. The difference between two dried weights would yield the mass of total volatile solids (TVS) in the sample. The difference between the mass of TVS and mass of free cells in the mixed liquor would yield the mass of immobilized cells as attached volatile solids (AVS). RESULTS AND DISCUSSION The startup conditions of four reactors were kept as identical as possible to allow a direct comparison. Reactors R2 and R3 achieved 99% removal of feed TOC at an organic loading of 6 g TOC/l-day 28 days after startup. They were capable of sustaining similar TOC removal performance at organic loadings as high as 36g TOC/l-day. Significant effervescence induced by substantial biogas production in reactors R2 and R3 was evident under high organic loading conditions (e.g. 151 CH4/day at 36g TOC/l-day). However, excessive detachment of immobilized cells in these two reactors was absent, and the average concentrations of effluent VSS of these two reactors were consistently less than 250 mg/l. Reactor R1 performed less satisfactory as compared with reactors R2 and R3, and it required 40 days to achieve similar performance. It was capable of removing 99% of feed TOC at organic loadings as high as 12g TOC/l-day. The average concentration of effluent VSS of reactor R l at this organic loading was approx. 500 mg/l, indicating that microcarrier R1 was less capable of retaining immobilized cells as compared with microcarriers R2 and R3. As would be expected, reactor R4 (sand reactor) was most susceptible to excessive loading increases during the startup phase, and it required 80 days to attain desired performance at an organic loading of 6 g TOC/l-day. The performance of reactor R1 deteriorated as the organic loadings exceeded 7 g TOC/l-day. These findings strongly suggest that

CARLJ. Y ~ et al.

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Table 3. Average pseudo-steady-state reactor performance at an TOC/I-day HRT (h) Feed TOC (mg/1) Effluent TOC (rag/l)

% TOC removal Immobilizedcell Concentration (g/I) Mass (g) % of total cell mass Free cell Concentration (rag/l) Mass (g) % of total cell mass Methane production Yield (1 CHUgTOC) % of bioges MCRT (days) Observed cell detachment rate (g vSS/g TOC)

loading of 6g

RI

R2

R3

R4

2O 5000 50 99

20 5000 40 99

20 5000 40 99

20 5000 150 97

15.4 6.7 91

40.0 17.4 99

20.7 9.0 98

5.0 2.2 80

338 0.68 9

147 0.29 I

92 0.18 2

278 0.56 20

0.95 70 226 0.026

1.05 65 188 0.025

1.01 65 38 0.055

the rough surface and internal pore space provided by the porous microcarriers had much to do with their relative success in startup performance.

Pseudo-steady-state experiments Table 3 summarizes the average pseudo-steadystate performance data. In general, TOC removal performance was excellent for all reactors (i.e. > 9 7 % ) during the pseudo-steady-state experimentation. Methane yield and biogas composition of these reactors were typical of anaerobic processes. The significance of surface roughness and internal pore space on the cell retention capacities of microcarriers is clearly demonstrated by the mass data of immobilized cells summarized in Table 3. More than 98% of total reactor cell mass was retained by porous microcarriers in reactors R2 and R3. Approximately 91% of total reactor cell mass was immobilized in the reactor Rl despite the fact it had the most surface area (Table 1). The cell retention capacity of Ottawa silica sand (i.e. 80% of total reactor cell mass) was rather poor as compared with those of porous microcarriers. Obviously, the reactors using porous microcarriers provided a more favorable environment for the proliferation of the slow-growing methanogenic bacteria consortia. A convenient parameter to quantify such effects is the mean cell residence time (MCRT), which can be defined as: M C R T (days) = (XV + XeV~)/QXe

orgen~

0.97 70 15 0.064

Reactors R2 and R3 attained extremely long MCRTs indicating that pore characteristics o f microcarriers R2 and R3 were highly conducive for better immobilization and retention of immobilized cells. These microcarriers provided large pores and ample internal pore space that allowed deep penetration and colonization of bacterial cells. As a result, excessive

