Psychrophilic (6-15 °C) High-Rate Anaerobic Treatment of Malting

Psychrophilic (6-15 °C) High-Rate Anaerobic Treatment of MaltingWastewater in a Two-Module Expanded Granular Sludge BedSystem

Salih Rebac,*,† Jules B. van Lier,† Piet Lens,‡ Joost van Cappellen,†Marc Vermeulen,† Alfons J. M. Stams,§ Freddy Dekkers,⊥Koen Th. M. Swinkels,⊥ and Gatze Lettinga†

Sub-department Environmental Technology, Wageningen Agricultural University, Bomenweg 2,6703 HD Wageningen, The Netherlands, Department of Molecular Physics, Wageningen Agricultural University,Dreijenlaan 3, 6703 HA Wageningen, The Netherlands, Laboratory of Microbiology, Wageningen AgriculturalUniversity, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands, and Bavaria B.V., Postbus 1,5737 ZG Lieshout, The Netherlands

Psychrophilic (6-15 °C) anaerobic treatment of malting wastewater was investigated.A two-module expanded granular sludge bed reactor system with a total volume of140 dm3 was used to treat malting wastewater having a soluble and total chemicaloxygen demand (COD) between 233 and 1778 mg dm-3 and between 317 and 4422mg dm-3, respectively. The removal efficiencies at 6 °C were 47 and 71% of the solubleand volatile fatty acids (VFA) COD, at organic loading rates (OLR) ranging between3.3 and 5.8 kg of COD m-3 day-1. The removal efficiencies at 10-15 °C were 67-78and 90-96% of the soluble and VFA COD at an OLR between 2.8 and 12.3 kg of CODm-3 day-1. The specific methanogenic activity of the sludge present in each moduleincreased 2-3-fold during system operation for 400 days. The relatively highconcentration of suspended solids in the influent (25% of the total COD) caused adeterioration of the sludge bed in the first reactor module. This was aggravated byexcessive growth of acidifying biomass, which persisted in the first module sludgebed and resulted in granular sludge flotation. However, the second module couldaccommodate the increased OLR, thus providing a very high effluent quality (solubleCOD < 200 mg dm-3) of the total system. The stability of module I concerningsuspended solids could be restored by presettling the wastewater.

Introduction

Dilute wastewaters from bottling, malting, soft drink,and brewery manufacturing generally have a low tem-perature (5-25 °C). Anaerobic treatment of these waste-waters can be problematic due to the presence of dis-solved oxygen and suboptimal operation temperatures.The direct anaerobic treatment of wastewaters at lowtemperatures could offer a significant reduction in op-erational costs, since a large portion (35%) of total energyof a mesophilic reactor is required for heat (1).

Since temperature strongly affects both the bioconver-sion rates and the bacterial growth rate, adaptation ofthe conventional process design is likely required in orderto apply high volumetric organic loading rates at subop-timal operation temperatures (2, 3). By using expandedgranular sludge bed (EGSB) reactors, efficient anaerobic

treatment can be achieved at temperatures rangingbetween 10 and 20 °C at reasonably high volumetricorganic loading rates (4, 5). The EGSB reactor designin particular is advantageous for anaerobic treatment ofdilute wastewaters, because the applied high upflowvelocities (4-10 m h-1) guarantee a good contact of thewastewater and the methanogenic sludge (6-8).

Moduling the anaerobic conversion process can alsoimprove the overall treatment efficiency of wastewatersthat are composed of nonacidified compounds (9) or toproduce an effluent with low volatile fatty acids (VFA)concentrations (3, 10).

This paper describes the use of a two-module EGSBreactor system for the psychrophilic treatment of maltingwastewater. The rationale for the module-separatedprocess is to create niches where biomass can adapt tothe prevailing conditions in each module with respect tosubstrates and intermediates. The performance of thesystem as a function of the chemical oxygen demand(COD) and suspended solids (SS) load is presented. Theeffects of the operational conditions on the biological andphysical-chemical sludge characteristics are also de-scribed.

Materials and MethodsExperimental Setup. Two stainless steel reactors in

series (internal diameter 0.15 m; height 3.0 m) were used

* To whom correspondence should be addressed. Telephone: +31 317 483851/483339. Fax: + 31 317 482108. Bitnet/E-mail:[email protected].

† Sub-department Environmental Technology, Wageningen Ag-ricultural University.

‡ Department of Molecular Physics, Wageningen AgriculturalUniversity.

§ Laboratory of Microbiology, Wageningen Agricultural Univer-sity.

⊥ Bavaria B.V.

856 Biotechnol. Prog. 1998, 14, 856−864

10.1021/bp980093c CCC: $15.00 © 1998 American Chemical Society and American Institute of Chemical EngineersPublished on Web 11/11/1998

in the two-module pilot-scale EGSB reactor system, witha total volume of 140 dm3, internal reactor settlersincluded (Figure 1). Each module was equipped with ascreen-type gas-liquid-solid (GLS) separator at the toppart (4). Biogas production was measured with a wettest gasmeter (Meterfabriek, Dordrecht, The Nether-lands) connected to the GLS separator via a water seal.The temperature in the module was measured at a heightof 1.5 m by using a Pt-100 electrode and a West 6700controller (West Instruments Ltd., Brighton, England).Each module was equipped with an external water circuitin which water of the desired temperature was pumpedthrough the jacket of the reactor. Both wastewater flowand effluent recirculation flow were provided bymonopumps (Seepex, Germany), with maximum flows of200 and 220 dm3 h-1, respectively. The wastewater flowand the recirculation flow were mixed at the bottom ofeach module, resulting in a total superficial upflowvelocity between 4 and 6 m h-1 inside the module.Influent samples were taken from the wastewater flowprior to mixing. Effluent samples from each module weretaken from the module outlets.

