Intrinsic kinetic parameters of substrate utilization by immobilized anaerobic sludge

This article presents a method for evaluating the intrinsic kinetic parameters of the specific substrate utilization rate (r) equation and discusses the results obtained for anaerobic sludge-bed samples taken from a horizontal-flow anaerobic immobilized sludge (HAIS) reactor. This method utilizes a differential reactor filled with polyurethane foam matrices containing immobilized anaerobic sludge which is subjected to a range of feeding substrate flow rates. The range of liquid superficial velocities thus obtained are used for generating data of observed specific substrate utilization rates (r(obs)) under a diversity of external mass transfer resistance conditions. The r(obs) curves are then adjusted to permit their extrapolation for the condition of no external mass transfer resistance, and the values determined are used as a test for the condition of absence of limitation of internal mass transfer. The intrinsic parameters r(max), the maximum specific substrate utilization rate, and K(s), the half-velocity coefficient, are evaluated from the r values under no external mass transfer resistance and no internal mass transfer limitation. The application of such a method for anaerobic sludge immobilized in polyurethane foam particles treating a glucose substrate at 30 degrees C resulted in intrinsic r(max) and K(s), respectively, of 0.330 mg chemical oxygen demand (COD) . mg(-1) volatile suspended solids (VSS) . h(-1) and 72 mg COD . L(-1). In comparison with the values found in the literature, intrinsic r(max) is significantly high and intrinsic K(s) is relatively low. (c) 1997 John Wiley & Sons, Inc.     

Performance of a horizontal-flow anaerobic immobilized biomass (HAIB) reactor and dynamics of the microbial community during degradation of pentachlorophenol (PCP)

The anaerobic biological treatment of pentachlorophenol (PCP) and methanol as the main carbon source was investigated in a horizontal-flow anaerobic immobilized biomass (HAIB) reactor at 30+/-1 degrees C, during a 220-day trial period. The reactor biomass was developed as an attached biofilm on polyurethane foam particles, with 24h of hydraulic retention time. The PCP concentrations, which ranged from 2.0 to 13.0 mg/L, were controlled by adding synthetic substrate. The HAIB reactor reduced 97% of COD and removed 99% of PCP. The microbial biofilm communities of the HAIB reactor amended with PCP, without previous acclimatization, were characterized by polymerase chain reaction (PCR) and amplified ribosomal DNA restriction analysis (ARDRA) with specific Archaea oligonucleotide primers. The ARDRA technique provided an adequate analysis of the community, revealing the profile of the selected population along the reactor. The biomass activities in the HAIB reactor at the end of the experiments indicated the development of PCP degraders and the maintenance of the population of methanogenic Archaea, ensuring the high efficiency of the system treating PCP with added methanol as the cosubstrate. The use of the simplified ARDRA method enabled us to monitor the microbial population with the addition of high concentrations of toxic compounds and highlighting a selection of microorganisms in the biofilm.     

High-rate treatment of terephthalate in anaerobic hybrid reactors.

The anaerobic degradation of terephthalate as sole substrate was studied in three anaerobic upflow reactors. Initially, the reactors were operated as upflow anaerobic sludge bed (UASB) reactors and seeded with suspended methanogenic biomass obtained from a full-scale down-flow fixed film reactor, treating wastewater generated during production of purified terephthalic acid. The reactors were operated at 30, 37, and 55 degrees C. The terephthalate removal capacities remained low in all three reactors (<4 mmolxL-1xday-1, or 1 g of chemical oxygen demand (COD)xL-1xday-1) due to limitations in biomass retention. Batch experiments with biomass from the UASB reactors revealed that, within the mesophilic temperature range, optimal terephthalate degradation is obtained at 37 degrees C. No thermophilic terephthalate-degrading culture could be obtained in either continuous or batch cultures. To enhance biomass retention, the reactors were modified to anaerobic hybrid reactors by introduction of two types of reticulated polyurethane (PUR) foam particles. The hybrid reactors were operated at 37 degrees C and seeded with a mixture of biomass from the UASB reactors operated at 30 and 37 degrees C. After a lag period of approximately 80 days, the terephthalate conversion capacity of the hybrid reactors increased exponentially at a specific rate of approximately 0.06 day-1, and high removal rates were obtained (40-70 mmolxL-1xday-1, or 10-17 g of CODxL-1xday-1) at hydraulic retention times between 5 and 8 h. These high removal capacities could be attributed to enhanced biomass retention by the development of biofilms on the PUR carrier material as well as the formation of granular biomass. Biomass balances over the hybrid reactors suggested that either bacterial decay or selective wash-out of the terephthalate fermenting biomass played an important role in the capacity limitations of the systems. The presented results suggest that terephthalate can be degraded at high volumetric rates if sufficiently long sludge ages can be maintained, and the reactor pH and temperature are close to their optima.     

