Method for producing microbial cellular biomass having flocculant properties

Abstract

The disclosure relates to a microbial cellular biomass having flocculant properties, to its obtaining process and to its use in the treatment by flocculation.

Claims

1. A process for producing a microbial cellular biomass having flocculant properties comprising the steps: a) introduction into a reactor: of a substrate comprising at least 30% by weight of monosaccharides, the resulting reaction medium having a C/N molar ratio of less than or equal to 10 and a dilution rate D of less than or equal to 0.35 h.sup.−1; b) stirring of the reaction medium obtained in a) and retention of the C/N ratio at a value less than or equal to 10 and the dilution rate D at a value less than or equal to 0.35 h.sup.−1; c) elimination of any filamentous bacteria and filamentous fungi by sequential decantation of the obtained biomass in b); and obtention of the microbial cellular biomass having flocculant properties.

2. The process according to claim 1, further comprising a ventilation of the reaction medium during the process.

3. The process according to claim 1, carried out in an open environment.

4. The process according to claim 1, carried out in a non-sterile environment.

5. The process according to claim 1, wherein the reaction medium of step a) comprises microorganisms selected from the group comprising the gammaproteobacteria, flavobacteria, alphaproteobacteria, and betaproteobacteria.

6. The process according to claim 1, further comprising an inoculation step of the reactor with one or more microorganisms, selected from the group comprising the gammaproteobacteria, flavobacteria, alphaproteobacteria and betaproteobacteria, prior to step a).

7. The process according to claim 1, wherein the substrate comprises a hydrolysate and/or monosaccharides.

8. The process according to claim 1, further comprising a step of injecting into the reactor a monosaccharide or mixture of monosaccharides.

9. The process according to claim 1, wherein the temperature in the reactor is from 4 to 55° C.

10. The process according to claim 1, wherein step c) is a decantation inside the reactor, the decanted solid being removed from the reactor or a decantation of the effluent leaving the reactor, the decanted solid being removed and the residue then being reinjected into the reactor.

11. The process according to claim 1, further comprising a wastewater treatment step, said wastewater treatment step comprising a step of contacting wastewater with a coagulant and the microbial cellular biomass having flocculant properties.

12. A microbial cellular biomass having flocculant properties obtained according to the process of claim 1.

13. The microbial cellular biomass according to claim 12 configured for the treatment of wastewater.

14. A process of treating wastewater comprising a step of contacting wastewater with a coagulant and a microbial cellular biomass according to claim 12.

15. The process according to claim 1, further comprising a microbial inoculum.

16. The process according to claim 1, comprising monosaccharides selected from the group comprising hexoses and pentoses.

17. The process according to claim 1, comprising nutrients for the microbial inoculum

Description

[0084] FIG. 1 is a schematic representation of the implementation of the process of the invention comprising a continuous reactor, a mineral and trace element supply system, a substrate supply system, source of phosphorus and nitrogen (CPN), an air supply system, a mechanical agitator, a purge line, a draw-off system, baffles and a device for controlling the dilution rate and the C/N and C/P ratios.

[0085] FIG. 2 represents a photograph of the called-so “jar-test” apparatus used to carry out the flocculation tests, the six beakers being filled with urban wastewater.

[0086] FIG. 3 represents an analysis of the performance of biomasses obtained by the process according to the invention. On the abscissa axis is plotted the gain of the commercial polymer (PAM AN 934 SH) compared to the coagulant FeCl.sub.3 alone (in percentage), and on the ordinate axis is plotted the gain (%) of the biomass according to the tested invention compared to FeCl.sub.3 alone.

[0087] FIG. 4 represents an enlarged view (Scale: 10 μm) of bacterial exopolysaccharides included in a microbial cellular biomass according to the invention. It can be observed, thanks to Indian ink staining, the relatively compact layer that surrounding the bacterial wall and constituting the capsule, which is formed of viscous molecules elaborated by the bacteria (it is also possible to use other stains such as Alcian blue staining, which, at pH 2.5, adheres to negatively charged macromolecules and then colors the acidic polysaccharides in blue).

[0088] FIG. 5 represents a comparison between A) a photograph of microscopic observations of consortium dominated by Sphaerotilus (selection process carried out without selective purging, that means without step c) of the process) and B) a photograph of microscopic observations of consortium dominated by biomass having flocculant properties (selection process carried out with selective purging of filamentous bacteria); enlarged view (Scale: 10 μm). This comparison shows that in the absence of process step c), filamentous bacteria and fungi are not eliminated.

[0089] The invention will be better understood upon reading the following non-limiting examples.

Example 1: Implementation of the Process According to the Invention

[0090] Inoculum and Culture Media

[0091] A reactor under stirring and ventilation is inoculated with activated sludge from the Ginestous water treatment plant, France) collected and maintained according to Cavaille et al. (Cavaille et al. 2016. “Understanding of Polyhydroxybutyrate Production under Carbon and Phosphorus-Limited Growth Conditions in Non-Axenic Continuous Culture.” Bioresource Technology 201 (February): 65-73. https://doi.org/10.1016/j.biortech.2015.11.003).