Inoculum. The first module was inoculated with freshmesophilic (20-24 °C) methanogenic granular sludge,originating from a 760 m3 full-scale UASB reactor of theBavaria brewery (Lieshout, The Netherlands). Thesecond module was inoculated with methanogenic granu-lar sludge cultivated in a 225.5 dm3 pilot-scale EGSB

reactor treating malting wastewater at 12-20 °C (5). Thelatter sludge had been stored unfed at 4 °C for about 9months prior to use. The total amounts of granularsludge inoculated on day 0 in modules 1 and 2 wereapproximately 30 and 33 g of volatile suspended solids(VSS) dm-3, respectively. For reasons described below,the first module of the system was reinoculated with 14and 27 g of VSS dm-3 of new mesophilic sludge at days234 and 337, respectively.

Wastewater. The malting wastewater (Table 1) origi-nated from the batch steep process of a malting factory(Bavaria B.V., Wageningen, The Netherlands). Previ-ously, the malting wastewater was found to be 73%anaerobically biodegradable at 15 °C (5). Sodium bicar-bonate (0.502-0.672 g dm-3) was supplied to the waste-water to ensure a reactor pH in the range of 6.5-7.5.Considering the wastewater composition, no extra nu-trients were added (5). The composition of the maltingwastewater depended on the type of barley used in thesteeping process and its growth conditions prior toharvesting. Fresh wastewater samples were collecteddaily after 3 h from the wet steeping process and storedin two 0.2 m3 tanks each during the first 30 days ofoperation (Figure 1, 2). Thereafter, two extra 0.6 m3

tanks were used (Figure 1, 1).System Operation. Feeding of the system was

started immediately after inoculation (day 0) at 13 °Cand at an organic loading rate (OLR) of 8 kg of COD m-3

day-1 and a hydraulic retention time (HRT) of 4.9 h. Inperiods I, III, and V, the malting wastewater containeda relatively high concentration of suspended solids (SS).In period VI (days 330-390), presettled malting waste-water was used as feed (Figure 1, 17). The volume ofthe settler was 240 dm3. During continuous operationof the system, duplicate samples of the influent and theeffluent of both modules were taken three times perweek, except for periods days 275-340 and days 345-390, when the samples were taken only once and twiceper week, respectively.

Activity Tests. Assessments of the specific substratedegradation rates at 10 °C were performed with the seedsludge and reactor sludge samples with acetate, a VFAmixture (acetate:propionate:butyrate ) 1:1.5:1.8, basedon COD), ethanol, or sucrose as the substrate, as de-scribed by Rebac et al. (5). The maximum specificsubstrate degradation rates (Amax) with acetate of 1.5 gof COD dm-3 and the apparent half-saturation constants(Km) were determined at 10 ( 1 °C in 2.5 dm3 batchreactors intermittently stirred for 10 s at 60 rpm, every6 min, and contained 1.5 g of VSS dm-3 sludge. Depletionof the acetate concentration was followed until thesubstrate concentration was below the detection limit(<0.1 mg of COD dm-3).

The apparent Km values were estimated from substratedepletion curves by using a Michaelis-Menten-derivedequation. The substrate conversion rate depends on thebiomass concentration and the specific activity of thebiomass according to

where X is the biomass concentration (g of VSS dm-3), Ais the substrate degradation rate (g of CODacet g-1 of VSSday-1), and S is the substrate concentration (g of CODacetdm-3). Integration of the combined eqs 1 and 2 then

Figure 1. Schematic diagram (not to scale) of the 140 dm3 two-module pilot-scale EGSB system used in this study: (1) waste-water tanks (2 × 600 dm3); (2) wastewater tanks (2 × 200 dm3);(3) sodium bicarbonate solution tank; (4) influent first stage;(5) EGSB reactors; (6) segmented drum screens; (7) gas-liquidseparators; (8) effluent module I ) influent module II; (9)intermediate tank (20 dm3); (10) recirculation flow; (11) biogas;(12) water seals; (13) wet gas meters; (14) cooling circulator;(15) Pt 100 electrode; (16) effluent second stage; (17) settler (240dm3).

dSdt

) -AX (1)

A ) AmaxS

Km + S(2)

Biotechnol. Prog., 1998, Vol. 14, No. 6 857

yields

where St is the substrate concentration at time t (g ofCODacet dm-3) and S0 is the initial substrate concentra-tion (g of CODacet dm-3).

Wu et al. (11) and van Lier et al. (12) successfully useda similar equation for estimating the apparent Km ofmesophilic and thermophilic sludge. The constants wereestimated by using a nonlinear regression routine forparameter estimation as described by Rebac et al. (1995).Changes in Amax and Km of the granular sludge, whichwas present at the bottom (0.2 m) as well as at the top(1.73 m) of each module of the EGSB system, weredetermined in the period between days 230 and 390.

Physical-Chemical Characterization of theGranular Sludge. The size distribution, settling prop-erties and granular strength of the sludge were deter-mined as described by Rebac et al. (5). Diffusion coeffi-cients were determined at 21 ( 2 °C by using pulsed-field gradient nuclear magnetic resonance (NMR) andsubsequent nonlinear least-squares (NLLS) monoexpo-nential analysis of the data as described by Lens et al.(13).

Analysis. Total and soluble COD, VFA, ethanol,sugars, glycerol, succinate, lactate, and citric acid andthe biogas composition (CH4, H2) were determined asdescribed by Rebac et al. (5). Total suspended solids(TSS), VSS, and the granule density were analyzedaccording to standard methods (14).

ResultsSystem Performance during the Treatment of

Cold Malting Wastewater. Period I (the first 33 days)can be considered as the start-up period of the system,when the mesophilic methanogenic granular sludge sup-plied in the first stage was allowed to acclimatize to thenew wastewater and the low temperature (11-15 °C).The system achieved a COD removal efficiency of about70% within 1 week at an OLR ranging from 2.4 to 8.6 kgof COD m-3 day-1 (Figure 2b). During this period, theSS content of the wastewater was high (CODss, Table 1).The SS accumulated in the reactor settler and was partlyentrapped in the sludge bed of the first stage (data notshown).

