CONTINUOUS METHOD FOR REDUCING THE AMOUNT OF ORGANIC COMPOUNDS IN WASTEWATER

Abstract

The present invention relates to a method for reducing the amount of organic compounds in wastewater, comprising providing a wastewater comprising NaCl in a concentration of at least 6% (w/v), contacting said hypersaline wastewater with a halophilic microorganism, and reducing the 5 amount of organic compounds by said halophilic microorganism in the presence of at least one substrate which has been added to the wastewater and which allows for the growth of said halophilic microorganism, wherein the reduction of the amount of organic components is carried out as a continuous process in bioreactor.

Claims

1.-15. (canceled)

16. A method for reducing the amount of organic compounds in wastewater, comprising (a) providing or obtaining a wastewater comprising NaCl in a concentration of at least 6% (w/v), (b) contacting said hypersaline wastewater with a halophilic microorganism, and (c) reducing the amount of organic compounds by said halophilic microorganism in the presence of at least one substrate which has been added to the wastewater and which allows for the continuous growth of said halophilic microorganism, wherein the reduction of the amount of organic components is carried out as a continuous process in bioreactor.

17. The method of claim 16, wherein the halophilic microorganism is an extremely halophilic microorganism such as Haloferax mediterranei.

18. The method of claim 16, wherein the biomass concentration in the bioreactor is at least 10 g/l, in particular wherein the biomass concentration in the bioreactor is 20 g/l to 40 g/l.

19. The method of claim 16, wherein the bioreactor is connected with a cell separation device which continuously separates the cells of the halophilic microorganism to obtain a filtrate of the treated wastewater, in particular wherein the cell separation device comprises a filter, in particular a membrane filter, which allows for the continuous separation of the cells of the halophilic microorganism from the first portion of the treated wastewater to obtain a filtrate of the treated wastewater.

20. The method of claim 16, wherein total organic content of the hypersaline wastewater obtained or provided in step a) is lower than 400 mg/l.

21. The method of claim 16, wherein the continuous process is controlled by the following parameters (i) concentration of the at least one substrate, (ii) recirculation rate R and (iii) dilution rate D.

22. The method of claim 16, wherein the recirculation rate R is 0.8 to 0.99, and/or the dilution rate D is equal to or larger than 0.05 h.sup.−1, and/or the continuous process is carried out under conditions which allow for a growth rate μ of the halophilic microorganism of larger than 0.008 h.sup.−1.

23. The method of claim 16, wherein the amount of at least one organic compound selected from the group consisting of nitrobenzene, formate, phenol, methylenedianiline, in particular 4,4′-Methylenedianiline (MDA), and aniline is reduced, and/or wherein the amount of total organic carbon (TOC) is reduced, in particular wherein the amount of the at least one organic compound or of the total organic carbon is reduced by at least 30%.

24. The method of claim 16, wherein the treated wastewater is concentrated after separation of the cells from the wastewater, and wherein the continuous process further comprises subjecting the treated wastewater or the concentrated treated wastewater to sodium chloride electrolysis, thereby producing chlorine and sodium hydroxide and optionally hydrogen, in particular wherein the sodium chloride electrolysis is selected from membrane cell electrolysis of sodium chloride, in particular membrane electrolysis using oxygen consuming electrodes and diaphragm cell electrolysis of sodium chloride.

25. The method of claim 24, wherein the bioreactor is connected via a cell separator to a device allowing for sodium chloride electrolysis of the treated wastewater.

26. A bioreactor comprising a hypersaline wastewater and at least one substrate which allows for the growth of cells of Haloferax mediterranei, wherein the biomass concentration of the cells of Haloferax mediterranei in the wastewater is a least 10 g/l.

27. The bioreactor of claim 26, wherein the bioreactor comprises at least 1000 1 hypersaline wastewater.

28. The bioreactor of claim 26, wherein the biomass concentration in the bioreactor is at least 20 g/l.

29. The bioreactor of claim 26, wherein the bioreactor is connected a cell retention device which allows for continuously separating the cells from the wastewater to obtain a filtrate of treated wastewater.