40 - ~

• R~

oR8

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• Ri R4

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5o ¢14~o $urfac¢ Area fm2)

4750

• R2

40

oR3

20

• Ri 0

I 80

I

I 120

I

I

16o

Total Pore Vol-me ( ~ )

(l)

where X is the immobilized cell concentration in g AVS/I, V is the expanded media bed volume in liters, Xe is the effluent VSS concentration in g/l, V, is the reactor liquid volume in liters and Q is the feed rate in l/day. Equation (l) assumes that (a) effluent VSS was originated from the detachment of immobilized cells, since the feed used was free of suspended matter; and (b) completely mixed conditions were prevalent in reactors, since the recycle ratio (Qr/Q, where Qr is the recycle rate) maintained during the pseudo-steady-state-experimentation was 850.

40 - ~ > o R 2

2o -

• R$

• Ri

0

0

I

I 2O

I

40

Mean Pore Diameter (microns)

Fig. 2. Effects of microcarrier pore characteristics on the cell retention capacity. (a) Surface area, (b) total pore volume and (c) mean pore diameter.

Micropore effects on methanogenic fluidized beds

•

-

•

•

•

penetrate or had no pores at all, had much lower cell retention capacities. Although microearrier R3 had the largest mean pore diameter, its cell retention capacity was fimited by its total pore volume. Therefore, surface area, total pore volume, and mean pore diameter concomitantly determine the cell retention capacity of porous microcarriers. It is interesting to note that the data illustrated in Fig. 2 also provide experimental evidence confirming the theoretical predictions on the cell retention capacity of porous microcarriers as reported by Wang and Wang (1989).

•

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O

0 6OO

O

O

O

3O0 0 5OO 4OO 30O _~ 2OO 100 0 8

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•

Batch substrate utilization kinetic experiments

A

A

A

A

A

AAAAA

A 6

I 0

1123

I 200

I

I 400

I

I 600

Time (mln) Fig. 3. T h e b a t c h kinetic data o f reactor RI at an initial

TOC concentration of 690 mg/l. CH4 production (ml/min), O; bulk-liquid TOC (rag/l), O; bulk-liquid VSS (mg/l), Ak; and bulk-liquid pH, A.

detachment of immobilized cells was virtually absent in reactors R2 and R3, as indicated by low etttuent VSS concentrations. By comparison, microcarrier RI and Ottawa silica sand were less effective in retaining immobilized cells thereby yielding shorter MCRTs. Bacterial cells were most likely immobilized on the exterior surfaces of these microcarriers and therefore, more susceptible to the hydraulic shearing effects. The relative superiority of the larger pored microcarriers is further examined as follows. Figure 2 presents the immobilized cell concentration as a function of three microcarrier pore characteristics: surface area, total pore volume, and mean pore diameter. The surface area of microcarrier R l was approx. 2 orders of magnitude greater than those of R2 and R3, and the surface area of Ottawa silica sand was over an order of magnitude smaller than those of R2 and R3 [Fig. 2(a)]. Yet for porous microcarriers, the one with the most surface area had the least amount of immobilized cells. This demonstrates that surface area alone is not a good parameter for assessing the cell retention capacity of a given porous microcarrier. Total pore volume is also insufficient by itself to quantify the amount of immobilized cells that a given microcarrier can retain, as indicated by the amount of immobilized cells retained by microcarrier RI [Fig. 2(b)]. The higher cell retention capacities provided by the larger pored microcarriers (R2 and R3) were most likely due to the accessibility of the pores to the bacterial cells [Fig. 2(c)]. Reactors R1 and R4, which either had pores too small for the bacterial cells to

A typical set of experimental data observed during the batch substrate utilization kinetic experimentation is illustrated in Fig. 3. Under all conditions tested zero-order TOC utilization kinetics were observed at bulk-liquid TOC concentrations > 10 rag/l, whereas first-order TOC utilization kinetics became evident at bulk-liquid TOC concentrations < 10 mg/1. Therefore, for bulk-liquid TOC concentrations > 10 rag/l,

dS /dt = - k S X ,

(2)

where S is the bulk-liquid TOC concentration in mg/l and k is the maximum TOC utilization rate in d a y - [ The term Xt is derived as follows:

(3)

X, = ( X V + X , E ) / V , .

