Culture method of purple light-nutrition bacteria and its application

Abstract

The present invention provides a method for culturing purple phototrophic bacteria (PPB), the method comprising the steps of: (a) feeding a mixed bacterial population comprising PPB and wastewater into a tubular reactor; (b) circulating the bacterial population and wastewater in the tubular reactor under near-infrared (NIR) illumination for a period of time and harvesting a portion of the resulting biomass; and (c) repeating step (b). The present invention also provides the biomass obtained from the method, which can be used for plant cultivation.

Description

Technical Field

[0001] This invention provides a method for culturing purple phototrophic bacteria. This invention also provides biomass obtained by this method. Background Technology

[0002] The previously published documents listed or discussed in this specification should not necessarily be regarded as an admission that such documents are part of the prior art or are common general knowledge.

[0003] With the global population growing and the demand for food becoming increasingly urgent, the development of edible plant cultivation has been driven. However, soil-borne plant pathogens pose a threat to all aspects of agriculture, impacting crop health, yield, and overall soil quality. These microorganisms, including bacteria, fungi, nematodes, and viruses, can cause plant diseases, reduce nutrient utilization, and impair the overall productivity of agricultural systems. Various methods, such as the application of fungicides and pesticides, have been tried to control soil pathogens. However, these methods face increasingly stringent scrutiny due to environmental concerns and the potential development of resistant strains. Therefore, developing effective and sustainable methods to control soil pathogens and improve agricultural yields is crucial.

[0004] In recent years, beneficial microorganisms (such as some bacteria or fungi) have been introduced into the soil to provide a natural form of control by competing with or directly attacking harmful pathogens. Among them, *Rhodopseudomonas palustris* (…) Rhodopseudomonas palustriPurple non-sulphur bacteria (PPB) have demonstrated superiority in improving agricultural production (see Sakarika et al., 2020. Purple non-sulphur bacteria and plant production: benefits for fertilization, stress resistance and the environment. Microb Biotechnol 13(5), 1336-1365). Due to their diverse metabolic functions, PPBs can utilize a wide range of substrates, including both organic and inorganic compounds (see Brown et al., 2022. Rhodopseudomonas palustris: A biotechnology chassis. Biotechnol Adv, 108001). Furthermore, they can convert low-value substrates into high-value products, such as plant hormones (indole-3-acetic acid) and vitamins, which can promote plant growth. Furthermore, it can produce 5-aminolevulinic acid (5-ALA), which can serve as an antimicrobial substance (see Capson-Tojo et al., 2020. Purple phototrophicbacteria for resource recovery: Challenges and opportunities. Biotechnol Adv43, 107567.; Dhar et al., 2023. Anoxygenic phototrophic purple non-sulfurbacteria: tool for bioremediation of hazardous environmental pollutants. World J Microbiol Biotechnol 39(10), 283). Routine cultivation of *Rhodopseudomonas palustris* is expensive and involves complex procedures due to the need for strict sterilization and the synthesis of substrates / sterile substrates. Simultaneously, the slow growth of biomass limits its widespread application. Despite the increasing interest in the use of PPB for plant growth, there are currently no existing methods for developing low-cost PPB culture methods or their practical applications in supporting plant growth.

[0005] Therefore, alternative and / or improved methods for culturing purple phototrophic bacteria (PPB) are needed. Summary of the Invention

[0006] The various aspects and embodiments of the present invention will now be described with reference to the following numbered clauses.

[0007] 1. A method for culturing purple phototrophic bacteria (PPB), the method comprising the following steps: (a) Feeding a mixed microbial community containing PPB and wastewater into a tubular reactor; (b) Under near-infrared (NIR) irradiation, the microbial community and wastewater in the tubular reactor are circulated for a period of time, and a portion of the resulting biomass is harvested; and (c) Repeat step (b).

[0008] 2. The method according to Clause 1, wherein the tubular reactor, which was in its initial state prior to step (a), was not sterilized.

[0009] 3. The method according to Clause 1 or Clause 2, wherein the surface area to volume ratio of the tubular reactor is 20 m². 2 / m 3 up to 70 m 2 / m 3 For example, 30 m 2 / m 3 up to 50 m 2 / m 3 For example, 33.3 m 2 / m 3 Up to 40 m 2 / m 3 .

