Anaerobic digestion of dairy wastewater by inverse fluidization: The inverse fluidized bed and the inverse turbulent bed reactors

Depósito de Investigación de la Universidad de Sevilla https://idus.us.es/ This is an Accepted Manuscript of an article published by Taylor & Francis in Environmental Technology, Volume 24, on November 2003, available at https://doi.org/10.1080/09593330309385687 © 2003 Selper Ltd. En idUS Licencia Creative Commons CC BY-NC-ND ANAEROBIC DIGESTION OF DAIRY WASTEWATER BY INVERSE FLUIDIZATION: THE INVERSE FLUIDIZED BED AND THE INVERSE TURBULENT BED REACTORS C. Arnaiz *1 , P. Buffiere 2 , S. Elmaleh 3 , J. Lebrato 4 and R. Moletta 2 . 1 Departamento de Ingeniería Química y Ambiental. Escuela Universitaria Politécnica. Universidad de Sevilla. Virgen de Africa 7, 41011 Sevilla, Spain. 2 Laboratoire de Biotechnologie de l'Environnement, INRA. Avenue des Etangs, 11100 Narbonne, France. 3 Groupe Génie des Procédés. Université de Montpellier II. CC 024, 34095 Montpellier Cedex 5, France. 4 Grupo Tratamiento de Aguas Residuales. Escuela Universitaria Politécnica. Universidad de Sevilla. Virgen de Africa 7, 41011 Sevilla, Spain. * Corresponding author: Carmen Arnaiz. Departamento de Ingeniería Química y Ambiental. Escuela Universitaria Politécnica. Universidad de Sevilla. Virgen de Africa 7, 41011 Sevilla, Spain. Fax: +34-95-4282777; e-mail: [email protected] ANAEROBIC DIGESTION OF DAIRY WASTEWATER BY INVERSE FLUIDIZATION: THE INVERSE FLUIDIZED BED AND THE INVERSE TURBULENT BED REACTORS ABSTRACT This paper describes the application of the inverse fluidization technology to the anaerobic digestion of dairy wastewater. Two reactors were investigated: the inverse fluidized bed reactor and the inverse turbulent reactor. In these reactors, a granular floating solid is expanded by a down-flow current of effluent or an up-flow current of gas, respectively. The carrier particles (Extendospheres (TM) ) were chosen for their large specific surface area (20,000 m 2 m -3 ) and their low energy requirements for fluidization (gas velocity of 1.5 mm s -1 , 5.4 m h - 1 ). Organic load was increased stepwise by reducing hydraulic retention time from more than 60 days to 3 days, while maintaining constant the feed COD concentration. Both reactors achieved more than 90% of COD removal, at an organic loading rate of 10–12 kg COD m -3 d -1 , respectively. The performances observed were similar or even higher than that of other previously tested fluidized bed technologies treating the same wastewater. It was found that the main advantages of this system are: low energy requirement, because of the low fluidization velocities required; there is no need of a settling device, because solids accumulate at the bottom of the reactor, so they can be easily drawn out and particles with high-biomass content can be easily recovered. Lipid phosphate concentration has been revealed as a good method for biomass estimation in biofilms since it only includes living biomass. Keywords: anaerobic process, wastewater treatment, granular floating carrier, inverse fluidization technology, phospholipid analysis NOMENCLATURE ALP attached lipid phosphate (nmol ml -1 ) ASBR anaerobic sequencing batch reactor COD chemical oxygen demand (kg m -3 ) CSTR continuously stirred tank reactor d p particle diameter (µm) FB up-flow fluidized bed reactor HRT hydraulic retention time (d) IFB inverse fluidized bed reactor ITB inverse turbulent bed reactor OHPA obligate hydrogen producing acetogens bacteria OLR organic loading rate (kg COD m -3 d -1 ) PVC polyvinyl chloride SLP suspended lipid phosphate (nmol ml -1 ) SSA specific surface area (m 2 m -3 ) TOC total organic carbon (kg m -3 ) TSS total suspended solids (mg ml -1 ) U mf minimum fluidization velocity (m h -1 ) VAS volatile attached solids (mg ml -1 CARRIER ) VFA volatile fatty acids (kg m -3 ) VSS volatile suspended solids (mg ml -1 ) INTRODUCTION Anaerobic digestion offers significant advantages over aerobic systems, like low energy consumption, reduced solids formation, low nutrient requirement and potential energy recovery from the methane