In period II, the CODsol removal efficiencies exceeded70% at OLRs ranging from 2.8 to 12.3 kg of COD m-3

day-1. About 45% of the malting wastewater was pre-acidified (Figure 3). As shown in Figure 3, a considerableconversion of COD to methane (up to 40% of CODsol)already occurred in module I.

In period III (days 182-217), the CODsol removalefficiencies of the system dropped to 54% (Figure 2c),which was due to the low level of wastewater preacidi-

fication (26%) (Table 1). An additional 20-25% of theCODsol was acidified in module I of the system (Figure3). Acidification also occurred in period III, characterizedby an increased level of suspended solids, due to changesin the schedule of the barley steeping which led to anaccumulation of SS in the wastewater. The excess of SScaused a severe wash-out of the methanogenic granularsludge from module I. At the end of period III (day 234),module I was reseeded with 14 g of VSS dm-3 of freshinoculum.

In period IV (days 234-265), the EGSB system wasexposed to a temperature of 6 °C, and the OLR remainedat 4.3 kg of COD m-3 day-1. The removal efficiency ofthe total system based on CODsol and CODvfa dropped to47 and 71%, respectively.

In period V (days 266-330), the temperature wasincreased from 6 to 12 °C (Figure 2a). The systemresponded immediately with a higher CODsol efficiencywhich was increased by 46% (Table 2). A decrease in theHRT (on day 310) from 4.9 to 3.5 h hardly affected theCODsol removal efficiencies. Period V was characterizedby a high SS content of the wastewater (Table 1),

Table 1. Characteristics of COD Load of the Malting Wastewater in the Different Periods of the Investigationa

period, daysCODtot

(mg of COD dm-3)CODss

(mg of COD dm-3)CODsol

(mg of COD dm-3)CODb

(mg of COD dm-3)CODvfa

(mg of COD dm-3) pH pHcc

I, 0-33 1115-4422 (2106) 176-2988 (1053) 485-1751 (1053) 241-830 (438) 70-708 (222) 6.3-6.7 (6.5) (7.0)II, 34-153 317-2089 (1157) 34-410 (234) 233-1778 (923) 70-796 (416) 31-196 (94) 5.9-6.4 (6.2) (6.9)III, 182-217 418-1593 (940) 92-386 (227) 276-1258 (713) 68-344 (229) 14-477 (83) 5.6-6.2 (5.9) (7.0)IV, 234-265 822-1387 (1076) 59-682 (196) 679-1176 (880) 197-505 (308) 30-265 (107) 6.4-6.6 (6.5) (7.1)V, 266-330 898-1336 (1118) 101-535 (303) 443-1235 (815) 125-425 (282) 7-206 (40) 5.8-6.4 (6.2) (7.2)VI, 337-393 621-1626 (1042) 109-237 (163) 494-1437 (879) 159-913 (410) 33-202 (82) 5.9-6.5 (6.3) (7.2)

aAverage values are given in parentheses. b COD identified compounds, without VFA: COD of sugars, ethanol, glycerol, lactate, succinate,citric acid. c pHc of wastewater corrected after dosing sodium bicarbonate (average values).

Km ln(St

S0) + St - S0 ) -AmaxXt (3)

Figure 2. Overall operational conditions and overall perfor-mance of the two-module EGSB system: (A) temperature (4)and HRT (s); (B) total organic loading rate based on CODtot(0) and CODsol (s); (C) removal efficiency based on CODsol (s)and conversion of CODsol to CODmeth (O).

858 Biotechnol. Prog., 1998, Vol. 14, No. 6

resulting in a continuous wash-out of methanogenicgranular sludge from module I.

At the start of period VI (day 337), module I wasreseeded for the second time with 27 g of VSS dm-3 freshmesophilic granular sludge. The feed from day 337 tothe end of the experiment consisted of presettled maltingwastewater (Figure 1, 17) in order to eliminate theperformance disturbances caused by high SS concentra-tions. The measurement of CODtot and CODsol of themalting wastewater before and after the settler showeda reduction in CODtot and CODss concentrations of 30-40 and 80-90%, respectively, whereas the CODsol re-mained almost unchanged (data not shown). The CODsolremoval efficiency achieved during period VI, averaging78%, was the highest over the whole experiment.

Performance of Module I versus Module II. Theperformance of each module of the pilot-scale EGSB

system is shown in Figure 4. Module I was exposed toextremely high variations in OLR from 4 to up to 24 kgof COD m-3 day-1 over the experimental periods (Figure4a1) affecting the CODsol removal efficiency (Figure 4b1).In periods III-VI, relatively more acidification (an ad-ditional 10-25% of influent CODsol) occurred in moduleI (Figures 3 and 5a,b), which resulted in an extensivegrowth of the acidogenic populations on the methano-genic sludge granules. It was observed that flotation ofgranular sludge occurred at sugar sludge loading rates(SLR) ranging from 0.023 to 0.036 g of COD g-1 VSSday-1 (based on the start-up value of the VSS content)and at up-flow velocities of 4-6 m h-1 (periods III, IV,and V). The fermentation of nonacidified compounds inmodule I led to higher hydrogen concentrations in thebiogas. In periods I-VI, hydrogen concentrations in thebiogas of module I were in the ranges (ppm) 18-54, 65-460, 344-468, 97-368, 29-367, and 101-450, respec-tively. The higher hydrogen concentrations were con-comitant with a retarded conversion of propionate inmodule I (Figure 5a,b). In module II, the H2 concentra-tions were in the ranges (ppm) 5-11, 10-38, 60-116,10-119, 20-634, and 30-83, respectively, in periodsI-VI. In module II, elevated H2 concentrations wereformed only at the end of periods III and V. The lattercan be due to the fact that a considerable acidificationalso took place in module II, as a result of the significantbiomass losses in module I.

