"MICROBIAL-BASED BIOHYBRID SYSTEM COMPRISING BIOGENIC

SEMICONDUCTOR NANOPARTICLES AND A METHOD TO PRODUCE THE SAME"

Technical field

This application relates to a microbial-based biohybrid system comprising biogenic semiconductor nanoparticles and a method to produce the same .

Background art

One of the most important challenges facing society is the development of clean and sustainable processes for production of fuels and chemicals . Semi-arti ficial photosynthesis is an exciting area of research targeting the production of fuels from light , where biocatalysts ( enzymes or microorganisms ) can be coupled with synthetic lightharvesting materials originating so-called biohybrid systems . So far, most biohybrids were developed using chemically produced semiconductors , which require environmentally unfriendly and expensive synthes is and in most of the systems an electron mediator was required for production of the fuels . One promising alternative is the use of biohybrids constructed using semiconductor nanoparticles that are biologically produced by the involved microorganisms ( called biogenic semiconductor ) .

The synthesis of biogenic semiconductor nanoparticles is an eco- friendly and cost-ef fective process to obtain functional nanomaterials with unique features . Biohybrids have been constructed based on biogenic nanoparticles of CdS and AgInS2/ In2S3 as semiconductors . Martins , M . et al . ¹ have disclosed enhanced light-Driven hydrogen production by self-photosensiti zed biohybrid systems of Desulfovibri o desulf uri cans , Ci trobacter freundii , and Shewanella oneidensi s , sel f-photosensiti zed with biogenic cadmium sul fide nanoparticles . With this system, H2 was continuously produced with a speci fic rate of 36 pmol gdcvP ¹ tn ¹ . High apparent quantum yields of 23 % and 4 % were obtained, with and without methyl viologen, respectively, exceeding values previously reported .

Document US2012164062 discloses a method of biosynthesi zing nanoparticles and quantum dots , comprising culturing photosynthetic cells and/or fungal cells of a multicellular fungus in a culture medium compris ing one or more species of metal in ionic or non-ionic form; and one or more counter elements to the one or more species of metal , or one or more compound comprising one or more counter elements to the one or more species of metal ; wherein the cells biosynthesi ze nanoparticles and quantum dots incorporating the metal . In the case of this technology, the composition of the nanoparticles and quantum dots includes elements selected from the periodic table groups 11 to 16 and focuses on the use of cyanobacteria .

Document US2014287483 discloses semiconductor nanoparticles , methods , systems , and compositions . Robust , reproducible production of large amounts of semiconductor nanoparticles , such as quantum dots , from bacterial cultures during continuous growth is provided, without a need for extensive post growth processing or modi fication . The result is a semiconductor of nanoparticle dimensions and quality that is suitable for commercial applications in lighting, display, imaging, diagnostics , photovoltaics and hydrogen generation . Even though bacteria are used to produce CdS , CdSe or Se nanoparticles and quantum dots , this technology aims to obtain them in an isolated state .

Summary

The present invention relates to a microbial-based biohybrid system comprising microorganisms coupled with biogenic CdSMoS2 nanoparticles doped with biogenic nanoparticles of metals selected from the platinum group, wherein the microorganisms are anaerobic bacteria capable of generating hydrogen sulphide .

In one embodiment the anaerobic bacteria are selected from D. desulf uricans _r D. vulgari s or S . oneidensi s .

In one embodiment the metal from the platinum group is selected from Pd, Pt , Rh or Ru .

In one embodiment the biogenic CdSMoS2 nanoparticles have a particle si ze distribution between 10 and 200 nm .

The present invention also relates to a method to produce the microbial-based biohybrid system comprising the following steps :

- Growing anaerobic bacteria with the capacity to generate H2S in a growth medium comprising a sulphur source ;

- Addition of a cadmium solution and a molybdenum solution;

- Incubation at a temperature between 35 and 37 ° C for a time between 2 and 3h to promote the synthesis of biogenic CdSMoS2 nanoparticles at the surface of the anaerobic bacteria ; - Harvesting the anaerobic bacteria comprising the biogenic CdSMoS2 nanoparticles at the surface ;

- Synthesis of biogenic nanoparticles of metals selected from the platinum group ;

- Incubating the harvested anaerobic bacteria comprising the biogenic CdSMoS2 nanoparticles at the surface , together with the biogenic nanoparticles of metals selected from the platinum group prepared in the previous step to produce the microbial-based CdSMoS2 biohybrids dopped with biogenic nanoparticles of metals selected from the platinum group .