produced [1]. This process is now widely used in many environmental applications, in different configurations and modes of operation. However, anaerobic digestion has a low growth rate of anaerobic bacteria and requires longer hydraulic retention time (HRT), which means longer residence time of the liquid phase, to achieve satisfactory degradation when compared to aerobic treatment [2]. In the last decades, the development of various techniques like fixed or fluidized beds for the retention of the anaerobic microorganisms inside the reactor has enabled the shortening of HRT and the increasing of organic loading rate (OLR). Literature is rich in successful reports on this technology. It is used either for aerobic treatment [3,4], or anaerobic treatment such as denitrification [5] and anaerobic digestion [6-7]. In the field of anaerobic digestion, however, it has not been extensively used at full-scale. In traditional fluidization or up-flow fluidization, the solid particles have a density greater than the liquid and they are fluidized by a liquid stream flowing in the opposite direction of gravity. The lack of industrial success of up-flow fluidized beds may result from a combination of several negative points: a high level of maintenance because of their complexity, the need for liquid recycling, or hydrodynamic problems [8]. More recently, investigations were carried out on inverse fluidized beds (IFB) or downflow fluidized beds, applied to aerobic wastewater treatment [3,9] or anaerobic wastewater treatment [10]. In this kind of reactors, particles with a density lower than the liquid that float when IFB is standing, are fluidized by a down-flow current of liquid when it is working (Figure 1a). This technology presents interesting features: the down-flow configuration enables overcoated particles to be recovered in the bottom of the bed. Moreover, the liquid and the produced biogas are flowing in opposite directions, which helps for bed expansion [11]. The expansion of a carrier with a specific density lower than the liquid is also possible under an up-flow current of gas only, resulting in a new reactor configuration: the inverse turbulent bed (ITB) (Figure 1b). The gas bubbles generate downward liquid motions and apparent bed expansion. This phenomenon (called pseudo-fluidization) has already been identified by several researchers [12-14]. If the density of the particles remains smaller than but close to that of the liquid, the fluidization can be achieved with only an upward gas flow, counter to the liquid flow [15]. Fluidization in gas-liquid-solid systems where the gas is the continuous phase is mainly a system of cross-current up-flow of the gas with the liquid. It has application in, for instance, biotechnology [16] and catalytical chemical processes. Theoretically, this kind of reactor may enable a stable control of the bed height, since the particles cannot settle below the gas injection zone because of their density. Additional data on hydrodynamics of IFB and ITB used in this study can be found in [8], [10] and [17]. This work compares an IFB and an ITB performance, both treating synthetic dairy wastewater, with a carrier material that allows low energy requirement for fluidization, providing also a good surface for biomass attachment and development. MATERIALS AND METHODS Laboratory-scale reactors The experimental set-up used in this study is showed in Figure 2. The reactors consist in a PVC tubular section of 0.08 m internal diameter and 1 m height with a conic bottom, with a total volume of 5 l. Initial volumic fractions of gas, liquid and solid phase were 0.22, 0.12 and 0.66, respectively. The system is equipped with a water jacket keeping the liquid temperature at 35 ºC ± 2. The biogas production is measured by a gas flow meter (Sho-Rate TM ). The reactors were monitored for pH and addition of NaOH is provided during the start-up period. Effluent is discharged through a port on the low part of the column, connected to an outlet tube that kept the liquid level in the reactor. Recycling of the liquid in the IFB and recycling of the biogas in