OPTIMAL OPERATING POLICY OF A FLUIDIZED BED BIOREACTOR USED FOR MERCURY UPTAKE FROM WASTEWATERS BY USING IMMOBILIZED P. PUTIDA CELLS

A model-based analysis of a three-phase continuously operated fluidized-bed bioreactor (TPFB) is developed in order to determine the multi-objective optimal and sustainable operating policy of a TPFB used for removing mercury ions from wastewater. More specifically, the analysis is focus on finding the optimal feeding policy of alginate porous beads of known particle size containing immobilized biomass (P. putida bacteria) that minimize the biomass consumption, while keeping a quasi-constant high mercury removal conversion, under quasi-stable reactor performances. The extended bioreactor model is accounting for the biomass growth, biodegradation, and its partial leakage and washout. Bioreactor dynamics prediction has been generated by using a simple Michaelis-Menten kinetic model adopted from literature. The resulted optimal feeding policy of the bioreactor points out the importance of the adoption of an extended and adequate process/reactor model able to solve difficult engineering operation problems by quickly adjusting the feeding conditions according to the time-varying characteristics of the biomass culture, and to the limited possibilities to control the process during the wastewater residence time in the bioreactor.


Introduction
The bioreactor optimal operation is one of the most difficult and challenging engineering problem, due to well-known bioprocess complexity and variability, besides the large number of variables influencing the bioprocess difficult to be accounted in a quite reduced kinetic model.  Mercury is considered a priority hazardous pollutant, due to its high toxicity. A maximum permissible concentration of 50 g/L is imposed to discharged wastewaters [1]. Moreover, the European Union stated, under Directive 2000/60/EC, the cessation or phasing out of discharges, emissions and losses of mercury within 2020, with a complete remediation of the polluted water bodies [3]. "Heavy metal pollution of the aquatic environment and particularly mercury pollution due to mining and industrial activities still represents a high worldwide concern. The main source of mercury is the combustion of fossil fuel and solid waste (Fig. 1), the fuel including traces of mercury up to 0.3 mg/kg in coal, and up to 3 mg/kg in municipal solid waste [3]. However, other significant sources of pollution can be also mentioned, such as the use of mercury as cathode in the chlor-alkali industrial electrolysis at large scale leading to significant mercury emissions (ca. 1 g Hg/t chlorine; [4]), and highly polluted wastewater (up to 7.6 mg/L [1]). Mercury is also used in numerous industrial and medical applications (fungicides, disinfectants, dental products, catalysts, igniters, dyes production, etc.)" [5][6][7].
The current treatment procedures ( Fig. 2) to remove mercury from wastewater register in the following categories [1]: i) Precipitation with hydrogen sulfide, with the disadvantage of handling large amounts of toxic H2S, and resulting large volumes of mercury contaminated sludge necessary to be deposited in special places, with no possibility of recycling the precipitated mercury; ii) Retention on the ion exchange columns, with the disadvantage of requiring costly adsorbents; it can be applied only for removing low loads of mercury from wastewater; besides, most of ion-exchange resins cannot be regenerated thus raising disposal problems, while the renewable resins are expensive; iii) Retention on various cheap sorbents, such as activated carbon, char from coal, volcanic tuff, modified cellulose or agro-based waste materials; iv) Renewable polymeric membranes also display promising sorption and filtration capabilities; v) Bioaccumulation into genetically modified bacteria cells by sequestering mercury via binding to metallothionein molecules or cytosolic chelating agents; this method is limited by the cell metabolic resources, e.g. by using cyanobacteria, Bacillus sp., or E. coli.
Microbial "detoxification" of wastewaters approached in this paper involves metabolic processes of certain cell cultures, that results in reducing the mercuric ions to elementary volatile mercury, which is less toxic for microorganisms and more easily to be recovered from liquid / gas (air) phases. Pseudomonas sp.; Aeromonas hydrophila; Escherichia coli are to be mentioned among the resistant strains used [1]. The process takes place inside the living cells with a high efficiency; the mer-reductase / NADPH used being continuously synthetized and regenerated during the cell growth (see the simplified reactions in Fig. 3).
This paper is aiming at using a multi-objective criterion and an elaborated bioreactor model to derive sustainable optimal operating policies of a TPFB used for mercury uptake from wastewaters by immobilized cultures of P. putida. The target optimization criteria concern the sustainability of the TPFB operation, leading to the concomitant optimality of: I) an economic criterion (maximum of mercury removal conversion); ii) a safety-stability criterion (quasi-stationary removal conversion over a defined time-horizon); iii) an environmental criterion (minimum wasted biomass with mercury traces leaving the TPFB).