Metabolic Characteristics of the Sludge. Table 3shows that the specific VFA degrading activities at 10°C of the mesophilic module I inoculum increased byfactors of 15 and 20, respectively, after 140 days ofcontinuous operation under psychrophilic conditions (10-13 °C). The activity with acetate and the VFA mixtureassessed at the end of period III (day 217) was slightlylower than that at day 140. Apparently the sludge wasdetrimentally affected by sludge wash-out and sludge beddeterioration that occurred in module I during period III.The high ethanol and sucrose acidifying activity of thesludge from module I at day 217 reflects the high degreeof anaerobic acidification in the module I during periodIII.

The specific acetate degrading activity of the sludgefrom the module II after 140 days of operation increasedby 58% compared to that of the seed sludge. However,its specific activity with the VFA mixture as the substratedid not increase. The latter observation corroborateswith the very low butyrate concentrations (<30 mg ofCODbut. dm-3) in the effluent of the first stage. Thespecific VFA degrading activity of the sludge sampledfrom module II on day 217 was lower than that of sludgesampled on day 140 (Table 3). This very likely can beattributed to the sludge wash-out from the first stage andsubsequent accumulation of these solids in the sludge bedof module II. Indicatively, the ethanol acidifying activityat day 217 of the sludge from module II had increasedby 95% compared to that at day 140.

Table 2. Performance Data of the Two-Module Psychrophilic Pilot-Scale EGSB System Treating Malting Wastewatera

period,days

OLRtot (kgof COD m-3

day-1)

OLRsol (kgof COD m-3

day-1)HRT(h)

temp(°C) pHR1,ef

b pHR2,efb

CODsol,out(mg of

COD dm-3)

VFAout(mg of

COD dm-3)

CODsolremoval

(%)

VFAremoval

(%)

I, 0-33 7.7-21.8 (10.5) 2.4-8.6 (5.2) 4.9 11-15 (13) (6.8) (6.9) 149-995 (464) 0-613 (178) 29-72 (59) -43-100 (67)II, 34-153 2.2-14.5 (8.0) 2.8-12.3 (6.4) 3.5 10-15 (11) (6.8) (6.9) 133-989 (308) 0-558 (59) 42-79 (67) 54-100 (90)III, 182-217 4.1-15.8 (9.3) 2.6-12.5 (7.1) 2.4 10-13 (12) (6.8) (6.9) 116-559 (319) 7-198 (64) 36-67 (54) 70-97 (86)IV, 234-265 4.0-6.8 (5.3) 3.3-5.8 (4.3) 4.9 6-7 (6) (6.9) (7.0) 332-721 (463) 30-322 (140) 39-57 (47) 46-87 (71)V, 266-330 4.1-9.0 (6.1) 2.2-7.1 (4.5) 3.5-4.9 10-14 (12) (7.0) (7.1) 140-367 (260) 0-66 (22) 49-82 (68) 91-100 (96)VI, 337-393 4.3-11.3 (7.2) 3.8 - 9.9 (6.1) 3.5 12-14 (13) (7.2) (7.1) 22-487 (202) 7-114 (29) 64-96 (78) 3-98 (95)

a Average values are given in parentheses. b pH of effluents from modules I and module II.

Figure 3. Fate of CODsol in the two-module EGSB system: firstbar, CODvfa of the influent; second bar, CODvfa-M1 + CODmeth-M1of the first module; third bar, CODvfa-M2 + CODmeth-M1 +CODmeth-M2 of the second module and CODmeth of the firstmodule, expressed as percentage of average influent CODsol inthe experimental periods. Note that only 73% of the CODsol isbiodegradable.

Figure 4. Organic loading rate and efficiency of each moduleof the moduled EGSB system: (A) CODsol organic loading rate(s); (B) CODsol removal efficiency (- - -). The numbers followingthe abbreviation refer to the module of the system.


The development of the maximum specific acetatedegrading activities and the apparent half-saturationconstant (Km) of both the sludge sampled at the bottomand the top from module I during period days 234-393(Table 4) was temporary prevented by the severe sludgewash-out occurring during period V (days 266-330).

Despite the fact that the fresh mesophilic sludge wasexposed instantaneously to psychrophilic conditions (6-7°C), its acetate degrading activity measured at 10 °Cdoubled in a period of about 60 days (days 234-295). Thespecific acetate degrading activities of the sludge fromthe top of module I were higher (7-32%) than those ofthe sludge from the bottom of that module.

The maximum acetate degrading activities of thesludge sampled from both the bottom and the top ofmodule II were similar, and they steadily increased intime between days 295 and 393. The apparent Km valuesof the sludge in the module II were identical over theheight of the reactor and remained unchanged during 159days of reactor operation. Interestingly, the specificactivity of the seed sludge did not change when itsactivity on day 0 was compared with that measured onday 234 (after 6 months of storage at 4 °C). It remainedstable at 0.09 g of COD g-1 of VSS day-1 (Table 4).

Physical Characteristics of the Sludge. Duringthe first 140 days of operation, the mean diameter of thegranules in module I increased significantly (Figure 6 A1and B1). This can be partly attributed to the extensivegrowth of an acidogenic population, as illustrated by itsdistinct increase in acidifying activity (Table 3). Incontrast, the diameter of the granules in module II hardlyincreased, probably due to fact that the feed of moduleII consisted merely of VFA (Figure 6A2 and B2). Thestrong effect of an acidifying population on the diameterof sludge granules is also confirmed by results from thesludge sample taken from module II on day 217. Inperiod III, due to a loss of biomass from module I, theacidogenic organisms grew in the second module, andtheir attachment to the granules resulted in largergranules in module II (Figure 6C2). The size distributionof the seed sludge, used for inoculation of module I onday 234, is depicted in Figure 6C1. Size distributionmeasurements could not be made on day 217 due to thesmall amount of sludge that remained in module I.