the ITB was ensured by a peristaltic pump (Masterflex Cole Parmer). Influent composition The substrate used in this work is a synthetic dairy wastewater containing lactoserum with a total organic carbon concentration (TOC) of 12 kg m -3 , equivalent to a chemical organic demand (COD) of 30 kg m -3 . Lactoserum is a substrate easily biodegradable which is quickly transformed in VFA by acidogenic bacteria. This is a characteristic of some wastewater from food industry. The average characteristics of the influent are given in Table 1. Physical properties of the carrier material Table 2 shows physical properties of the support material used in this work. Commercially-available Extendosphere (TM) (provided by PQ Hollowsphere, Ltd.) is a light mineral granular material mainly composed of silica. The shape of the particle is perfectly spherical, and the surface of the material present small crevices. Average particle diameter is 175 µm. Reactors inoculation and start-up The reactors were filled with the solid carrier material up to 40% of their active volume (working volume). It requires a low gas velocity for being expanded (1.5 mm s -1 ). The reactors were inoculated with sludge from an anaerobic pond treating dairy wastewater up to 25% of the reactor total volume. Synthetic influent was kept in a refrigerator to avoid fermentation and it was constantly agitated by a magnetic stirrer to ensure homogenization. During the first two weeks of acclimatization of the ITB reactor, a constant liquid recycling from the bottom to the top of the reactor was kept in order to provide a good contact between the (settling) biomass and the (floating) solid carrier and was shut down when a consistent biogas production began. The reactors were monitored for temperature, flow rate, pH and biogas production. Biogas composition, total suspended solids (TSS), volatile suspended solids (VSS), suspended lipid phosphate (SLP), volatile attached solids to the carrier (VAS), attached lipid phosphate to the carrier (ALP), volatile fatty acids (VFA) and TOC were routinely analyzed. HRT, based on expanded bed volume, was fixed at 66 days and it was reduced stepwise to 3 days at the end of the study. Measurements and analysis VFA and TOC of the discharged effluent and biogas composition were determined daily through off-line analysis. Liquid samples were centrifuged at 10,000 rpm for 10 min before analysis to remove suspended solids. VFA analysis was done using a gas chromatograph with a flame ionization detector Chrompack CP 9000, nitrogen being the carrier gas (335 kPa). The column was a semi capillar Econocap FFAP (15 m length and 0.53 mm diameter). Injector and detector temperatures were 250 °C and 275 °C respectively. The temperature of the oven was programmed to rise from 80 °C to 120 °C during the analysis with an elevation of 10 °C per minute. The chromatograph was coupled with an integrator Shimadzu CR3A. TOC was titrated by UV oxidation with a Dohrman DC 80 apparatus. Carbon compounds were oxidized in potassium persulfate at low temperature and the formed carbon dioxide was detected by infrared absorption. Samples were diluted twice with orthophosphoric acid at 10%. The carbon dioxide contained in the samples was previously eliminated by bubbling oxygen gas for 2 min. Gas was analyzed by gas chromatography with a Shimadzu GC-8A apparatus with argon carrier (3 bar) using a catharometer detector (90 mA). CO 2 and NO 2 were separated in a Hayesep Q precolumn (80 ¯ 100 mesh, 2 m×1/8 inch); O 2 , H 2 , N 2 and CH 4 were separated in a second column with a molecular sieve 5 Å (80 ¯ 100 mesh, 2 m × 1/8 inch). Oven temperature was 35 °C; temperature of both injector and detector was 100 °C. The chromatograph was coupled to a Shimadzu CR5A integrator. pH was measured with a Mettler Toledo 1100 Calimatic pH meter. Biomass determination TSS and VSS in the effluent were measured according to Standard Methods [18], understanding that TSS is the portion of total solid retained by the filter and that VSS is the residue of TSS after ignition. Biofilm development within the reactor was measured by