Bioreactor model and optimization problem formulation
To simulate the TPFB reactor performances and to derive the optimization problem solution, an extended dynamic ideal model of the bioreactor was adopted ( Fig. 5 [1]), including a simple Michaelis-Menten kinetics of the bioprocess derived from a structured extended model (Fig. 4, [7][8][9]), thus allowing its use for bioprocess optimization. The model includes the main characteristics of the bioprocess on the solid support, and the solid-liquid-gas interfacial transport of the mercury, even if additional calculations are necessary to derive the particle effectiveness and to solve the mass flux equality at solid-liquid-gas interface during the transient regime. The extended bioreactor model is accounting for the biomass growth, biodegradation, and its partial leakage and washout (Fig. 4, [7 -9]). The nominal operating conditions of the TPFB are presented in Fig. 3.
The TPFB model main hypotheses are presented by [1]. In short, due to the well-mixing conditions, the mercury ions diffuse from bulk phase through the external diffusional film surrounding the particles and then through the internal pores until reaching the immobilized biomass where the mercury is crossing the cell membrane, and then it is reduced to metallic mercury (see the simplified reactions in Fig. 3). The resulted metallic mercury from the cell cytosol diffuses through the cell membrane and then it is transported as micro-drops (of ca. 5 m diameter [3]), or dissolved (solubility of 26 g/L at 26 o C) to the gas-liquid interface from where it passes as vapors into the homogeneous gas phase (air). Low amounts of mercury bio-accumulated into bacteria are neglected in the model. Mercury mass balance in the liquid and gas phases includes the inlet-outlet and transport terms (Fig. 5).

Solving the optimization problem, and results
To solve this complex multi-objective optimization problem, the "weighting function method" has been used (joint function  in Fig. 6-right), by associating to each criterion (j, j=1-3) weighting factors (giving less weight to the biomass consumption: Hg x w , σ w , and X w in this case), chosen depending on the relative importance given to each objective. The formulated normalized three objective functions for the optimization problem, accounts for (Fig. 6): i) an economic criterion, that is maximum of the average mercury ion reduction conversion ( Hg x ), over the considered running-time interval t[0, 300] min.; ii) a safety criterion, that is a minimum standard deviation (denoted by ) of Hg x from its average value over the considered running-time interval; iii) an environmental minimum impact criterion, that is minimum biomass consumption over the considered running-time interval.
The optimization problem variables are the concentration of the feeding biomass [  (Fig.7-8) [1]. The best TPFB reactor operating policy B (Fig.8) was obtained for the relative weights: . by giving less weight to the biomass consumption, thus ensuring a maximum conversion, but also quite-uniform over the running time, with the expense of a higher consumption of biomass. This operating policy is preferred because the biomass is cheap and renewable, while the removal of mercury from wastewater is a very important ecological issue deserving lots of investments, and operational costs.

Conclusions
Derivation of alternative optimal operation policies of a TPFB bioreactor used for testing mercury removal efficiency by using immobilized bacteria on a suitable porous support (alginate beads) is an essential engineering analysis to be used in further process scale-up and control.
In the simulated case study, the very effective bacteria metabolism allows an efficient reduction of the cytosolic mercury (92-99% conversion), excretion and transport of the volatile metal to the gas phase for a high pollutant load in the wastewater. By keeping a reasonable small size of alginate particles (1-2 mm), the most important control parameters appear to be the biomass load in the bioreactor and the wastewater/biomass feed flow rate.
The use of an accurate Michaelis-Menten kinetic model of the reduction bioprocess, and of a reasonable extended bioreactor model are essential steps for deriving satisfactory and interpretable results, that is prediction of various optimal operating alternatives. The study points-out the high importance of considering a detailed biomass mass balance in the bioreactor model for ensuring a satisfactory precision of the results.
Finally, the use of immobilized (modified) resistant bacteria in fluidizedbed aerators is proved as being a viable, effective, and less costly alternative for mercury removal from wastewaters at a large-scale. The optimal choice of the operating parameters, and especially the optimal biomass continuous addition policy can be ranged to ensure a high and quasi-stationary process conversion (around 99%), eventually leading to mercury loads in the bioreactor effluent lower than the regulations' threshold, with the expense of a moderate biomass consumption. This disadvantage is compensated by the use of a cheap and renewable biomass, and by the tremendous ecological importance of removing the very toxic mercury from wastewaters, or from surface waters.