30. A purification system comprising a bioreactor comprising a hypersaline wastewater and cells of at least one halophilic microorganism, said bioreactor being connected to a cell retention device which allows for continuously separating the cells from the wastewater to obtain a filtrate of treated wastewater, wherein said cell retention device is connected to a device allowing for sodium chloride electrolysis of the treated wastewater.

Description

IN THE FIGURES

[0188] FIG. 1 Process scheme. Saline wastewater (1) is supplemented with media components. Second substrate is added (2) to guarantee stability of the continuous process. A constant bleed stream (3) is taken to ensure constant biomass concentration in steady state. Treated cell free harvest (4) shows low residual TOC concentrations compared to original feed. The corrosion resistant bioreactor (6) is equipped with a pump that ensures constant loop flow (5), so that harvest can be separated continuously using membrane module (7).

[0189] FIG. 2 Influence of dilution rate and biomass concentration on the quality of the harvest. Both factors have a linear influence on residual TOC.

[0190] FIG. 3 TOC reduction by extreme halophilic cells in continuous process in %. Feed represents saline wastewater. Harvest 1-4 represent biologically treated samples after processing without Co-feeding (Phase 1 and 3) and with Co-feeding.

[0191] FIG. 4 Control concept of the process. The Biomass concentration and the specific growth rate μ can be changed by altering the parameters dilution rate D, Recirculation Rate R and substrate in Feed S.sub.in.

[0192] All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

[0193] The invention will be merely illustrated by the following Examples. The said Examples shall, whatsoever, not be construed in a manner limiting the scope of the invention.

Exemplary Embodiments

EXAMPLE 1

Continuous Processing with High Productivity

[0194] Cultivation was established in a bioreactor to show feasibility of TOC reduction in brines containing very low amounts of organic carbon. It should be investigated how process parameters influence the quality of the harvest. The developed process uses continuous cultivation process and cell retention. HFX cells were cultivated in brine containing 15% w/v NaCl. Process parameters pH, stirrer speed, dilution rate and reactor temperature were controlled. The process is run continuously using a cell retention system with a polysulfone (PSU) hollow fibre membrane. It has an area of 420 cm.sup.2 and a pore size of 0.2 μm.

[0195] The special non-corrosive Labfors PEEK (Infors, AG, Switzerland) reactor was utilized with the following specifications: [0196] 1 L Borosilicate glass culture vessel with cooling/heating jacket [0197] Borosilicate glass exhaust gas cooling [0198] Special corrosion resistant Polymer (PEEK) bioreactor top lid [0199] Special corrosion resistant Polymer (PEEK) thermometer holder [0200] Borosilicate glass sampling tube and gas inlet tube [0201] Special corrosion resistant agitator [0202] Loop pump constantly circulating cell suspension out of vessel and back into vessel [0203] Hollow fibre membrane module attached to loop [0204] Bleed pump attached to loop [0205] Harvest pump attached to membrane module [0206] Feed pump

[0207] Online Analytics of: [0208] Exhaust gas CO.sub.2 [0209] Exhaust gas O.sub.2 [0210] Glass pH probe [0211] pO.sub.2 probe and [0212] Thermal Mass flow controller for incoming air

[0213] The following media components were added to the brine: KCl 1.66 g/l, NH.sub.4Cl 1.5 g/l, KH.sub.2PO.sub.4 0.15 g/l, MgCl.sub.2.6H.sub.2O 1.3 g/l, MgSO.sub.4.7H.sub.2O 1.1 g/l, FeCl.sub.3 0.005 g/l, CaCl.sub.2.2H.sub.2O 0.55 g/l, KBr 0.5 g/l, Mn stock 3 ml and trace elements 1 ml. Trace elements containing Fe, Cu, Mn, Co, Zn. Amount of glycerol as co-substrate was calculated in a way that the resulting growth rate was constant for all experiments, independent from dilution rate. [0214] Temperature: 37° C. [0215] pH: 7.0 (controlled using 0.5 M HCl and 0.5 M NaOH)

[0216] Using a multivariate design of experiments it was shown what influence the process parameters dilution rate and biomass concentration have on harvest quality. Dilution rate was varied in the range of 0.1 to 0.6 h.sup.−1, Biomass concentration in the range of 2 to 5 g/L.