In one embodiment the growth medium comprises a sulphur source selected from sulphate for sulphate-reducing bacteria, thiosul fate , cysteine or/and another organic sulphur source for other anaerobic bacteria .

In one embodiment the cadmium solution is added in a concentration between 2 . 5 and 3 mM .

In one embodiment the molybdenum solution is added in a concentration between 0 . 8 and 1 mM .

In one embodiment the metal selected from the platinum group is added in a concentration between 90 and 100 mg/L .

In one embodiment the anaerobic bacteria and the metal selected from the platinum group are incubated for 15 to 20 mins at 35 to 37 ° C and then flushed with H2 for 5 to 10 mins , and then are incubated at 37 ° C overnight with an overpressure of 1 bar H2 originating the biogenic platinum group metal nanoparticles . In one embodiment the step of incubating the harvested anaerobic bacteria comprising the biogenic CdSMoS2 nanoparticles at the surface with the biogenic nanoparticles of metals selected from the platinum group being centri fuging the anaerobic bacteria comprising the biogenic CdSMoS2 nanoparticles and incubating them with the biogenic nanoparticles of metals selected from the platinum group between 5- 10 min at a temperature between 22-25 ° C .

In one embodiment the microbial-based biohybrid system is for use in the production of H2 or other compounds such as acetate , ethanol , formate , methane or lactate , using light as energy source .

Detailed Description

The present invention discloses a microbial-based biohybrid system comprising microorganisms coupled with biogenic CdSMoS2 nanoparticles doped with biogenic nanoparticles of metals selected from the platinum group metals .

The microorganisms of the biohybrid system are selected from anaerobic bacteria capable of generating hydrogen sulphide (H ₂ S ) .

The biogenic CdSMoS2 nanoparticles of the present invention have a much-improved H2 production performance with light as energy source , from 3 to 17- fold more than previously described biohybrids constructed with only CdS .

This is a green technology since the metal nanoparticles are produced using microorganisms under mild operation conditions relative to chemically synthesi zed metal nanoparticles that require high temperatures and pressure for the synthesis .

This technology can use light as the only energy source for H2 production as opposite to biohybrids developed by Wang et 2017 ² ' ³ that also require glucose .

Moreover, this technology does not need an electron mediator to be used during production of H2 or other compounds , as opposite to the biohybrid systems using chemically synthesi zed semiconductor nanoparticles .

The microbial-based biohybrid system is obtained by using microorganisms such as anaerobic bacteria grown in media with a sulphur source and incubated with cadmium and molybdenum . These two metals react with the H2S while this is produced by the bacteria originating CdSMoS2 nanoparticles that are biogenically synthesi zed . After that , the microbial CdSMoS2 are dopped with nanoparticles of platinum group metals , also produced biogenically by the anaerobic bacteria .

In the end microbial-metal dopped CdSMoS2 biohybrids are obtained, composed by anaerobic bacteria coupled to the two types of biogenic nanoparticles .

These characteristics make the present technology a green and low-cost process for H2 production from light , for example from solar energy .

Furthermore , the presently disclosed microbial-based biohybrid system can also be used to produce other value compounds such as acetate, ethanol, formate, methane, or lactate from light.

Brief description of drawings

For easier understanding of this application, figures are attached that represent the preferred forms of implementation which nevertheless are not intended to limit the technique disclosed herein.

Figure 1 describes the photocatalytic process involved in the production of hydrogen from light (A) using cysteine (B) as sacrificial electron donor, producing cystine (C) .