Evaluation of the microbial diversity in a horizontal-flow anaerobic immobilized biomass reactor treating linear alkylbenzene sulfonate

The purpose of this work was to assess the degradation of linear alkylbenzene sulfonate (LAS) in a horizontal-flow anaerobic immobilized biomass (HAIB) reactor. The reactor was filled with polyurethane foam where the sludge from a sanitary sewage treatment was immobilized. The hydraulic detention time (HDT) used in the experiments was of 12 h. The reactor was fed with synthetic substrate (410 mg l⁻¹ of meat extract, 115 mg l⁻¹ of starch, 80 mg l⁻¹ of saccharose, 320 mg l⁻¹ of sodium bicarbonate and 5 ml l⁻¹ of salt solution) in the following stages of operation: SI—synthetic substrate, SII—synthetic substrate with 7 mg l⁻¹ of LAS, SIII—synthetic substrate with 14 mg l⁻¹ of LAS and SIV—synthetic substrate containing yeast extract (substituting meat extract) and 14 mg l⁻¹ of LAS, without starch. At the end of the experiment (313 days) a degradation of ∼35% of LAS was achieved. The higher the concentration of LAS, the greater the amount of foam for its adsorption. This is necessary because the isotherm of LAS adsorption in the foam is linear for the studied concentrations (2 to 50 mg l⁻¹). Microscopic analyses of the biofilm revealed diverse microbial morphologies, while Denaturing Gradient Gel Eletrophoresis (DGGE) profiling showed variations in the population of total bacteria and sulphate-reducing bacteria (SRB). The 16S rRNA gene sequencing and phylogenetic analyses revealed that the members of the order Clostridiales were the major components of the bacterial community in the last reactor operation step.     

BTEX removal in a horizontal-flow anaerobic immobilized biomass reactor under denitrifying conditions

Because benzene, toluene, ethylbenzene, and xylenes (BTEX) and ethanol are important contaminants present in Brazilian gasoline, it is essential to develop technology that can be used in the bioremediation of gasoline-contaminated aquifers. This paper evaluates the performance of a horizontal-flow anaerobic immobilized biomass (HAIB) reactor fed with water containing gasoline constituents under denitrifying conditions. Two HAIB reactors filled with polyurethane foam matrices (5 mm cubes, 23 kg/m³ density and 95 % porosity) for biomass attachment were assayed. The reactor fed with synthetic substrate containing protein, carbohydrates, sodium bicarbonate and BTEX solution in ethanol, at an Hydraulic retention time (HRT) of 13.5 h, presented hydrocarbon removal efficiencies of 99 % at the following initial concentrations: benzene 6.7 mg/L, toluene 4.9 mg/L, m-xylene and p-xylene 7.2 mg/L, ethylbenzene 3.7 mg/L, and nitrate 60 mg N/L. The HAIB reactor fed with gasoline-contaminated water at an HRT of 20 h showed hydrocarbon removal efficiencies of 96 % at the following initial concentrations: benzene, 4.9 mg/L; toluene, 7.2 mg/L; m-xylene, 3.7 mg/L; and nitrate 400 mg N/L. Microbiological observations along the length of the HAIB reactor fed with gasoline-contaminated water confirmed that in the first segment of the reactor, denitrifying metabolism predominated, whereas from the first sampling port on, the metabolism observed was predominantly methanogenic.     