[0217] The results showed that TOC concentration in the wastewater was decreased from 84 ppm down to a range of 11-35 ppm. Both dilution rate and biomass concentration have a strong linear influence on the harvest quality. The higher the biomass in the fermenter, the lower is the residual TOC in the harvest. On the other hand the effect of dilution rate on TOC and harvest quality is negative. Lowest residual TOC was achieved using high biomass concentration and low dilution rate. The results also show us that high dilution rates can be compensated by higher biomass concentration.

[0218] The data gained in that experiment were used to calculate a linear regression model with biomass concentration and dilution rate as input. Model data were analysed using MODDE software. The model showed very good correlation (R.sup.2 0.97, Q.sup.2 0.82, Validity 0.35, Reproducibility 0.99). Depending on a predefined quality of the harvest and requested dilution rate of the process the biomass concentration can be read of the model. Process can be controlled by defined addition of co-substrate, recirculation rate and dilution rate.

EXAMPLE 2

Stabilizing Effect of Co-Substrate

[0219] A stable process is characterized by a state that shows little if any change. Experiments should show if stable continuous cultivation is possible without use of a co-substrate.

[0220] The bioreactor system described in Example 1 was used for those experiments. The following media components were added to the brine: KCl 1.66 g/l, NH.sub.4Cl 1.5 g/l, KH.sub.2PO.sub.4 0.15 g/l, MgCl.sub.2.6H.sub.2O 1.3 g/l, MgSO.sub.4. 7H.sub.2O 1.1 g,/l FeCl.sub.3 0.005 g/l, CaCl.sub.2.2H.sub.2O 0.55 g/l, KBr 0.5 g/l, Mn stock 3 ml and trace elements 1 ml. Trace elements containing Fe, Cu, Mn, Co, Zn. In case of ‘co-feeding’ the medium was supplemented with 0.69 g/L Glycerol. [0221] Temperature: 37° C. [0222] pH: 7.0 (controlled using 0.5 M HCl and 0.5 M NaOH)

[0223] The experiment was performed with a biomass concentration of 1.7 g/L Haloferax mediterranei. Cultivation was done in continuous mode using a constant feed of 100 mL/h resulting in a dilution rate D=0.1 h-1. The bioreactor was set in total cell retention, which means the bleed is set to 0 mL/h.

[0224] Samples of the harvest were taken to investigate TOC and residual formate concentration. Concentration of glycerol and organic acids was determined using HPLC.

[0225] The experiment was performed in three phases: 1) without co-feeding (100 h), 2) with co-feeding (40 h), 3) without co-feeding (100 h).

[0226] Results showed that formate concentration in this experiment was reduced 70-88% of the original concentration and TOC content was degraded 65-79% by treatment with extreme halophilic Haloferax mediterranei. Co-feeding had a high influence on quality of the treated brine.

[0227] Both TOC and residual formate concentration increased over time when no second substrate was fed (Harvest 1 taken after 50 h, Harvest 2 taken after 100 h). It shows that the process is not stable without co-feeding, cells reduce in activity and harvest quality is constantly decreasing. Cells could not utilize organic components in brine for formation of biomass.

[0228] When cells were co-fed with glycerol quality of the harvest increased (Harvest 3). Cells formed fresh biomass and metabolized co-substrate and organic impurities simultaneously. When supply of co-substrate was stopped, quality of harvest decreased again (Harvest 4 taken 70 h after stop of co-feeding).

EXAMPLE 3

Process Control and Start-Up Strategy

[0229] It could be derived from example 1 that biomass concentration has a high influence on the formate degradation. Consequently the amount of biomass in the fermenter is proportional to the productivity of the process. Hence, controlling the biomass concentration means controlling the degradation process.