Figure 2 shows the characterization of D. desulf urlcans-

CdSMoS2 by Scanning Electron Microscopy (SEM) (Fig. 1A) , transmission electron microscopy (Fig. IB and C) and by SEM-

EDS (Fig. ID) .

Figure 3 shows the characterization of biogenic palladium nanoparticles (BioPd) by Scanning Electron Microscopy (SEM) (Fig. 2A) , transmission electron microscopy (Fig. 2B and C) and by SEM-EDS (Fig. 2D) .

Figure 4 shows the profile of H2 production from light by the previously reported D. desulf urlcans-CdS biohybrids and by the novel D. desulfur! cans-CdSMoS2+BioPd biohybrid.

Figure 5 shows the H2 production from light by microbial biohybrids constructed using D. desul furlcans and the binary metal composites of CdSWS2, CdSMoS2 and CdSNiS self-produced by the bacteria. Figure 6 shows the profile of H2 production from light by microbial biohybrids composed by di f ferent microorganisms and the sel f-produced (biogenic ) CdSMoS2+BioPd nanoparticles .

Figure 7 shows the profile of CO2 photoreduction by microbial-CdSMoS2+BioPd .

Description of the embodiments

Now, preferred embodiments of the present application will be described in detail with reference to the annexed drawings . However , they are not intended to limit the scope of this application .

The present invention discloses a microbial-based biohybrid system comprising microorganisms coupled with biogenic CdSMoS2 nanoparticles doped with biogenic nanoparticles of metals selected from the platinum group metals .

In one embodiment , the microorganism is selected from anaerobic bacteria with the capacity to generate H2S . In one embodiment , the anaerobic bacteria are selected from D. desulf uricans _r D. vulgari s or S . oneidensi s .

The biogenic CdSMoS2 nanoparticles are doped with a metal from the platinum group metals selected from Pd, Pt , Rh or

Ru .

In one embodiment the biogenic CdSMoS2 nanoparticles have a particle si ze distribution between 10 and 200 nm . The present application also discloses a method to produce the microbial-based biohybrid system comprising the following steps :

Growing anaerobic bacteria with the capacity to generate H2S in a growth medium comprising a sulphur source ;

Addition of a cadmium solution and a molybdenum solution;

Incubation at a temperature between 35 and 37 ° C for a time between 2 and 3h to promote the synthesis of biogenic CdSMoS2 nanoparticles at the surface of the anaerobic bacteria ;

Harvesting the anaerobic bacteria comprising the biogenic CdSMoS2 nanoparticles at the surface ;

Synthesis of biogenic nanoparticles of metals selected from the platinum group ;

Incubating the harvested anaerobic bacteria comprising the biogenic CdSMoS2 nanoparticles at the surface , together with the biogenic nanoparticles of metals selected from the platinum group prepared in the previous step to produce the microbial-based CdSMoS2 biohybrids dopped with biogenic nanoparticles of metals selected from the platinum group .

In one embodiment , the cadmium solution is added in a concentration between 2 . 5 and 3 mM .

In one embodiment , the molybdenum solution is added in a concentration between 0 . 8 and 1 mM .

In one embodiment , the synthesis of CdSMoS2 nanoparticles occurs in a growth medium that comprises a sulphur source , for example , sulphate for sulphate-reducing bacteria, thiosul fate , cysteine or/ and another organic sulphur source for other anaerobic bacteria . During this step, H2S is gradually produced by the bacteria that will be involved in the synthesis of CdSMoS2.

In one embodiment, in the end of the exponential growth phase of the anaerobic bacteria, between 2.5 and 3 mM of a cadmium chloride solution is added slowly to the culture followed by the addition also slowly of between 0.8 and 1 mM of the molybdenum solution. Then, the anaerobic bacteria cells are incubated at a temperature between 35 and 37 °C for a time between 2 and 3h, originating the biogenic CdSMoS2 nanoparticles at the surface of the anaerobic bacteria.