High efficiency of a coupled aerobic-anaerobic recycling biofilm reactor system in the degradation of recalcitrant chloroaromatic xenobiotic compounds

Chloroaromatic compounds are xenobiotics that cause great concern. The degradation of a model molecule, 3,4-dichlorobenzoate (3,4-DCB), was studied using three aerobic (AE)-anaerobic (AN) biofilm reactor systems: a coupled aerobic-anaerobic recycle biofilm reactor (CAR) system, an in-series anaerobic-aerobic biofilm reactor (SAR) system; and an independent aerobic and anaerobic biofilm reactor (IAR) system. In all three systems the inlet substrate concentration was 2.0 g/l and the dilution rates ranged from 0.045 to 0.142 per hour. The results show that the degradation efficiency of the CAR system (expressed as dechlorination and xenobiotic disappearance efficiencies, and biomass yield), was higher at all dilution rates tested than in both SAR and IAR systems. Moreover, dechlorination and xenobiotic disappearance efficiencies for resting suspended aerobic and anaerobic cells or mixed aerobic-anaerobic growing cells under anaerobic conditions were higher than under aerobic conditions. These results suggest that a “cooperative metabolism” between aerobic and anaerobic bacteria (caused by an exchange of cells and metabolites between AE and AN reactors) in the CAR system overcame the metabolic and kinetic limitations of aerobic and anaerobic bacteria in the AE and AN reactors of IAR and SAR systems. Therefore, the degradation efficiency of persistent and recalcitrant chloroaromatic xenobiotic compounds could be enhanced by using a CAR system. Received: 1 March 1999 / Received revision: 11 May 1999 / Accepted: 16 May 1999     

Kinetics of BTEX degradation in a packed-bed anaerobic reactor

The ever-increasing diversity of industrial activity is responsible for the discharge of compounds that are toxic or difficult to degrade into the environment. Some of the compounds found in surface and ground waters, usually deriving from the contamination of oil-based products, are benzene, toluene, ethylbenzene and xylenes (BTEX). To remove these compounds from contaminated water, a bench-scale horizontal-flow anaerobic immobilized biomass reactor, containing anaerobic biomass from various sources immobilized in polyurethane foam matrices, was employed to treat a synthetic substrate composed of protein, carbohydrates and BTEX solution in ethanol, as well as a BTEX solution in ethanol as the sole carbon source. The reactor removed up to 15.0 mg/l of each BTEX compound over a hydraulic detention time of 11.4 h. A first-order kinetic model fitted the experimental data well, showing correlation coefficients higher than 0.994. The apparent first-order coefficient values, , ranged from 8.4±1.5 day⁻¹ for benzene to 10.7±1.4 day⁻¹ for o-xylene in the presence of ethanol, protein and carbohydrates, and from 10.0±2.0 day⁻¹ for benzene to 13.0±1.7 day⁻¹ for o-xylene in the presence of ethanol. The BTEX degradation rates estimated here were 10- to 94-fold higher than those found in reports on microcosm studies.     

The effect of low temperature (5–29 °C) and adaptation on the methanogenic activity of biomass

The influence of low temperature (5–29 °C) on the methanogenic activity of non-adapted digested sewage sludge and on temperature/leachate-adapted biomass was assayed by using municipal landfill leachate, intermediates of anaerobic degradation (propionate) and methane precursors (acetate, H₂/CO₂) as substrates. The temperature dependence of methanogenic activity could be described by Arrhenius-derived models. However, both substrate and adaptation affected the temperature dependence. The adaptation of biomass in a leachate-fed upflow anaerobic sludge-blanket reactor at approximately 20 °C for 4 months resulted in a sevenfold and fivefold increase of methanogenic activity at 11 °C and 22 °C respectively. Both acetate and H₂/CO₂ were methanized even at 5 °C. At 22 °C, methanogenic activities (acetate 4.8–84 mM) were 1.6–5.2 times higher than those at 11 °C. The half-velocity constant (K _s) of acetate utilization at 11 °C was one-third of that at 22 °C while a similar K _i was obtained at both temperatures. With propionate (1.1–5.5 mM) as substrate, meth‐anogenic activities at 11 °C were half those at 22 °C. Furthermore, the residual concentration of the substrates was not dependent on temperature. The results suggest that the adaptation of biomass enables the achievement of a high treatment capacity in the anaerobic process even under psychrophilic conditions. Received: 23 December 1996 / Received last revision: 18 June 1997 / Accepted: 23 June 1997     

Method for estimating the kinetics of substrate degradation in horizontal-flow anaerobic immobilized sludge reactors

Apparent and intrinsic kinetic parameters of substrate decomposition were estimated from the profiles of chemical oxygen demand (COD) and volatile fatty acids (VFA) concentrations along the length of the horizontal-flow anaerobic immobilized sludge (HAIS) reactor. The first order substrate utilization kinetic model describes well the experimental data.     