The difference in the mean diameter between thesludge samples from the bottom and the top of themodule I (Table 5) reveals the occurrence of sludgegranule segregation over the height of the sludge bed.The sludge samples from module I on days 350 and 393(Tables 4 and 5) originate from the second seed sludgeused for the reinoculation of module I on day 337. Theapparent diffusion coefficients of the sludge sampled atthe top and the bottom of module II were very similarduring the period between days 295 and 393 (Table 5).In contrast, the sludge samples from the bottom ofmodule I had an apparent diffusion coefficient that wasmarkedly lower between days 350 and 393 (Table 5). Thedensity of the sludge from modules I and II ranged

Table 3. Maximum Specific Substrate Degrading Activities at 10 °C of the Inoculum and the EGSB Sludgea

maximum specific substrate degrading activity (g of COD g-1 of VSS day-1)

module I module II

time acetate VFA mixture ethanolb sucroseb acetate VFA mixture ethanolb sucroseb

day 0c 0.009(0.002) 0.005(0.001) 0.127(0.025) ndd 0.099(0.009) 0.113(0.011) 0.241(0.107) ndday 140 0.143(0.007) 0.091(0.007) 0.472(0.036) nd 0.169(0.003) 0.103(0.001) 0.188(0.019) ndday 217 0.104(0.013) 0.082(0.000) 0.594(0.003) 0.874(0.033) 0.171(0.009) 0.090(0.000) 0.354(0.071) 0.373(0.061)

a Standard deviation is given in parentheses. b Ethanol and sucrose acidifying activity. c Seed sludge after 5 (first module) and 14(second module) months of storage at 4 °C. d nd ) not determined.

Figure 5. VFA concentration in the influent and effluent ofeach module: (A) influent, module I; (B) effluent, module I; (C)effluent, module II. CODacet (s), CODprop (]).

Table 4. Maximum Specific Acetate DegradingActivities (Amax) and Apparent Half-Saturation Constant(Km) at 10 °C of the Inoculum and the EGSB Sludgea

maximum specific acetate degrading activity(g of COD g-1 of VSS day-1) and

apparent Km (g of COD dm-3)

module I module II

time Amax Km Amax Km

day 0 0.009(0.002) ndd 0.099(0.009) ndday 234b 0.021(0.001) 0.012(0.004) 0.093(0.002) 0.018(0.012)

Bottom Sludgeday 295 0.043(0.001) 0.020(0.009) 0.229(0.003) 0.142(0.064)day 350 0.035c(0.001) 0.018c(0.001) 0.241(0.003) 0.169(0.025)day 393 0.069(0.009) 0.048(0.009) 0.260(0.020) 0.123(0.026)

Top Sludgeday 295 0.057(0.000) 0.026(0.003) 0.241(0.002) 0.092(0.011)day 350 0.042c(0.003) 0.016c(0.003) 0.247(0.002) 0.130(0.009)day 393 0.074(0.007) 0.059(0.017) 0.274(0.005) 0.116(0.009)

a Standard deviation is given in parentheses. b Module I, firstreinoculation with new mesophilic sludge at day 234; module II,seed sludge after 20 months of storage at 4 °C. c 15 days aftersecond reinoculation with new mesophilic sludge at day 337. d nd) not determined.


between 1029 and 1049 and between 1022 and 1034 kgm-3, respectively. The strength of the granules presentin module I had increased compared to the inoculum, butit decreased during the course of the experiment. Thestrength of the sludge samples from the module IIdecreased to a small extent during the course of theexperiment.

DiscussionReactor Performance with More Acidified Waste-

water (Periods II and VI). The results obtained in thisstudy show the feasibility of direct anaerobic treatmentof malting wastewater under psychrophilic conditions atrelatively high volumetric organic loading rates. Whentreating presettled, more acidified wastewater, the two-module EGSB system achieved higher and more constant

CODsol removal efficiencies of 78% at 13 °C (Table 2,period VI), compared to 66% obtained in a single-moduleEGSB system operating at 20 °C, treating the same typeof malting wastewater (5). The two-module EGSB sys-tem accommodated 3-5 times higher OLRs of acidifiedwastewater, compared to those reported so far for otherpsychrophilic anaerobic wastewater treatment systems(2, 8, 15-17). Moreover, the COD removal efficiencieswere very high and comparable to the maximum ef-ficiency of 85% found during the mesophilic anaerobictreatment of brewery wastewaters at 30 °C at an OLRof 20 kg of COD m-3 day-1 and a HRT of 2.1 h (18).Similar results were found even at 37 °C, where anefficiency of 80% was obtained in a three-stage systemat an OLR of 25 kg of COD m-3 day-1 and a HRT of 6 h(19). The removal efficiencies achieved in this study are

Figure 6. Development of the size distribution of mixed granular sludge from each module expressed as percentage of the biomassweight of the granules. (A) start of the experiment; (B) at day 140, and (C2) day 217. (C1) Reinoculation of the first stage at day 234.Duplicate samples are presented by the bars. The figures following the abbreviation refer to the module of the system.

Table 5. Physical Characteristics of the Inoculum and the Psychrophilic EGSB Sludgea

density, apparent diffusion coefficient, strength, and mean diameter of granular sludge

module I module II

timedensity(kg m-3)

diff coeff(10-9 m2 s-1)

strength(kN m-2)

mean diameter(mm)

density(kg m-3)

diff coeff(10-9 m2 s-1)

strength(kN m-2)

mean diameter(mm)

day 234b 1042(5.6) 1.82(0.03) 95(25.4) 1.47(0.17) 1027(1.1) 1.33(0.03) 88(3.5) 1.78(0.09)

Bottom Sludgeday 295 1044(0.1) 1.19(0.01) 113(9.6) 2.03(0.05) 1029(7.6) 1.47(0.03) 77(1.7) 2.40(0.10)day 350 1049c(4.3) 1.49c(0.14) 143c(1.7) 2.03(0.15) 1030(1.5) 1.31(0.02) 72(7.0) 2.40(0.04)day 393 1042(9.7) 0.99(0.23) 108(0.0) 2.09(0.02) 1030(2.4) 1.28(0.06) 70(3.5) 2.43(0.16)