[0010] 4. The method according to any one of the preceding clauses, wherein the NIR optical density in the tubular reactor is 10 W / m². 2 Up to 40 W / m 2 For example, 15 W / m 2 Up to 30 W / m 2 For example, approximately 21 W / m 2 .

[0011] 5. The method according to any one of the preceding clauses, wherein the purple phototrophic bacteria (PPB) comprises Rhodopseudomonas palustris (… Rhodopseudomonas palustris Optionally, after 5 to 7 days of operation of the method, the relative abundance of Rhodopseudomonas palustris is 50% to 90% of the total bacterial community, for example 70% to 85%, for example about 80%.

[0012] 6. The method according to any one of the preceding clauses, wherein the wastewater is food processing wastewater, and optionally the food processing wastewater includes soybean wastewater.

[0013] 7. The method according to any one of the foregoing clauses, wherein one or more of the following apply: (i) The tubular reactor comprises 5 to 30 tubular vessels, such as 10 to 14 horizontal tubular vessels, wherein the tubular vessels are optionally connected in series. (ii) The diameter of each tubular container is 10 mm to 100 mm, for example 20 mm to 60 mm, for example 28 mm to 60 mm; and (iii) The length of each tubular container is from 500 mm to 5,000 mm, for example from 1,000 mm to 2,800 mm.

[0014] 8. The method according to any one of the preceding clauses, wherein the wavelength of the near-infrared (NIR) irradiation is from 800 nm to 1,000 nm, for example, about 850 nm.

[0015] 9. The method according to any one of the preceding clauses, wherein the time period is 5 to 7 days.

[0016] 10. The method according to any one of the preceding clauses, wherein the method is operated in a semi-continuous mode with a hydraulic retention time (HRT) of 10 to 40 days, for example 15 to 30 days, for example about 20 days, optionally wherein one to three cleaning operations are performed per HRT cycle to remove bacteria adhering to the inner wall of the tubular reactor.

[0017] 11. The method according to any one of the foregoing clauses, wherein one or more of the following apply: (ai) Total chemical oxygen demand (TCOD) is 5 g / L to 20 g / L, for example 6 g / L to 15 g / L, for example about 7 g / L, for example 7.04 g / L; (aii) Soluble chemical oxygen demand (SCOD) is 0.01 g / L to 10 g / L, for example 0.05 g / L to 7 g / L, for example 4.2 g / L to 5 g / L, for example about 4.28 g / L, for example 0.1 g / L to 0.5 g / L, for example about 0.1 g / L; (aiii) The organic loading rate in step (b) is from 0.1 g COD / L / d to 5 g COD / L / d, for example from 0.5 g COD / L / d to 2 g COD / L / d, for example about 0.56 g COD / L / d; (aiv) from ammonia (NH4) + The nitrogen concentration provided by (-N) is from 10 mg / L to 200 mg / L, for example 20 mg / L to 100 mg / L, for example 25 mg / L to 50 mg / L, for example about 28 mg / L; (av) is composed of phosphate (PO4) 3--P) provides phosphorus concentrations from 50 mg / L to 200 mg / L, for example from 70 mg / L to 100 mg / L, for example about 72.97 mg / L; (avi) The biomass concentration before harvest is at least 2.0 g total soluble solids (TSS) / L; (avii) Biomass production ranging from 0.5 g total solids / g COD consumed to 1 g total solids / g COD consumed; and (aviii) The ratio of COD consumption to ammonia is 10:1 to 100:1, for example 20:1 to 40:1, for example about 30:1.

[0018] 12. The method according to any one of the preceding clauses, wherein the biomass obtained by step (b) comprises one or more of 2-hydroxyindole-3-acetic acid, indole-3-acetic acid (IAA), L-tryptophan, L-phenylalanine and indole-3-carboxaldehyde, and more particularly, comprises 5-aminolevulinic acid, optionally wherein the biomass obtained by step (b) comprises 2-hydroxyindole-3-acetic acid, indole-3-acetic acid (IAA), L-tryptophan, L-phenylalanine and indole-3-carboxaldehyde and 5-aminolevulinic acid.