Estimation of intrinsic kinetic parameters in immobilised cell systems for anaerobic wastewater treatment

Summary Intrinsic kinetic parameters for anaerobic sludge immobilised in polyurethane foam matrices treating a glucose based substrate were estimated under negligible mass transfer resistance in shaker incubator experiments. Micro-granules detachment due to high rotational agitation speeds was found to affect the results.     

Treatment of cyanide-containing wastewater from the food industry in a laboratory-scale fixed-bed methanogenic reactor

During the process of producing cassava starch from Manihot esculenta roots, large amounts of cyanoglycosides were released, which rapidly decayed to CN⁻ following enzymatic hydrolysis. Depending on the varying cyanoglycoside content of the cassava varieties, the cyanide concentration in the wastewater was as high as 200 mg/l. To simulate anaerobic stabilization, a wastewater with a chemical oxygen demand (COD) of about 20 g/l was prepared from cassava roots and was fermented in a fixed-bed methanogenic reactor. The start-up phase for a 99% degradation of low concentrations of cyanide (10 mg/l) required about 6 months. After establishment of the biofilm, a cyanide concentration of up to 150 mg CN⁻/l in the fresh wastewater was degraded during anaerobic treatment at a hydraulic retention time of 3 days. All nitrogen from the degraded cyanide was converted to organic nitrogen by the biomass of the effluent. The cyanide-degrading biocoenosis of the anaerobic reactor could tolerate shock concentrations of cyanide up to 240 mg CN⁻/l for a short time. Up to 5 mmol/l NH₄Cl (i.e. 70 mg N/l = 265 mg NH₄Cl/l) in the fresh wastewater did not affect cyanide degradation. The bleaching agent sulphite, however, had a negative effect on COD and cyanide removal. For anaerobic treatment, the maximum COD space loading was 12 g l⁻¹ day⁻¹, equivalent to a hydraulic retention time of 1.8 days. The COD removal efficiency was around 90%. The maximum permanent cyanide space loading was 50 mg CN⁻ l⁻¹ day⁻¹, with tolerable shock loadings up to 75 mg CN⁻ l⁻¹day⁻¹. Under steady-state conditions, the cyanide concentration of the effluent was lower than 0.5 mg/l. Received: 15 August 1997 / Received revision: 10 October 1997 / Accepted: 14 October 1997     

Effects of support material on the pattern of volatile fatty acid accumulation at overload in anaerobic digestion of semi-solid waste

Anaerobic degradation of a semi-solid waste with a total solids content of 4% particulate matter, much of it insoluble, was investigated in four laboratory-scale reactors. Two of the reactors were equipped with different textile materials for immobilisation of microorganisms, while the other two were used as continuously-stirred-tank reactor references. A constant organic loading rate and hydraulic retention time were used in the start-up period; the hydraulic retention time was then decreased and the effects of this change were monitored. Volatile fatty acid (VFA) concentration and pH were chosen as indicators of the microbial status in the reactors. The reactors with support material showed a greater resistance to overload than did the continuously-stirred-tank reactors. This is in agreement with many studies undertaken on the anaerobic treatment of wastewater. However, no problems with clogging occurred, showing that a support material is also applicable in systems treating waste containing large amounts of insoluble, particulate matter. The pH was comparable to VFA for indicating an approaching process failure. However, the pattern of VFA accumulation was qualitatively different between the reactors with and without support material. Obviously the metabolic pattern of mixed cultures changes when the microorganisms are immobilised. Received: 3 December 1996 / Received revision: 7 February 1997 / Accepted: 14 February 1997     

Immobilization of Burkholderia cepacia in polyurethane-based foams: embedding efficiency and effect on bacterial activity