[0230] The cofeeding strategy that was developed for this process gives the unique possibility to control this high-throughput degradation. By adding a defined concentration of additional substrate (here glycerol) the amount of biomass in the steady state condition is defined.

[0231] The automated control strategy of the process is composed of two phases. In phase I (the start-up phase) the goal is to expand the cells to a target biomass concentration. The phase II (steady state phase) is the phase of the biological treatment.

[0232] The start-up phase is planned as batch, where substrate concentration is determined according to the target biomass concentration. Biomass concentration can then be estimated as following:

x=x.sub.0.Math.e.sup.μmax.Math.(t-t.sup.0)   Equation 1

[0233] where x.sub.0 is the biomass concentration at start time t.sub.0, μmax is the maximum specific growth rate and x is the biomass concentration at time t. For growth of HFX on glycerol maximum specific growth rate was determined μ.sub.max=0.067 h.sup.−1. Substrate consumption follows the correlation

s=Y.sub.x/s.Math.x   Equation 2

[0234] The yield Y.sub.x/s defines the amount of substrate needed to form a certain amount of biomass. For growth of HFX on glycerol it was determined Y.sub.x/s=0.75 mol/mol under batch conditions.

[0235] When the target biomass concentration is reached, the process is switched to phase II (steady state phase). The process will get to steady state condition when the final biomass concentration is reached. In this phase a constant feed is added and cell-free harvest as well as the cell containing bleed are constantly removed from the reactor. The steady state condition is defined by the process parameters D (dilution rate [h.sup.−1]), R (recirculation rate []) and s.sub.in (Substrate concentration in the feed [g/L]) according to Equation 3, 4 and 5. The closer the biomass concentration in phase I is to target concentration, the shorter this adaptation phase will be.

[00002]$\begin{array}{cc}x=\frac{Y_{X\text{/}\mathrm{Gly}}.Math.S_{\mathrm{in}\mathrm{Gly}}+Y_{X\text{/}\mathrm{For}}.Math.S_{\mathrm{in}\mathrm{For}}}{1-R}& \mathrm{Equation}3\\ R=\frac{\mathrm{Harvest}\mathrm{flow}}{\mathrm{Feed}\mathrm{flow}}& \mathrm{Equation}4\\ \mu =\left(1-R\right).Math.D& \mathrm{Equation}5\end{array}$

[0236] Y.sub.x/Gly and Y.sub.x/For are the yields that describe how much biomass x is formed per g Glycerol or

[0237] Formate. We could determine Y.sub.x/Gly=0.63 Cmol/Cmol and Y.sub.x/For=0.44 Cmol/Cmol. Equation 3 was derived from equations that are available in literature and from findings that were made as results of our research.

[0238] The bioreactor setup described in example 1 was used for this experiment. The following components were used to prepare a synthetic medium: NaCl 150 g/L, KCl 1.66 g/l, NH.sub.4Cl.5 g/l, KH.sub.2PO.sub.4 0.15 g/l, MgCl.sub.2.6H.sub.2O 1.3 g/l, MgSO.sub.4. 7H.sub.2O 1.1 g/l, FeCl.sub.3 0.005 g/l, CaCl.sub.2.2H.sub.2O 0.55 g/l, KBr 0.5 g/l, Mn stock 3 ml and trace elements 1 ml. Trace elements containing Fe, Cu, Mn, Co, Zn.

[0239] Biomass was estimated using a softsensor. Detailed description can be found in the literature.

[0240] A combination of different R and s.sub.in were used to achieve defined biomass concentrations and growth rates. Dilution rate D was kept constant at 0.1 h.sup.−1 for the whole process. FIG. 4 shows that the biomass concentration could be controlled on different levels—steady state conditions could be reached. The specific growth rate μ and the biomass concentration x can be controlled independently by altering the parameters D, R and s.sub.in.