determining VAS to the carrier on washed samples of bioparticles. The procedure used in this study in order to determine SLP and ALP was a modification of that found in [19]. For ALP, samples of biomass-laden particles are withdrawn from the reactor and gently washed with distilled water to remove any unattached biomass. The procedure consisted of: (a) 3 ml of effluent for SLP and 0.5-1 ml of bioparticles for ALP, were added into 70 ml screw-cap test tubes. Then, 20 ml of chloroform, 20 ml of methanol and 20 ml of deionized water were added to the samples. The extraction mixture was gently shaken for 10 minutes and allowed to stand up to complete phase separation. (b) To facilitate recovery of the chloroform, the aqueous (upper) phase was aspirated from the test tubes with the aid of a vacuum pump and subsamples of 5 ml of the chloroform layer were transferred into 10 ml screw-cap test tubes. At this point, lipids can be stored at –20 ºC. (c) The chloroform was removed under a stream of nitrogen and phosphate was liberated from lipids by adding 2.7 ml of a potassium persulfate solution (5 g added to 100 ml of 0.36 N H 2 SO 4 ) and the sealed test tubes were heated in an oven at 105 ºC for 1 h. (d) Phosphate release by persulfate digestion was determined by adding 0.6 ml of a ammonium molybdate solution (2.5% of (NH 4 ) 6 Mo 7 O 24 ⋅ 4H 2 O in 5.72 N H 2 SO 4 ) (allowed to stand for 10 min) and 2.7 ml of a malachite green solution absorbed organic matter on flocs and biofilms. Phospholipids, present on bacterial membrane up to 90-98%, do not form part of cell reserves and are easily degrade during bacteria lysis [30]. Therefore, their estimation only includes living biomass. When relation between these two measures is calculated (VAS/ALP and VSS/SLP), an estimation of living biomass with regards to total biomass is obtained. As it shows in Figure 6a-6b, the amount of attached biomass for the IFB (up to 8 mg ml - 1 ) was higher than for ITB (never above 6 mg ml -1 ) at similar OLR, which could result in a thinner biofilm in the ITB (probably because of a higher level of biomass attrition) and higher diffusion rate. In the same way, suspended biomass within IFB (up to 25.0 mg ml -1 ) was higher than within ITB (never above 5.0 mg ml -1 ) at similar OLR (Figure 6c-6d), which could result in blocking of pipes in full-scale reactors. In fact, several problems of clogging took place in IFB during the study. We also concluded from these data that volatile solid is a gross method of biomass estimation since higher solids concentration does not means higher living biomass or higher biological activity. It is important to point out a better overall carbon removal rate in ITB in spite of a total biomass (measured as volatile solids or lipid-phosphate concentration) much lower than in the IFB. It can be explain if a more active biomass is admitted. On the other hand, not always there has to be a relation between the concentration of biomass and the parameters of performance of a reactor. A priori, it seems clear that in a system in which the organic matter is degraded only and exclusively by the action of microorganisms, the consumption rate of substratum and the growth or production rate of microbial cell mass must be deeply related. Nevertheless, several factors make these rates not proportional: it is necessary to take into account that the microorganisms can not be considered chemical reactors since they present the capability of adaptation, and population displacements can occur. This aspect can be very important in industrial wastewater treatment, in which very specific substrata could select very specific groups of bacteria, progressively more active. An important point of the relationship between total biomass amount measured as volatile solids and total biomass amount measured as lipid-phosphate concentration is that is not of y = ax type, as it could be expected, but of y = ax + b type (Figure 6a-6d): when living biomass is completely stabilized, there is still into the reactors volatile solids in suspension and within the biofilm. Biomass consist of a biodegradable