Immobilization of the trichloroethylene-degrading bacterium Burkholderia cepacia was evaluated using hydrophilic polyurethane foam. The influence of several foam formulation parameters upon cell retention was examined. Surfactant type was a major determinant of retention; a lecithin-based compound retained more cells than pluronic- or silicone-based surfactants. Excessive amounts of surfactant led to increased washout of bacteria. Increasing the biomass concentration in the foam from 4.8 to 10.5% dry weight per wet weight of foam resulted in fewer cells being washed out. Embedding at reduced temperature did not significantly affect retention, while the use of a silane binding agent gave inconsistent results. The optimal formulation retained all but 0.2% of total embedded cells during passage of 2 L of water through columns containing 2 g of foam. All foam formulations tested reduced the culturability of embedded cells by several orders of magnitude, but O₂ consumption and CO₂ evolution rates of embedded cells were never less than 50% of those of free cells. Nutrient amendments stimulated an increase in cell volume and ribosomal activity in immobilized cells as indicated by hybridization studies using fluorescently labeled ribosomal probes. These results indicate that, although immobilized cells were mostly nonculturable, they were metabolically active and thus could be used for biodegradation of toxic compounds. Received 23 December 1996/ Accepted in revised form 13 March 1997     

Effect of feed composition and upflow velocity on aggregate characteristics in anaerobic upflow reactors

Two upflow anaerobic hybrid reactors treated lactose and a mixture of ethanol, propionate and butyrate, respectively, at a volumetric loading rate of 3.7 kg chemical oxygen demand (COD) m⁻³day⁻¹, a hydraulic retention time of 5 days and a liquid upflow velocity of 0.01 m/h. Under steady-state conditions, the lactose-fed sludge had much higher (20%–100%) specific methanogenic conversion rates than the volatile-fatty acid␣(VFA)/ethanol-fed sludge for all substrates tested, including VFA. In both reactors, a flocculant sludge developed, although a much higher content of extracellular polysaccharide was measured in the lactose-fed sludge [1900 μg compared to 305 μg uronic acid/g volatile suspended solids (VSS)]. When the liquid upflow velocity of a third, VFA/ethanol-fed reactor was increased to 0.5 m/h, granulation of the sludge occurred, accompanied by a large increase (200%–500%) in the specific methanogenic conversion rates for the syntrophic and methanogenic substrates studied. Granulation reduced the susceptibility of the sludge to flotation. Glucose was degraded at a high rate (100 mg glucose gVSS⁻¹h⁻¹) by the sludge from the third reactor, despite not having been exposed to a sugar-containing influent for 563␣days. Received: 7 June 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Transformation of 1,1,1-trichloroethane in an anaerobic packed-bed reactor at various concentrations of 1,1,1-trichloroethane, acetate and sulfate

Biotransformation of 1,1,1-trichloroethane (CH₃CCl₃) was observed in an anaerobic packed-bed reactor under conditions of both sulfate reduction and methanogenesis. Acetate (1 mM) served as an electron donor. CH₃CCl₃ was completely converted up to the highest investigated concentration of 10 μM. 1,1-Dichloroethane and chloroethane were found to be the main transformation products. A fraction of the CH₃CCl₃ was completely dechlorinated via an unknown pathway. The rate of transformation and the transformation products formed depended on the concentrations of CH₃CCl₃, acetate and sulfate. With an increase in sulfate and CH₃CCl₃ concentrations and a decrease in acetate concentration, the degree of CH₃CCl₃ dechlorination decreased. Both packed-bed reactor studies and batch experiments with bromoethanesulfonic acid, an inhibitor of methanogenesis, demonstrated the involvement of methanogens in CH₃CCl₃ transformation. Batch experiments with molybdate showed that sulfate-reducing bacteria in the packed-bed reactor were also able to transform CH₃CCl₃. However, packed-bed reactor experiments indicated that sulfate reducers only had a minor contribution to the overall transformation in the packed-bed reactor. Received: 22 January 1997 / Received revision: 12 May 1997 / Accepted: 19 May 1997     

Influence of the starting microbial nucleus type on the anaerobic granulation dynamics