[0092] The sludge is centrifuged at 4100 g for 15 minutes and the supernatant is then removed. The pellet is recovered, and aliquots are frozen in liquid nitrogen and stored at −20° C.

[0093] The inoculum thus prepared, before the seeding of the reactor, an aliquot is thawed for 15 hours at a temperature of 4° C. and then rinsed with a saline solution (0.9% w/v NaCl) by means of three successive centrifugations (4100 g-15 min). Before inoculation of the reactor, the sludges are fed-batch for 24 h with additions of 1.8 g/L of an equimolar mixture of fructose and glucose.

[0094] The reactor is seeded with an initial activated sludge concentration of 1.5 g/L of suspended volatile matter.

[0095] The culture medium is composed of a first solution containing the source of carbon, nitrogen and phosphorus. This is a feed containing a carbon source (in the example an equimolar mixture of glucose and fructose), a phosphate buffer solution and ammonium added according to the degree of nitrogen limitation chosen, here so as to obtain a (C/N) ratio consumed of 8.5 (molar).

[0096] A second solution is added containing the mineral salts and trace elements required for microbial cultures (Table 1).

TABLE-US-00001 TABLE 1 Compound Concentration Ferric Ammonium Citrate Solution at 250 g/L 0.1 mL/L (C.sub.6H.sub.5 + 4yFe.sub.xN.sub.y0.sub.7 with approximately 28% Fe) MgSO.sub.4•7H.sub.2O Solution at 250 g/L 0.2 mL/L KOH Solution at 250 g/L 0.07 mL/L H.sub.2SO.sub.4 96% v/v 0.035 mL/L Solution “traces of elements” (0.3 g/L H.sub.3BO.sub.3, 0.1 mL/L 0.21 g/L CoCl.sub.2 6H.sub.2O, 0.11 g/L ZnSO.sub.4 7H.sub.2O, 0.04 g/L MnCl.sub.2 4H.sub.2O, 0.03 g/L Na.sub.2MoO.sub.4 2H.sub.2O, 0.02 g/L CuSO.sub.4 5H.sub.2O et 0.01 g/L NiCl.sub.2 6H.sub.2O)

[0097] In order to evaluate the effect of constant microbial contamination on the efficiency of microbial selection, a continuous flow of diluted activated sludge is injected into the reactor. This flow rate is chosen to bring a flow of contaminating cells corresponding to 5% of the cell production of the reactor (5% of r.sub.x (g/(L.Math.h)−0.1 g/L/h), that is approximately 0.005 g/L/h under stable conditions with a CODp around 0.8 g/L in the reactor. Despite this continuous supply of contaminating cells, selection of cellular biomass with flocculant properties occurs.

[0098] Production of Cellular Biomass with Flocculent Properties

[0099] The substrate used includes the elements in Table 2 below:

TABLE-US-00002 TABLE 2 Mass concentration Molar concentration Compound in the reactor (g/L) in the reactor (mol/L) Glucose 0.766 0.025 Fructose 0.766 0.025 N (essentially NH.sub.4Cl) 0.32 0.0059 P (phosphate buffer solution) 0.033 0.001 C/N 8.5 C/P 50

[0100] The microorganisms selected by the process are essentially of the Comamonadaceae type. These microorganisms produce bacterial EPS.

[0101] A reactor of 8 L of useful volume (10 L in total) (FIG. 1) equipped with a double jacket was used. Stirring fixed at 130 RPM is provided by Rushton turbines and two counter blades of 5 cm wide. To regulate the dissolved oxygen concentration at (2.5±0.5) mg/L, flow meters (EL-FLOW®, Bronkhorst High-Tech B.V., France) were used. The temperature was regulated at 25° C.±1° C., and the pH was maintained at 7.0±0.3 with the addition of potassium hydroxide (KOH, 1M). The operating conditions and on-line data acquisition (dissolved oxygen concentration, pH and temperature) are controlled via an acquisition software.

[0102] Twice a month, the reactors are emptied for cleaning in order to avoid the formation of biofilms on the walls, on the probes and on the blades. When deemed necessary, feeder hoses are replaced.

[0103] The reactors are operated in a sequential manner, with a feed and stirring phase (55 min) followed by a decantation phase (4 min 30 sec) and then a purge phase (30 sec). The purging of the reactors is carried out thanks to 3 taps at the bottom of the tank, connected to recovery tanks. At each cycle, the fed volume is equivalent to the volume purged of 960 mL (purge rate of 1.9 L/min). Since the useful volume is 8 L, the hydraulic retention time is 8.33 h (dilution rate D=0.12 h.sup.−1).

[0104] The microbial cellular biomass having flocculant properties A is obtained.

Example 2: Evaluation of the Flocculent Properties of Microbial Cellular Biomasses Having Flocculent Properties According to the Invention

[0105] The flocculent properties are evaluated on a representative effluent, here an urban wastewater (UW).