Top Sludgeday 295 1029(28.6) 1.18(0.00) 97(0.8) 1.78(0.20) 1023(0.8) 1.35(0.01) 73(4.4) 2.02(0.13)day 350 1046c(4.9) 2.05c(0.03) 121c(7.4) 1.26(0.01) 1022(0.8) 1.38(0.02) 71(1.8) 2.19(0.08)day 393 1041(2.5) 1.44(0.10) 104(7.0) 1.54(0.03) 1034(0.2) 1.28(0.08) 68(0.0) 2.15(0.05)

a Standard deviation is given in parentheses. b Module I, first reinoculation with new mesophilic sludge at day 234; module II, seedsludge after 20 months of storage at 4 °C. c 15 days after second reinoculation with new mesophilic sludge at day 337.


even comparable to the results of Perez et al. (20), whoreported an 82% COD removal by a thermophilic (55 °C)anaerobic fluidized bed reactor operated at an OLR of32 kg of COD m-3 day-1 and an HRT of 11 h.

When treating more acidified wastewater, module Iachieved a high methanogenic capacity (methanizationof about 40% of influent soluble COD), whereas moduleII served as a scavenger of residual VFA from module I(Figure 3). Comparison of part b of Figure 5 with part cshows that, when the VFA concentrations entering themodule II are below 200 mg of COD dm-3 (Figure 5b,c),the treatment efficiency of that module was still verysatisfactory (period II). This might be attributed to thevery low apparent Km (18 mg of COD dm-3) of the sludge(Table 4). These low Km values once again support theadvantages of using EGSB reactors in anaerobic treat-ment of low strength wastewaters, as previously foundby Kato et al. (8) and Rebac et al. (4). Propionate, knownas the most difficult VFA intermediate during methano-genic anaerobic degradation (21), was efficiently removedby the moduled psychrophilic reactor system (Figure5b,c). The second module offered excellent conditions forpropionate removal, as inhibitory effects of oxygen,hydrogen, and other VFAs were minimal in this module.This confirms the good propionate removal efficiency ofmoduled psychrophilic (8 °C) laboratory-scale reactorstreating a VFA-sucrose mixture (3).

Reactor Performance with More NonacidifiedWastewater (Periods III, IV, and V). When treatingmore nonacidified wastewaters, obviously module I re-ceives the highest OLR and the highest wastewatercomplexity (Table 1, periods I, III, and V), This leadsmore rapidly to a reactor imbalance, as evidenced by thesludge wash-out, the low COD removal capacity (Figure4a1 versus 4b1), and the elevated H2 concentrations inthe biogas. In contrast, the performance of module II wasstable, and the module remained highly efficient, as thefeed of this module consisted of a wastewater with amuch more constant composition, with substantiallymore acidified COD (Figure 5b).

The high content of nonacidified matter in the waste-water resulted in flotation of granules, as this substrateleads to the formation of a layer of acidifying sludgearound granules. This fluffy coating reduces the settlingvelocity and can lead to gas entrapment inside thegranule. Subsequently, flotation of the granules occursunder the high hydraulic regime, prevailing in EGSBreactors. Flotation of mesophilic granular sludge occursat a sugar SLR of 0.325 g of COD g-1 of VSS day-1 andat a liquid up-flow velocity of 0.5 m h-1 (22). Flotationof granular sludge under psychrophilic conditions isenhanced by the very high biomass yield (0.22 g of VSS-COD g-1 of CODremoved of glucose at 10 °C, (3)) and theextremely low starvation rate (1.57 × 10-5 h-1 at 10 °C(3)) of the acidifying bacteria. Also, the presence ofoxygen (up to 10 mg dm-3) in the malting wastewater(steeping process-aeration process) may support theformation of a fluffy coating around the granules. Oxy-gen enables fast growing facultative anaerobic bacteria,which have 10 times higher growth yields compared tostrict anaerobes, to develop in the sludge (23). Flotationof granules with a fluffy acidifying outer layer in moduleI is a persistent problem which requires further researchon the growth and granulation rate of acidifiers underpsychrophilic conditions.

The degradation of SS in module I can hardly beexpected because the hydrolysis rate of SS drops sharplyat low temperatures and even approximates zero forvarious types of solids (24). An effective and plain

solution for the problems of these SS was presettling ofthe solids (period VI, Table 1).

Biomass Characteristics. In the period betweendays 295 and 393, Amax values were in the range of thosefound at 10 °C (Table 4) for sludge cultivated in a one-step EGSB reactor fed with VFA for 12 months (4).Theacetate degrading activity (Tables 3 and 4) of the sludgepresent in module I developed slowly at 10 °C, probablydue to high inflow of nonacidified COD yielding aci-dogenic bacteria and suspended solids, which diluted theacetoclastic biomass. Table 4 shows that, at low operat-ing temperatures (<10 °C), longer periods are requiredto achieve an acetate degrading activity comparable tovalues obtained at 15 °C (5). This is supported by resultsof continuous-flow experiments, where a temperatureincrease from 6 to 12 °C (period V) significantly increasedthe COD removal rate in module I (Figure 4 b1). Hence,the methanogenic activity of the mesophilic seed sludgeis of utmost importance for the start-up of high-ratepsychrophilic anaerobic reactor (<15 °C), when consider-ing that at 10 °C, fresh active mesophilic sludge expressesonly 10-12% of its methanogenic activity at 30 °C (4,25).

Obviously of considerable practical importance is theobservation that the methanogenic sludge preserved itsachieved methanogenic activity at 10 °C, even after 6months of storage at 4 °C (see Table 4, second module,day 0 versus day 234). This indicates that the starvationrate at 4 °C of methanogens grown at these low temper-atures is extremely low. Apparently, the cultivation ofsludge on low strength wastewater under psychrophilicconditions enables slow but stable enrichment of amethanogenic consortium.