The synthesis of biogenic nanoparticles of metals selected from the platinum group can be performed by the same type of anaerobic bacteria used to synthesize the biogenic CdSMoS2 nanoparticles. These metal nanoparticles are synthesized biologically in the presence of 90-100 mg/L of a metal from the platinum group using the method described by Martins M, Mourato C, Sanches S, Noronha JP, Crespo MTB, Pereira TAG* 2017, Biogenic platinum and palladium nanoparticles as new catalysts for the removal of pharmaceutical compounds Water Res., 108, 160-168. Briefly, 40-45 mL of cells at the end of the exponential growth phase (Od=0.6-0.7) are collected, harvested with sterile anaerobic 20-40 mM Tris-HCl buffer pH 6.8-7.8 and added to anaerobic metal solution, 90-100mg/L metal, pH 2-3.

In one embodiment, the anaerobic bacteria and the metal selected from the platinum group are incubated for 15 to 20 mins at 35 to 37°C and then flushed with H2 for 5 to 10 mins. In one embodiment the cells were incubated at 35 to 37 °C overnight with an overpressure of 1 bar H2 originating the biogenic platinum group metal nanoparticles. In one embodiment the metal from the platinum group is selected from Pd, Pt, Rh or Ru.

In one embodiment, the biogenic metal nanoparticles selected from the platinum group is added between 2 and 10 ml of suspension .

In one embodiment, the step of incubating the harvested anaerobic bacteria at room tempersature (22-25°C) during 5- 10 min comprising the biogenic CdSMoS2 nanoparticles at the surface, together with the biogenic nanoparticles of metals from the platinum group, comprises the doping of the CdSMoS2 biohybrids with biogenic platinum-group metal nanoparticles. In one embodiment this can be performed by using 20 to 25 mL of anaerobic bacteria comprising the biogenic CdSMoS2 nanoparticles at the surface, which are centrifuged and resuspended in 400 to 600 pl of a solution of 20 to 50 m Tris-HCL pH 6.8 to 7.8, 2 to 3 mM HCL-Cys teine , and 0.5 to 1.0 pM resazurine. In parallel 2 to 10 ml of a suspension of the biogenic nanoparticles from metals selected from the platinum group is also centrifuged and resuspended in 100 to 300 pl of the same solution and added to the concentrated bacteria-CdSMoS2 biohybrids, thus generating the metal-doped biohybrids of the present invention.

The total volume of the metal-doped biohybrids was introduced in anaerobic serum bottles containing 5 to 8 mL of a photocatalytic solution comprising 20 to 50 mM Tris-HCl pH= 6.8 to 7.8, 0.5 to 1 pM resazurin as anaerobic indicator and 20 to 100 mM cysteine as sacrificial electron donor and a pH of 2 to 3 ± 0.3. For the photocatalytic process the serum bottles containing the biohybrids were irradiated with light. The source of light can be violet LED, sunlight or other light source that emits visible light.

Example :

The sulphate reducing bacteria can be grown in modified Postgate C medium containing 1 g.L ^-1 NH4CI, 2.5 g.L ^-1 Na ₂ SO4, 0.06 g.L- ¹ CaCl ₂ .2H ₂ O, 0.06 g.L ^-1 MgSO ₄ .7H ₂ O, 1 g.L ^-1 yeast extract, 0.0071 g.L ^-1 FeSO4.7H ₂ O, 0.3 g L ^-1 sodium citrate tribasic dehydrate, 0.1 g.L ^-1 ascorbic acid, 0.1 g.L ^-1 sodium thioglycolate, 4.5 g L ^-1 sodium lactate, 0.16 mg In ¹ resazurin, nickel chloride (1 pM) and sodium selenite (1 pM) . Bacterial growth was carried out at 37 °C for a time between 16 and 19 hours in static conditions. Other bacteria can be used like Shewanella oneidensis, or others. In this case a suitable medium should be used to assure the anaerobic growth of these bacteria.

The doping of CdSMoS ₂ nanoparti cles with biogenic platinumgroup metal nanoparticles was performed using 20-25 mL of cells-CdSMoS ₂ nanoparticles and 2-10 ml of biogenic platinum- group metal nanoparticles, which were centrifuged, resuspended in a small volume (100-200 ul) and joined to form the biohybrids .