 The influence of four different granulation precursors, syntroph-enriched methanogenic consortia, Methanosaeta-enriched, Methanosarcina-enriched nuclei and acidogenic flocs, on the time course of complex granule development and the lag time for start-up was investigated in four upflow anaerobic sludge-bed and filter reactors. Although the operational conditions allowed the maintenance of the same specific growth rate of biomass in the four reactors, granulation proceeded rapidly with syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei. However, granulation was significantly retarded when acidogenic flocs were used as precursors. The granule mean Sauter diameter increased rapidly in the reactor inoculated with syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei and reached, at the end of the experiment, 3.1, 2.7 and 2.4 mm compared to 1.1 mm in that inoculated with acidogenic flocs. This corresponded to a rate of granule size increase of 31, 21, 18 μm/day in syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei, respectively, compared to 7 μm/day in acidogenic flocs. Biomass specific activities (i.e. acidogenic, syntrophic and methanogenic activities) increased stepwise in all reactors with time, especially in those inoculated with syntroph/methanogenic consortia and Methanosaeta nuclei. From these results it appears that syntrophs and Methanosaeta spp. play an important role in the anaerobic granulation process. Received: 25 January 1996 / Received revision: 3 September 1996 / Accepted: 13 September 1996     

Bioreactor with a semi-fixed packing: anaerobic lactose to lactic acid fermentation

The bioreactor with a semi-fixed packing consists of frames with stretched “Raschell”-type sacks on them. This construction enables the attainment of a larger interfacial area of the biofilm carrier per unit reactor volume as well as the easy removal of the exausted biomass at certain biofilm thickness. In the present paper the batch anaerobic conversion of lactose to lactic acid in this reactor with immobilized bacteria Lactobacillus casei is studied. The dilution rate of the feeding substrate solution was 1?mm/s. Comparison of our experimental results with data, obtained for free cells and other construction of bioreactors with immobilized cells is made. It is shown that the used immobilised biocatalyst is superior to free bacterial cells, or attached to the inner pores of polyurethane foam.     

Presence or absence of picophytoplankton in the western Ross Sea during spring 1994: a matter of size definition?

The occurrence of picophytoplankton in the Ross Sea was investigated during the cruise ROSSMIZE leg 1 (November/December 1994) using epifluorescence microscopy. This study was focused on the methodological aspects dealing with size definition of picophytoplankton. The results showed that a small difference (1 μm) in the definition of the upper size limit of picophytoplankton (2.0 vs 3.0 μm) may have significant implications for the relative importance of this component in the Ross Sea ecosystem. Moreover, the lack of correlation between autotrophs 0.2–2 μm in size and larger autotrophic cells (2–3 μm) in the marginal ice zone and in the closed pack-ice zone suggests that these two components have different ecological roles that are emphasized in these environmental conditions. Received: 25 August 1997 / Accepted: 5 January 1998     

Transformation of tetrachloroethene in an upflow anaerobic sludgeblanket reactor

  Reductive dechlorination of tetrachloroethene was studied in a mesophilic upflow anaerobic sludge blanket reactor. Operating the reactor in batch mode the dynamic transformation of tetrachloroethene, trichloroethene and dichloroethene (DCE) was monitored. Tetrachloroethene was reductively dechlorinated to trichloroethene, which again was dechlorinated at the same rate as DCE was produced. DCE showed a lag period of 40 h before transformation was observed. During normal reactor operation trans-1,2-DCE was the major DCE isomer, followed by cis-1,2-DCE. Small amounts of 1,1-DCE but no vinyl chloride were detected. When the influent tetrachloroethene concentration was increased from 4.6 μM to 27 μM, the transformation rate increased, indicating that the system was not saturated with tetrachloroethene. The main organic component in the effluent was acetate, indicating that the aceticlastic methane-producing bacteria were inhibited by the chlorinated ethenes. Received: 29 July 1996 / Received revision: 13 September 1996 / Accepted: 13 September 1996     

Granular sludge growth under different reactor liquid surface tensions in lab-scale upflow anaerobic sludge blanket reactors treating wastewater from sugar-beet processing

The formation of anaerobic granular sludge on wastewater from sugar-beet processing was examined in upflow anaerobic sludge blanket reactors. Two strategies were investigated: addition of high-energy substrate, i.e. sugars, and varying the reactor liquid surface tension. When there were insufficient amounts of sugars i.e. less than 7% of the chemical O₂ demand of the influent, no granulation was observed; moreover lowering the reactor liquid surface tension below 48 mN/m was found to increase biomass wash-out. On the other hand, when there were sufficient sugars, granular sludge growth occurred; moreover operating the reactor at a low reactor liquid surface tension reduced biomass wash-out and increased granular yield.