The increase of the apparent Km of the sludge inmodule I at days 234 and 393 compared to that at days295 and 350 (Table 4, 5) might be attributed to theformation of extracellular polymers (26, 27), or to exten-sive growth of acidifying bacteria on the surface ofmethanogenic granules (28). Interestingly, the observedincrease in Km was accompanied by a severe drop in theapparent diffusion coefficient (Table 5). The largergranules that formed segregated to the bottom (Table 5,mean diameter) and had a lower activity (Table 4),possibly due to substrate diffusion limitation (29, 30). Incontrast, small changes in apparent Km and apparent Diffcoefficient of the sludge present in module II occur overa long period of time (days 295-393, Tables 4 and 5) asresults of the slow growth of methanogens at low tem-perature.

The strength of the granules in module I initiallyincreased (Table 5), possibly as a result of the fastadditional formation of extracellular polymers inside thegranules (26). However, later the strength of the gran-ules dropped, probably due to the formation of anattached fluffy acidifying layer (31). Compared to theseed material, also the granules from the module IIdecreased in strength, which confirms previous findingswith mesophilic granules fed with ethanol (8) and acidi-fied wastewaters (18).

Conclusions

1. Cold (6-15 °C), low strength (<1000 mg of CODdm-3), acidified (60-70%) wastewaters can be efficientlytreated in a “high-rate” anaerobic EGSB reactor config-uration. The results of the present investigations clearlyreveal the prospects of full-scale application of anaerobictreatment for highly acidified low strength wastewaterunder psychrophilic conditions.


2. When applying a two-module EGSB system, anaer-obic treatment of acidified malting wastewater in thetemperature range 8-12 °C can be accomplished at anOLR up to 12 kg of COD m-3 day-1 and at an HRT of 3.5h. Moduling the EGSB system markedly improves theperformance and stability of the system compared tothose of a one-step process. When operated properly, amoduled system provides a remarkable long-term stableperformance at low temperatures, even when the systemis imposed with strong variations in OLR between 3 and12 kg of COD m-3 day-1.

3. The maximum specific conversion rate of themethanogenic seed sludge in the second module increasedby a factor 2.5 within 393 days of operation at 6-15 °C.This study clearly indicates that mesophiles grow andmetabolize at suboptimal temperatures. Newly grownviable methanogenic biomass is sufficiently well retainedin the second module. The methanogenic sludge pre-served its methanogenic activity achieved under psy-chrophilic conditions, even after prolonged unfed storageperiod (6 months at 4 °C).

4. Granular sludge flotation comprises a problem inthe first module for insufficiently nonacidified waste-waters, as it leads to excessive growth of acidogenic andfacultative anaerobic populations on the methanogenicgranules. It is, therefore, needed to apply a preacidifi-cation step, of which the design criteria should beassessed.

5. High suspended solids concentrations in the influ-ent negatively affect the sludge and sludge bed charac-teristics of the EGSB reactor. At least a part of thesesuspended solids present in the original wastewatershould be removed prior to feeding the wastewater in theEGSB system.

NotationAmax specific substrate degrading activity (g of COD

g-1 of VSS day-1)COD chemical oxygen demand (g O2 dm-3)HRT hydraulic retention time (h)Km apparent half-saturation constant (g of COD

dm-3)OLR organic loading rate (g of COD dm-3 day-1)SLR sludge loading rate (g of COD g-1 of VSS day-1)SS suspended solidsv up-flow velocity (m h-1)VFA volatile fatty acids (g dm-3)VSS volatile suspended solids (g dm-3)

AcknowledgmentThe financial support of Bavaria B.V., Lieshout, The

Netherlands, is gratefully acknowledged. We thank JimField for his critical remarks on the manuscript, J. vander Laan and I. Bennehey for their technical assistancewith the chromatographs, and S. Hobma for his as-sistance in performing some experiments.

References and Notes(1) Mills, P. J. Minimisation of energy input requirements of

an anaerobic digester. Agric. Wastes 1979, 1, 57-66.(2) Banik, G. C.; Dague, R. R. ASBR treatment of dilute

wastewater at psychrophilic temperatures. Proceedings of the69th Annual Water Environmental Conference, Dallas, TX,1996; pp 235-246.

(3) Lier van, J. B.; Rebac, S.; Lens, P.; Bijnen van, F.; OudeElferink, S. J. W. H.; Stams, A. J. M.; Lettinga, G. Anaerobictreatment of partly acidified wastewater in a two-stage

expanded granular sludge bed (EGSB) system at 8 °C. WaterSci. Technol. 1997, 36 (6-7), 317-324.

(4) Rebac, S.; Ruskova, J.; Gerbens, S.; van Lier, J. B.; Stams,A. J. M.; Lettinga, G. High-rate anaerobic treatment ofwastewater under psychrophilic conditions. J. Ferment. Bioeng.1995, 5, 15-22.

(5) Rebac, S.; van Lier, J. B.; Janssen, M. G. J.; Dekkers, F.;Swinkels, K. T. M.; Lettinga, G. High-rate anaerobic treat-ment of malting wastewater in a pilot-scale EGSB systemunder psychrophilic conditions. J. Chem. Technol. Biotechnol.1997, 68, 135-146.

(6) Man de, A. W. A.; van der Last, A. R. M.; Lettinga, G. Theuse of EGSB and UASB anaerobic systems for low strengthsoluble and complex wastewaters at temperatures rangingfrom 8 to 30 °C. Proceedings of the Fifth InternationalSymposium on Anaerobic Digestion; Hall, E.R., Hobson, P.N., Eds.; Bologna, Italy, 1988; pp 197-209.