Equation (3) assumes that the activities of free cells and immobilized cells are identical. Since the duration of each batch kinetic experiment was very short (i.e. < 10 h), Xt remained constant. Therefore, integration of equation (2) between t --0 and t = t yields: (4)

S = So - kXtt

where So the initial bulk-liquid TOC concentration in" mg/l. Figure 4 presents the measured k values as a function of initial bulk-liquid TOC concentrations. A relatively constant k value (0.5 day-l) was observed among three porous microcarriers which was independent of initial bulk-liquid TOC concentrations ranging from 200 to 1000 mg/l. A higher k value

3

2

........

1

~""

V~ ,., V,., -vi~---ig--.i# .........

0

I

0

I

400

I

I

800

V O-o-I

1200

S, (rag TOClL) Fig. 4. The observed k values as a function of initial TOC concentrations.

1124

CARLJ. Y ~ et al.

(2.25 day -l ) was observed in the sand reactor which was also independent of initial bulk-liquid TOC concentrations. The observed discrepancy in k values indicates the difference in predominant bacterial consortia established between reactors RI and R3 and reactor R4. Microscopic examinations performed during this study showed that free cells in reactors R1 to R3 were predominantly Methanothrix type bacterial consortia, whereas free cells in the reactor R4 were a mixture of Methanothrix and Methanosarcina.

tested, were low for porous microcarriers as compared to sand (0.5 vs 2.25 day-l). This suggests that different predominant bacterial consortia might have developed in reactors RI to R3 and reactor R4. Microscopic examinations performed revealed that free cells in reactors RI to R3 were predominantly Methanothrix type bacterial consortia, whereas free cells in the reactor R4 were a mixture of Methanothrix and Methanosarcina.

CONCLUSIONS The cell retention capacities of three porous microcarriers with diversified pore characteristics (R1, R2 and R3) and Ottawa silica sand (R4) were studied during startup and pseudo-steady-state operations of methanogenic fluidized bed reactors with acetic acid as the sole substrate. Furthermore, batch substrate utilization kinetics were also studied at different initial bulk-liquid substrate concentrations. The following conclusions can be drawn: (!) The experimental data reveal that, under similar startup conditions, porous microcartiers were capable of reducing the startup times by more than 50% as compared to the sand. The startup times required to achieve 99% removal of feed TOC at 5000 mg/l at an organic loading of 6 g TOC/I-day were 40, 28, 28, and 80 days for reactors RI, R2, R3 and R4, respectively. (2) At an organic loading of 6 g TOC/l-day, porous microcarriers were capable of retaining at least three times more immobilized cell mass as compared to sand. Furthermore, more than 90% of total reactor cell mass was immobilized on porous microcarriers as opposed to 80% on sand. As a result, the porous microcarriers were capable of sustaining very long MCRTs (in this case, from 38 to 228 days) which were conducive for better proliferation of slow-growing methanogenic bacterial consortia. (3) Analysis of the effects of microcarrier pore characteristics on the cell retention capacity revealed that neither surface area nor total pore volume were adequate in quantifying these effects. Instead, surface area and total pore volume should be used in conjunction with the mean pore diameter to provide better insight into the cell retention capacity of a given porous microcarrier. (4) Analysis of batch data on substrate utilization kinetics revealed that mass transfer limitations were absent at the bulk-liquid TOC concentrations >10mg/i. The observed maximum substrate utilization rates (i.e. k values), which were independent of initial bulk-liquid TOC concentrations

Acknowledgements--Tlfis study was supported by grants

from the National Science Foundation (ECE85-02763) and the Research Foundation of the University of Pennsylvania. CJY was supported with a minority fellowship from the US Department of Education during this study.

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Micropore effects on methanogenic fluidized beds

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