[0019] 13. A biomass obtained by the method according to any one of claims 1 to 12, wherein the biomass comprises one or more of 2-hydroxyindole-3-acetic acid, indole-3-acetic acid (IAA), L-tryptophan, L-phenylalanine, and indole-3-carboxaldehyde, and more particularly, comprises 5-aminolevulinic acid, optionally wherein the biomass obtained by step (b) comprises 2-hydroxyindole-3-acetic acid, indole-3-acetic acid (IAA), L-tryptophan, L-phenylalanine, and indole-3-carboxaldehyde, and 5-aminolevulinic acid.

[0020] 14. The biomass as described in Clause 13, wherein the biomass is provided in dried form or as a bacterial solution.

[0021] 15. A method for cultivating plants, the method comprising: (bi) Provide biomass obtained under any of Clauses 1 to 12 or biomass described under Clause 13 or Clause 14, and plants in soil or liquid culture medium; and (bii) Adding the biomass to one or both of the plant and soil, or to one or both of the plant and liquid culture medium, to increase the growth rate of the plant.

[0022] 16. Use of biomass obtained under any of Clauses 1 to 12 or biomass described under Clause 13 or Clause 14 for the purpose of increasing the growth rate of plants. Attached Figure Description

[0023] Figure 1 This is a photograph of a laboratory-scale tubular photoreactor device (10L) according to Embodiment 1 of this disclosure.

[0024] Figure 2 Includes (a) a photograph of a pilot-scale tubular photoreactor device (250L), (b) a graph of soluble chemical oxygen demand (SCOD) (mg / L) of influent and effluent relative to time (days) according to Example 2 of this disclosure, and (c) bacterial abundance in the photoreactor according to Example 2 of this disclosure.

[0025] Figure 3 The following are the cultivation results of (a) amaranth and (b & c) rice after the addition of PPB solution, according to Example 3 of this disclosure. Figure 3 In (c), CK represents Hogland nutrient solution, T1 represents Hogland nutrient solution + 5% PPB (by volume), and T2 represents Hogland nutrient solution + 10% PPB (by volume). Detailed Implementation

[0026] It has been unexpectedly discovered that by feeding a mixed microbial community containing purple phototrophic bacteria (PPB) and wastewater into a tubular reactor, circulating the microbial community and wastewater within the reactor under energy-efficient near-infrared (NIR) light-emitting diodes (LEDs, e.g., 850 nm), and harvesting a portion of the resulting biomass, a method can be obtained that reduces the cost of PPB cultivation and increases biomass yield. Most unexpectedly, this method allows the use of non-sterile conditions, significantly reducing the cost of PPB cultivation compared to conventional methods that require sterilization of the system. Furthermore, food processing wastewater can be used as a substrate instead of synthetic substrates, further reducing costs. Tubular reactors with a high surface area-to-volume ratio can be used to increase biomass yield. The harvested biomass can also be used for plant cultivation, as demonstrated by the embodiments disclosed herein, which improve plant growth rates.

[0027] Therefore, in a first aspect of the present invention, a method for culturing purple phototrophic bacteria (PPB) is provided, the method comprising the following steps: (a) Feeding a mixed microbial community containing PPB and wastewater into a tubular reactor; (b) Circulating the microbial community and wastewater in the tubular reactor under near-infrared (NIR) irradiation for a period of time, and harvesting a portion of the resulting biomass; and (c) Repeat step (b).

[0028] In the embodiments described herein, the word "comprising" may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word "comprising" may also refer to a situation where only the listed components / features are intended to be present (e.g., the word "comprising" may be replaced by the phrases "consisting of" or "substantially consisting of"). It is clearly anticipated that both broader and narrower interpretations can be applied to all aspects and embodiments of the invention. In other words, the word "comprising" and its synonyms may be replaced by the phrases "consisting of" or "substantially consisting of" or their synonyms, and vice versa.

[0029] The phrase "basically composed of..." and its derivatives in this article can be interpreted as referring to materials that may contain small amounts of impurities. For example, the purity of the material can be greater than or equal to 90%, such as greater than 95%, greater than 97%, greater than 99%, greater than 99.9%, greater than 99.99%, greater than 99.999%, or 100%.

[0030] As used herein, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” include plural references. Thus, for example, reference to “a condensation polymer” includes a mixture of two or more such compositions; reference to “the catalyst” includes a mixture of two or more such catalysts, and so on.