(7) Frankin, R. J.; Koevoets, A. A.; van Gils, W. M. A.; van derPas, A. Application of the BIOBED upflow fluidized bedprocess for anaerobic wastewater treatment. Water Sci.Technol. 1992, 25, 373-382.

(8) Kato, T. M.; Field, J. A.; Versteeg, P.; Lettinga, G. Feasibilityof Expanded Granular Sludge Bed Reactors for the anaerobictreatment of low strength soluble wastewaters. Biotechnol.Bioeng. 1994, 44, 469-479.

(9) Komatsu, T.; Hanaki, K.; Matsuo, T. Prevention of lipidinhibition in anaerobic processes by introducing a two-phasesystem. Water Sci. Technol. 1991, 23, 1189-1200.

(10) Lier van, J. B.; Boersma, F.; Debets, M. M. W. H.; Lettinga,G. High-rate thermophilic anaerobic wastewater treatmentin compartmentalized upflow reactors. Water Sci. Technol.1994, 30, 251-261.

(11) Wu, W.-M.; Thiele, J. H.; Mahendra, K J.; Zeikus, J. G.Methabolic properties and kinetics of methanogenic granules.Appl. Microbiol. Biotechnol. 1993, 39, 804-811.

(12) van Lier, J. B.; Sanz Martin, J. L.; Lettinga, G. Effect oftemperature on the anaerobic thermophilic conversion ofvolatile fatty acids by dispersed and granular sludge. WaterRes. 1996, 30, 199-207.

(13) Lens, P.; Hulshoff Pol, L.; Lettinga, G.; Van As, H. 1H NMRstudy of water transport processes in sulfidogenic granularsludge. Water Sci. Technol. 1997, 36 (6-7), 157-163.

(14) American Public Health Association. Standard methods forthe examination of water and wastewaters, 16th ed.; APHA:Washington, DC, 1985.

(15) Grant, S.; Lin, K. C. Effects of temperature and organicloading on the performance of upflow anaerobic sludgeblanket reactors. Can. J. Civ. Eng. 1995, 22, 143-149.

(16) Matsushige, K.; Inamori, Y.; Mizuochi, M.; Hosomi, M.;Sudo, R. The effects of temperature on anaerobic filtertreatment for low-strength organic wastewater. Environ.Technol. 1990, 11, 899-910.

(17) Switzenbaum, M. S.; Jewell, W. J. Anaerobic attached filmexpanded bed reactor treatment of dilute organics. In Pro-ceedings of the 51st Annual WPCF Conference, Anaheim, CA,1978; pp 1-164.

(18) Pereboom, J. H. F. Methanogenic granule development infull scale internal circulation reactors. Water Sci. Technol.1994, 30, 211-221.

(19) Stadlbauer, E. A.; Oey, L. N.; Weber, B.; Jansen, K.; Weidle,R.; Lohr, H.; Ohme, W.; Doll, G. Anaerobic purification ofbrevery wastewater in biofilm reactors with and without amethanation cascade. Water Sci. Technol. 1994, 30, 395-404

(20) Peres, M.; Romero, L. I.; Sales, D. Thermophilic anaerobicdegradation of distillery wastewater in continuous-flow fluid-ized bed bioreactors. Biotechnol. Prog. 1997, 13, 33-38

(21) Lettinga, G. Anaerobic digestion and wastewater treatmentsystems. Antonie van Leeuwenhoek 1995, 67, 3-28.

(22) Alphenaar, A. Anaerobic granular sludge: characterization,and factor affecting its functioning. Ph.D. Thesis, AgriculturalUniversity Wageningen, The Netherlands, 1994; pp 93-112.

(23) Shen, C. F.; Guiot, S. R. Long-term impact of dissolved O2on the activity of anaerobic granules. Biotechnol. Bioeng.1996, 49, 611-620.

(24) Zeeman, G.; Sanders, W. T. M.; Wang, K. Y.; Lettinga, G.Anaerobic treatment of complex wastewater and waste


activated sludge. In Proceedings of the Conference on Ad-vanced Wastewater Treatment, Amsterdam, The Netherlands,1996; pp 225-232.

(25) Henzen, M.; Harremoes, P. Anaerobic treatment of wastewater in fixed film reactors-literature review. Water Sci.Technol. 1983, 15, 1-102.

(26) Vanderhaegen, B.; Ysebaert, E.; Favere, K.; Van Wanbeke,M.; Peeters, T.; Panic, V.; Vandenlangenbergh, V.; Verstraete,W. Acidogenesis in relation to in-reactor granule yield. WaterSci. Technol. 1992, 25 (7), 21-30.

(27) Bhatti, Z. I.; Furukawa, K.; Fujita, M. Comparativecomposition and characteristics of methanogenic granularsludges treating industrial wastes under different conditions.J. Ferment. Bioeng. 1995, 3, 273-280.

(28) Yoda, M.; Nishimure, S. Controling granular sludge flota-tion in UASB reactors. Water Sci. Technol. 1997, 36 (6-7),165-173.

(29) Wijffels, R. H.; Englund, G.; Hunik, J. H.; Leenen, E. J. T.M.; Bakketun, AÄ .; Obon de Castro, J. M., Tramper, J. Effectsof limitation on immobilized nitrifying microorganisms at lowtemperatures. Biotechnol. Bioeng. 1995, 45, 1-9.

(30) Dolfing, J. Kinetics of methane formation by granularsludge at low substrate concentrations. The influence of masstransfer limitation. Appl. Microbiol. Biotechnol. 1985, 22, 77-81.

(31) Hulshoff Pol, L. W.; van de Worp, J. J. M.; Lettinga, G.;Beverloo, W. A. Physical characterization of anaerobic granu-lar sludge. In Proceedings of the International Conference onAnaerobic Treatment: A Grown-up Technology, Amsterdam,The Netherlands, 1986; pp 89-101.

Accepted October 5, 1998.

BP980093C


Documents

Psychrophilic (6-15 °C) High-Rate Anaerobic Treatment of Malting