[0241] The control concept uses the parameters (i) concentration of co-substrate, (ii) recirculation rate and (iii) dilution rate for control of quantity and quality of the treated waste water. However, due to interdependencies between the parameters (see Equation 4-Equation 5), some of the requirements set for an optimized process are contradicting (see Table 1). It is desired to reduce the amount of substrate that is added to the feed for reduction of operational costs. This leads to a low biomass concentration unless retention rate R is high. High retention rate, however, results in low specific growth rates μ and thus a low degradation rate for formate. On the other hand a high retention rate is desired for the low amount of cell waste that is produced and that causes additional costs for disposal.

[0242] Table 2 shows the application of the control concept for an example process. The basic process (case 1) is simulated at a dilution rate D=0.1 h.sup.−1, a retention rate of R=0.90, a Glycerol concentration of 2 g/L and a formate concentration of 0.23 g/L. If substrate concentration is reduced (case 2) for reduced operational costs, this results in lower biomass concentration. Example 1 showed that low biomass concentration reduces the formate degradation capacity. To increase Biomass concentration, the cell retention rate can be increased (case 3). As a result of the higher retention rate, the specific growth rate is reduced according to Equation 5. Case 4 shows a process with an increased dilution rate in comparison to the basic process. As a result of the higher dilution rate, also the growth rate increases. However, example 1 showed that high dilution rates have a negative impact on the quality of the treated waste water. The examples in

[0243] Table 2 illustrates that optimization of the process is always a balance between quality of the treated waste water on one hand and quantity as well as process costs on the other hand. Optimal setpoints for this process are low substrate concentration in feed, low amount of cell waste and high formate degradation rate.

TABLE-US-00001 TABLE 1 Requirements of the process and its consequences. Requirement Action Side effect Low substrate expenditure Low s.sub.in Low x unless R is high High formate degradation rate High μ Little R Low cell waste Low Bleedflow High R

TABLE-US-00002 TABLE 2 Example for the control concept. Biomass concentration X and specific growth rate μ are controlled using Dilution rate D, retention rate R, substrate concentration s.sub.in. To show the interdependencies of the parameters a basic process (1) is changed by different actions (2-4) (see Table 1). Y.sub.Gly/S = 0.54 g/g and Y.sub.For/S = 0.25 g/g is assumed for calculations. D R S.sub.in [g/L] S.sub.in [g/L] μ X [1/hr] [ ] Glycerol Formate [1/hr] [g/L] Description 1) 0.1 0.85 5.0 0.23 0.015 18.5  Basic process 2) 0.1 0.85 2.5 0.23 0.015 9.4 Low substrate expenditure, but low Biomass concentration 3) 0.1 0.92 2.5 0.23 0.008 18.5  High retention rate, but low growth rate 4) 0.2 0.85 5   0.23 0.030 18.5  Higher dilution rate, high growth rate, lower quality (See example 1)

EXAMPLE 4

Degradation of Formate, MDA (4,4′-Methylenedianiline), Nitrobenzene, Aniline and Phenol in Actual Residual Water in Continuous Bio-Processing Using Cell Retention System

[0244] Bioreactor Setup with Cell Retention

[0245] Continuous degradation of formate, MDA, Nitrobenzene, Aniline and Phenol in actual brine was performed using a cell retention system. The industrial brine was supplemented with media components given in table 1 and glycerol as co-substrate. The amount of glycerol in the medium was adjusted in such a way to achieve a specific growth rate of 0.026 h-1. Cultivation was established in the bioreactor as described for shake flask experiments. Fermentation in the bioreactor was performed at 450 rpm agitation and 37 ° C. The cell retention system was set in the bioreactor using a polysulfone (PSU) hollow fiber microfiltration membrane cartridge having an area of 420 cm.sup.2 and a pore size of 0.2 μm. Feed flows of 130 to 610 g/h led to a dilution rate of 0.1 to 0.6 h.sup.−1. By adjusting the feed flow in relation to the cell containing bleed flow and the cell free harvest, a constant biomass in the fermenter could be achieved.

[0246] Turbidity as indicator for cell density and HPLC analytics of the residual formate, acetate and glycerol were measured during the entire process. Residual MDA, Nitrobenzene, Aniline and Phenol were also measured by HPLC. It was shown that the amounts of formate, MDA, Nitrobenzene, Aniline and Phenol were reduced.