[0031] When used herein, the term "tubular reactor" refers to any suitable reactor for culturing PPB that is tubular or substantially tubular in shape. In some embodiments, the tubular reactor, in its initial state prior to step (a), may not have been sterilized (e.g., by autoclaving, by solvent (e.g., ethanol or isopropanol), by chemical sterilization, or by UV, X-ray, or gamma irradiation). Advantageously, this reduces the cost of culturing PPB compared to conventional methods that require sterilization.

[0032] Tubular reactors can have any suitable surface area to volume ratio. However, those with high surface area to volume ratios (e.g., >10 m²) are believed to be suitable. 2 / m 3 This can be beneficial because it can increase the interaction between the microbial community and NIR light. In some embodiments, the surface area to volume ratio of the tubular reactor can be 20 m². 2 / m 3 up to 70 m 2 / m 3 For example, 30 m 2 / m 3 up to 50 m 2 / m 3In some exemplary embodiments, the surface area to volume ratio of the tubular reactor can be 33.3 m². 2 / m 3 up to 40m 2 / m 3 For example, approximately 33.3 m 2 / m 3 or about 40 m 2 / m 3 Advantageously, the high surface area to volume ratio increases the photosynthetic efficiency of purple phototrophic bacteria, which in turn increases biomass production.

[0033] As used herein, the term “about” may allow for a degree of variability in a value or range, for example, within 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% of the limits of the value or range, and includes the exact value or range.

[0034] Tubular reactors can be configured as a single tubular vessel, but this may be impractical for large-scale reactions and for vessels manufactured where a smaller footprint than a linear tube would occupy is desired (e.g., by bending the tube itself). With this in mind, tubular reactors can be formed from multiple tubular vessels. These vessels can be connected in series, or they can operate individually in parallel with each other, or a combination of both. In some embodiments, multiple tubular vessels can be connected in series. In some embodiments that may be mentioned herein, the tubular reactor can comprise 5 to 30 tubular vessels, such as 10 to 14 horizontal tubular vessels. As mentioned above, these tubular vessels can be connected in series. As will be understood, the interconnections can be any suitable interconnection that allows the tubular reactor to operate, and there are no particular limitations, as long as they connect the tubular vessels together.

[0035] When used herein, each tubular vessel forms part of a tubular reactor. These tubular vessels may have any suitable diameter, which may be selected to allow NIR light to substantially penetrate the vessel. In this context, "substantially" can mean that the NIR light penetrates into the interior of the tubular vessel to achieve a depth sufficient for the desired selection and growth of PPB within the operating time range of the process. As will be understood, the actual diameter chosen will depend on the intensity of the light source under discussion and can therefore vary depending on the reactor designer's design for a particular reactor. However, in some embodiments that may be mentioned herein, the diameter of each tubular vessel may be from 10 mm to 100 mm, for example from 20 mm to 60 mm, or for example from 28 mm to 60 mm.

[0036] Aside from practical considerations, there are no particular limitations on the length of each tubular reactor. For example, the length of each tubular vessel can be from 500 mm to 5,000 mm, or from 1,000 mm to 2,800 mm.

[0037] Advantageously, these sizes increase biomass yield and the scalability of the method for industrial-scale applications (e.g., plant cultivation).

[0038] It should be understood that tubular vessels (and therefore essentially tubular reactors) should be constructed of materials that have the highest possible transmittance to NIR (e.g., transmittance >95%, e.g., >99%, e.g., >99.9%).

[0039] When used herein, the term "near-infrared (NIR)" refers to the near-infrared region of the electromagnetic spectrum (e.g., 780 nm to 2500 nm, such as 850 nm). In some embodiments, the wavelength of near-infrared (NIR) irradiation may be from 800 nm to 1000 nm. In some exemplary embodiments, the wavelength of NIR irradiation may be about 850 nm.

[0040] To provide adequate growth and selection for PPB, NIR should be provided with sufficient optical density, which can be easily determined by a technician. Any suitable NIR optical density can be used. For example, the NIR optical density in a tubular reactor could be 10 W / m². 2 Up to 40 W / m 2 For example, 15 W / m 2 Up to 30 W / m 2 In some exemplary embodiments, the optical density of NIR in a tubular reactor can be approximately 21 W / m². 2 .

[0041] When used in this article, the term "purple phototrophic bacteria (PPB)" refers to a group of phototrophic Gram-negative proteobacteria that produce their own food through photosynthesis. PPBs are colored by bacterial chlorophyll a or b, as well as various carotenoids, which gives them colors ranging from purple, red, brown, and orange.