After that, the biohybrid is introduced in anaerobic serum bottles (with a total volume of 11 mL) containing 6 mL of photocatalytic solution composed by 20 mM Tris-HCl, 1 ± 0.2 pM resazurin as anaerobic indicator and 20-100 mM cysteine as sacrificial electron donor and a pH of 2.5 ± 0.3. For the photocatalytic process the serum bottles containing the biohybrids are irradiated with light. The source of light can be violet LED, sunlight or other light source that emits visible light .

Figure 1 describes the process involved in the production of added-value products from light by the biohybrid composed by D. desulfur! cans-CdSMoS2 dopped with BioPd . The biogenic CdSMoS2 dopped with BioPd captures light energy generating low redox potential high-energy electrons that are trans ferred to the bacterial cells . At the surface of these cells there are redox proteins that capture these high energy electrons and trans fer them across the external membrane to hydrogenases located in the periplasm of the cel ls . These enzymes use the electrons and couple them with protons to produce H2 that freely crosses the membrane and is released . These electrons could be also trans ferred to other enzymes like formate dehydrogenases allowing to produce formate in the presence of light and CO2 . BioPd works as a secondary cocatalyst which increases H2 production by lowering the activation energy for hydrogen production . It can also contribute by capturing the photogenerated electrons which contributes to suppress electron-hole recombination .

Figure 2 shows the characteri zation of D. desulf uri cans- CdSMoS2 by SEM and SEM-EDS . These images show the formation of CdSMoS2 nanoparticles at surface of the cel ls . Some agglomerates were also observed on the surface of the cells . The SEM-EDS demonstrated that these precipitates are composed by Mo , S and Cd, showing the success ful synthesis of D. desulfur! cans-CdSMoS2. The Osmium used in the preparation of the samples for SEM-EDS analysis ( the protocol for preparation of cells for SEM-EDS is described by Martins et al , 2021 ) was also detected by SEM-EDS . Figure 3 shows the characteri zation of BioPd, the biogenic platinum-group metal nanoparticles produced by the bacteria, by SEM and SEM-EDS . These images show the formation of round precipitates at the surface of the cells . Some agglomerates were also observed on the surface of the cells . The SEM-EDS demonstrated that these precipitates are composed by only Pd demonstrating the success ful synthesis of BioPd .

Figure 4 shows results of hydrogen production by the di f ferent biohybrids . The H2 production is performed under irradiation with a violet LED which emits visible light (X= 445nm) with an irradiance of 0 . 4 W nr ² . In this study 81 mM of cysteine is used as sacri ficial electron donor . The H2 production is evaluated using 23 ml of D. desulf url cans- CdSMoS2 and 2 mL of BioPd . The results demonstrate the high improvement on H2 production by the new biohybrid D. desulfur! cans-CdSMoS2+BioPd . After 140 h o f light irradiation the biohybrid D. desulf url cans-CdS i s able to produce 60 pmol H2 while the D. desulf url cans-CdSMoS produce 100 pmol H2 . A further improvement is achieved when the D. desulf url cans-CdSMoS is dopped with BioPd allowing to produce 140 pmol of H2 after this time . The BioPd works as a cocatalyst for H2 production and by improving the charge separation of the semiconductor CdSMoS . Moreover, BioPd may also be involved in accelerating the trans fer of electrons to the cells enhancing the performance of the biohybrid .

Figure 5 shows data of H2 production by biohybrid dopped with di f ferent biogenic metals namely Nickel sul fide , tungsten sul fide and molybdenum sul fide . The data are obtained after 44 h of light incubation with a violet LED (X= 445nm, 0 . 4 W nr ² ) . In this study 15 mM of cysteine are used as sacri ficial electron donor . The H2 production is evaluated using 23 ml of each biohybrid. The biohybrids are synthesized with 3 mM Cd (cells-CdS ₂ ) , 3mM Cd+0.1 mM Ni ( cells-CdSNiS ) , 3 mM Cd+0.5 mM W (cells-CdSWS ₂ ) and 3 mM Cd+0.1 mM Mo (cells-CdSMoS ₂ ) . These results demonstrate that besides the combination of CdS with M0S2, other metal sulfides biologically produced can be combined with CdS and used to improve the H2 production performance of microbial-CdS biohybrids.