fraction and by an inert fraction, which cannot be biologically degradated. This inert fraction is constituted by dead-end products [31,32] and rest of extracellular matrix. Based on data of this study, biomass inert fraction would be represented by the ordinate at the y-axe of straight lines showed in Figure 6. It would explain that the higher ordinates were those obtained for attached biomass in both reactors, since exopolymer matrix is more abundant in biofilms, up to 80% of volatile solids [33]. Theoretical ratio between lipid phosphate concentration and volatile solids concentration is not calculated in this work. This theoretical ratio must be obtained from enrichment cultures of free cells dettached from the biofilms [34]. However, selective enrichments involve cell reproduction and only some of the total active cells are susceptible to regrowth. In addition, the reproducibility of results depends on incubation conditions, biomass type (free cultures or cells from biofilms) and the choice of nutritive medium. Therefore, theoretical ratio obtained by these methods has nothing to do with biomass into the reactors studied [35-37]. However, the similarity between the specific slopes in both reactors (Figure 6) suggests the possibility that these specific slopes are the theoretical ratio, between lipid phosphate concentration and volatile solids concentration, of the specific microbial cell mass into the reactors in the experimental conditions of this work. More data in this sense are needed for general conclusions. CONCLUSIONS This work compared two types of fluidized bed reactors: the inverse fluidized bed (IFB) reactor and the inverse turbulent reactor (ITB). Both of them are characterized by the use of a floating solid carrier for microbial adhesion. Fluidization is ensured by a down-flow current of effluent and by an up-flow current of biogas, respectively. COD removal attained by the IFB and the ITB systems treating synthetic dairy wastewater, can be compared with those obtained from some up-flow anaerobic fluidized bed reactors in similar conditions, and even better than the performance of other anaerobic reactor configurations (10–12 kg COD m -3 d -1 ). However, there is a limit in treating high OLR of synthetic dairy wastewater because of a combined effect of acetic and propionic acid accumulation. The ITB presented several advantages compared to the IFB. First, the bottom of the reactor can be used as a settler for recovering the sludge or the overcoated particles. Second, a gas injection in a reactor is easier than a liquid recycling: no clogging problems were met. Lipid phosphate concentration has been revealed as a good method for biomass estimation since it only includes living biomass. This point is of particular importance in biofilms, in which exopolymer matrix constitutes an important fraction of volatile solids. Future approach of this study concern the relationship between the hydrodynamic conditions in the reactors, the biomass growth onto the solid carrier, the biomass attrition into the reactors, active biomass and their influence on overall reactor performance. ACKNOWLEDGMENTS This work was supported by research grant No. IN92-D28480461-92 from the Ministry of Education and Cultures of Spain to the first author. REFERENCES 1. 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Lactoserum composition Feed composition Phosphorus 1.10 g 100 g -1 Nitrogen 2.35 g 100 g -1 Lactoserum 30 g l -1 COD 100.2 g 100 g -1 NaHCO 3 15 g l -1 TOC 40.65 g 100 g -1 Solution 1 8.5 ml Proteins 13 g 100 g -1 Solution 2 4 ml Lactose 65 g 100 g -1 Solution 3 8.5 g l -1 Lactic acid 2 g 100 g -1 Solution 1 Solution 2 NaCl 50 g l -1 Na 2 MoO 4 . 2H 2 O 0.4 g l -1 CaCl 2 . 2H 2 O 10 g l -1 H 3 BO 3 0.06 g l -1 NH 4 Cl 50 g l -1 FeCl 2 . 4H 2 O 0.3 g l -1 MgCl 2 . 6H 2 O 10 g l -1 CoCl 2 . 4H 2 O 0.04 g l -1 HCl 0.01N up to 1 l MnCl 2 . 4H 2 O 0.06 g l -1 Solution 3 ZnCl 0.007 g l -1 K 2 HPO 4 50 g l -1 NiCl 2 . 6H 2 O 0.06 g l -1 Table 2. Apparent density (kg m -3 ) Density (kg m -3 ) SSA (m 2 m -3 ) U mf (m h -1 ) d p (µm) Shape 400 690 20,000 5.40 175 spheres