[0042] In some embodiments, the purple phototrophic bacteria (PPB) may include Rhodopseudomonas palustris. As demonstrated in the Examples section of this disclosure, after operating the method for 5 to 7 days, the relative abundance of Rhodopseudomonas palustris can be 50% to 90% of the total bacterial community, for example 70% to 85%, for example about 80%.

[0043] When used herein, the term "wastewater" refers to any suitable wastewater that has been used for various applications (e.g., domestic, commercial, or as part of an industrial process such as an agricultural process) but can still be used for PPB cultivation. In some embodiments, the wastewater may be food processing wastewater. In some exemplary embodiments, food processing wastewater may include soybean wastewater. In some preferred embodiments, the wastewater may contain organic material for PPB growth, nitrogen (from ammonia), and phosphorus (from phosphate).

[0044] As will be understood, a skilled worker may determine the timeframe for implementing the method based on their knowledge of the art and whether training has been deemed complete. In some implementations, the timeframe may be 5 to 7 days.

[0045] As demonstrated in the Embodiments section of this disclosure, the method can be operated in a semi-continuous mode with a hydraulic retention time (HRT) of 10 to 40 days, for example 15 to 30 days, such as about 20 days. As will be understood, operators can vary the HRT outside these values ​​based on their experience. When used herein, the term "hydraulic retention time (HRT)" refers to the time that wastewater resides in the tubular reactor. In some embodiments, one to three cleaning operations may be performed per HRT cycle to remove bacteria adhering to the inner walls of the tubular reactor.

[0046] In some implementation schemes, one or more of the following may be applicable: (ai) Total chemical oxygen demand (TCOD) is 5 g / L to 20 g / L, for example 6 g / L to 15 g / L, for example about 7 g / L, for example 7.04 g / L; (aii) Soluble chemical oxygen demand (SCOD) is 0.01 g / L to 10 g / L, for example 0.05 g / L to 7 g / L, for example 4.2 g / L to 5 g / L, for example about 4.28 g / L, for example 0.1 g / L to 0.5 g / L, for example about 0.1 g / L; (aiii) The organic loading rate in step (b) can be from 0.1 g COD / L / d to 5 g COD / L / d, for example from 0.5 g COD / L / d to 2 g COD / L / d, for example about 0.56 g COD / L / d; (aiv) from ammonia (NH4) + The nitrogen concentration provided by (-N) is from 10 mg / L to 200 mg / L, for example 20 mg / L to 100 mg / L, for example 25 mg / L to 50 mg / L, for example about 28 mg / L; (av) is composed of phosphate (PO4) 3--P) provides phosphorus concentrations from 50 mg / L to 200 mg / L, for example from 70 mg / L to 100 mg / L, for example about 72.97 mg / L; (avi) The biomass concentration before harvest is at least 2.0 g total soluble solids (TSS) / L; (avii) Biomass production ranging from 0.5 g total solids / g COD consumed to 1 g total solids / g COD consumed; and (aviii) The ratio of COD consumption to ammonia is 10:1 to 100:1, for example 20:1 to 40:1, for example about 30:1.

[0047] As will be understood, the parameters above are suggestions only, and skilled operators may vary them beyond the provided range when necessary.

[0048] As will be understood, various determinations of wastewater (TCOD, SCOD, nitrate concentration, phosphorus concentration, and biomass concentration, etc.) can be readily determined by those skilled in the art using conventional techniques and equipment (e.g., titration, UV-Vis spectrophotometry, or commercially available test kits / equipment).

[0049] When used herein, the term "biomass" refers to the biological content in a reactor and may include material produced by the growth of a mixed microbial community, plus the bacteria themselves (e.g., PPB) and their metabolites, such as hormones. In some embodiments, the biomass obtained by step (b) may comprise one or more of 2-hydroxyindole-3-acetic acid, indole-3-acetic acid (IAA), L-tryptophan, L-phenylalanine, and indole-3-carboxaldehyde, and more particularly, 5-aminolevulinic acid. In some exemplary embodiments, the biomass obtained by step (b) may comprise 2-hydroxyindole-3-acetic acid, indole-3-acetic acid (IAA), L-tryptophan, L-phenylalanine, and indole-3-carboxaldehyde, as well as 5-aminolevulinic acid.