[0106] The flocculation tests are performed in jar-tests (Lavibond®—Amesbury, United Kingdom, Floc-Tester model, SN: 1013/61444) under identical conditions (stirring velocities, duration of the phases) for each coagulant/flocculant combination. A photograph of the so-called jar-tester system is shown in FIG. 2.

[0107] The coagulant used is ferric chloride. For each test, 6 beakers filled with 900 mL of UW are used. The first three beakers are used as reference (n.sup.o 1: control (no addition), n.sup.o 2: FeCl.sub.3 20 mg/L alone, n.sup.o 3 FeCl.sub.3 20 mg/L+polyacrylamide AN934SH (named PAM) 0.2 mg/L). The three remaining beakers are used for the evaluation of biomass A at 3 different concentrations (n.sup.o 4, n.sup.o 5, n.sup.o 6 FeCl.sub.3 20 mg/L+biomass A). Biomass A is used directly without prior extraction or purification. The dosage is evaluated by measuring the total chemical oxygen demand (COD). Generally, concentrations of 1, 6 and 12 mgCOD/L are implemented in the three test beakers.

[0108] After stirring and measuring the initial turbidity (Turb 0), the protocol includes three main steps: (i) addition of coagulant (FeCl.sub.3— 20 mg/L) in all beakers except the control and stirring at 150 rotations per minute (RPM) for 3 min; (ii) addition of flocculant (PAM 0.2 mg/L—Biomass A 1-6-12 mgCOD/L in beakers 4 to 6) and stirring at 50 RPM for 15 min; (iii) settling for 1 min and sampling at 2.2 cm from the surface for turbidity measurement (Turbx)

[0109] The results are analyzed in terms of the purification yields (in %) calculated as follows:

Purification yield (% r)=(Turb0−Turbx)/Turb0(*100)

[0110] The purification yields obtained are related to the reference containing only ferric chloride (beaker 2). When the purification yield obtained with the biomass according to the invention is higher than that obtained with ferric chloride alone, a positive gain is calculated as follows:

GainBiomassX (vs FeCl.sub.3)=(% r.sub.x−% r.sub.FeCl3)/% r.sub.FeCl3

GainPAM (vs FeCl.sub.3)=(% r.sub.PAM−% r.sub.FeCl3)/% r.sub.FeCl3

[0111] Finally, the performances obtained are systematically compared to those obtained with PAM AN 934 SH: Performance compared to PAM=GainBiomassX/GainPAM. This result analysis allows to get rid of the great variability of wastewater in terms of composition and their behavior towards the coagulants and flocculants used.

[0112] The method of representation is given in FIG. 3.

[0113] Four levels of performance are defined: poor (less than 20% of MAP gain), average (between 20% and 50% of MAP), good (between 50% and 100% of MAP) and excellent (greater than MAP gain).

[0114] Table 3 presents the results obtained with biomasses according to the invention (reference and variant 1 and 2), namely excellent flocculation performance (i.e., superior to that of PAM AN 934 SH). The operating conditions and results of two variants are also presented, which also provide satisfactory performance.

TABLE-US-00003 TABLE 3 Reference Variant 1 Variant 2 Operating conditions inoculum Activated sludge Activated sludge Consortia Reference Substrate Glucose/fructose Glucose/fructose Hydrolysate Mainly selected Mainly Mainly Mainly microorganisms Comamonadaceae Comamonadaceae Comamonadaceae C/N 8.5 9.5 8.5 C/P 48 48 48 D (h.sup.−1) 0.12 0.12 0.12 Cycle duration 1 h 1 h 1 h (steps b + c) Decantation duration 4 min 30 sec 4 min 30 sec 4 min 30 sec (step c) Results Flocculating properties 101% à 490% 110% à 200% 450 à 550% on UW du AN 934 SH (or GainPAM vs FeCl3) Flocculating properties Excellent Good Excellent on UW of the biomass according to the invention Flocculating properties n/d Excellent (72%) n/d on kaolinite of the biomass according to the invention (value of AN 934 SH) Flocculating properties n/d Good (465%) n/d on diluted digestate of the biomass according to the invention (value of AN 934 SH) n/d = not determined.

[0115] In variant 2, the hydrolysate is derived from enzymatically hydrolyzed screenings rejects (hemicellulases, endo-β-1,4-glucanases, endo-β-D-xylanase).

Example 3: Counterexamples

[0116] Table 4 shows some operating conditions that did not lead to the stable production of biomass with flocculent properties (counterexamples 1 and 2).

TABLE-US-00004 TABLE 4 Reference Counterexample 1 Counterexample 2 Operating conditions inoculum Activated sludge Activated sludge Activated sludge Substrate Glucose/fructose Glucose/fructose Glucose/fructose C/N 8.5 9 (to 17) 9.5 C/P 48 48 250 .fwdarw. 350 D (h.sup.−1) 0.12 0.12 0.12 Cycle duration 1 h n/a n/a (a step b + c) Decantation duration 4 min 30 sec 0 0 (a step c) Results Flocculent properties Excellent Poor Poor on UW n/a = not applicable.