Figure 6 shows the profile of H2 production of biohybrids constructed with different microorganisms. The data are obtained when the biohybrids are incubated with a violet LED (X= 445nm, 0.4 W 1m ² ) . The H2 production is evaluated using 23 ml of each biohybrid dopped with 2 mL of BioPd. The biohybrids Desulfovibrio desulf uricans and Desulfovi bri o vulgaris are synthesized with 3 mM Cd and 0.1 mM Mo while Shewanella oneidensis is synthesized with 1 mM Cd and 0.1 mM Mo. The results demonstrate that independently of the microorganism used as biocatalyst, the microbial- CdSMoS2+BioPd has an improved performance for H2 production from light comparing with a simple microbial-CdS biohybrid. An improvement of 3-fold, 17-fold and 6-fold was observed for D. desulf uricans , Desulfovi bri o vulgaris and Shewanella oneidensis biohybrids, respectively, after 70 h of light irradiation .

Figure 7 shows the performance of the new biohybrid for the production of value products other than H2. Theses assays are conducted under irradiation with a violet LED with an irradiance of 3.8 W 1m ² and a pH= 7.410.2. In this study 81 mM of cysteine are used as sacrificial electron donor and 100 mM of bicarbonate as CO2 source. The CO2 reduction is evaluated using 23 ml of D. desulfur! cans-CdSMoS2 and 2 mL of BioPd. These results demonstrate that besides the potential for H2 production the biohybrid system of the present invention can also be used to produce other valuable compounds namely formate and acetate from CO2 and light. In fact, the new biohybrid is able to produce simultaneously 9 mM of formate 5 mM acetate and 3.5 pM hydrogen. These results indicate that the photo-exited electrons can be transferred to different enzymes inside the cell.

Example 1 : D. desulfuricans-CdSM.oS2+BioPd biohybrid

1) Synthesis of D. desulf uricans-CdSMoS2 :

D. desulf uricans is first grown in medium containing 40 mM lactate as carbon source and 20 mM sulfate to produce the H2S required for the synthesis of CdSMoS2. After that, 3 Mm cadmium is slowly added followed by the slow addition of 0.1 mM molybdenum. The cells are then incubated during 3 hours at 37°C originating the D. Desulfur! cans-CdSMoS2 biohybrid. The D. desulfur! cans-CdSMoS2 is constructed in the presence of 3 mM of Cd and 0.1 mM Mo.

2) Synthesis of BioPd

Palladium nanoparticles (Pd°) are synthesized biologically by the same microorganism, originating the BioPd (using the method described by Martins et al, 2017) . BioPd is produced in the presence of 100 mg/L of Pd (II) .

This description is of course not in any way restricted to the forms of implementation presented herein and any person with an average knowledge of the area can provide many possibilities for modification thereof without departing from the general idea as defined by the claims. The preferred forms of implementation described above can obviously be combined with each other. The following claims further define the preferred forms of implementation.

Bibliography

1 - Martins, M., Toste, C., Pereira, I.A.C., 2021. Enhanced light-driven hydrogen production by self-photosensitized biohybrid systems. Angew. Chem. Int. Ed. 60, 2-10;

2 - Wang, B., Zeng, C., Chu, K.H., Wu, D., Yip, H.Y., Ye, L., Wong, P.K., 2017. Enhanced Biological Hydrogen Production from Escherichia coli with Surface Precipitated Cadmium Sulfide Nanoparticles. Adv. Energy Mater. 7, 1700611;

3 - Jiang, Z . , Wang, B., Yu, J.C., Wang, J., An, T., Zhao, H., Li, H., Yuan, S., Wong, P.K., 2018. AglnS2/In2S3 heterostructure sensitization of Escherichia coli for sustainable hydrogen production. Nano Energy 46, 234-240.