[0050] As demonstrated in the Embodiments section of this disclosure, purple phototrophic bacteria have been found to secrete (plant) growth hormones. Therefore, in a second aspect of the invention, a biomass obtained by the method disclosed above is provided, wherein the biomass comprises one or more of 2-hydroxyindole-3-acetic acid, indole-3-acetic acid (IAA), L-tryptophan, L-phenylalanine, and indole-3-carboxaldehyde, and more particularly, 5-aminolevulinic acid. In some exemplary embodiments, the biomass may comprise 2-hydroxyindole-3-acetic acid, indole-3-acetic acid (IAA), L-tryptophan, L-phenylalanine, and indole-3-carboxaldehyde, as well as 5-aminolevulinic acid.

[0051] In some embodiments, the biomass may be provided in dried form or as a bacterial solution. As will be understood, the precise method of preparing the biomass, whether in dried form or as a bacterial solution, is not particularly important and will be apparent to those skilled in the art.

[0052] As demonstrated in the Embodiments section of this disclosure, purple phototrophic bacteria have been found to enhance plant growth by providing nutrients, secreting growth hormones, and increasing stress resistance, while inhibiting the growth of plant pathogens while increasing the abundance of probiotics. Therefore, in a third aspect of the invention, a method for cultivating plants is provided, the method comprising: (bi) Provide biomass obtained according to the above-described method of this disclosure, and plants in soil or liquid culture medium; and (bii) Adding biomass to one or both of the plant and the soil, or to one or both of the plant and the liquid culture medium, to increase the plant's growth rate.

[0053] Other aspects and embodiments of the invention will now be discussed with reference to the following non-limiting examples.

[0054] Example

[0055] Example 1 - PPB culture (laboratory-scale performance (10 L))

[0056] PPB can simultaneously assimilate organic matter, nitrogen, and phosphorus in waste streams through photoheterotrophic growth under anaerobic / photothermal conditions. Therefore, light absorption efficiency is an important factor in biomass yield. A continuous-mode fermenter was successfully operated to cultivate PPB. However, the fermenter's low surface area-to-volume ratio (m²) was a challenge. 2 / m 3 This hinders processing capacity and limits its biomass yield. This is due to the low surface area exposed to the light source, resulting in low photosynthetic efficiency. Therefore, a high surface area-to-volume ratio (40 m²) is required. 2 / m 3 A tubular photoreactor was designed for this application. This tubular photoreactor consists of multiple horizontal tubular containers with a total working volume of 10 L. Figure 1 As shown, raw wastewater collected from a local food processing plant will be used as feed to the photobioreactor. The total chemical oxygen demand (TCOD) in the wastewater is 7.04 g / L, the soluble chemical oxygen demand (SCOD) is 4.28 g / L, and the soluble chemical oxygen demand (SOD) is composed of ammonia (NH4+). + The nitrogen concentration provided by (-N) is 28 mg / L and is provided by phosphate (PO4). 3-The phosphorus concentration provided by the -P reactor was 72.97 mg / L. A total of 0.8 L of digestate was removed daily and replaced with an equal volume of fresh 0.8 L of raw wastewater, resulting in a total organic loading rate (OLR) of 0.56 g COD / L / d. The results showed that the biomass concentration in the reactor stabilized at 2.0 g total suspended solids (TSS) / L, with a biomass yield of approximately 0.7 g total solids (TS) / g COD consumed. The tubular reactor exhibited a significantly higher OLR than other reactor types, demonstrating its superiority in cultivating PPB biomass.

[0057] Example 2 - PPB Culture (Pilot-scale Performance (250 L))

[0058] like Figure 2 As shown in Figure a, the PPB pilot-scale system comprises a 200-liter tubular photoreactor for cultivating PPB. This reactor includes 14 stacked tubes (dimensions: 60 mm diameter × 2800 mm length), a 50-liter buffer tank, and a programmable logic controller (PLC) control panel. The surface area to volume ratio of the tubular photoreactor is approximately 33.3 m². 2 / m 3 The reactor uses a wavelength of 850 nm and an optical density of 21 W / m². 2 An infrared (IR) light source was used to support its operation and PPB enrichment. The reactor operated in a semi-continuous mode with a hydraulic retention time (HRT) of 20 days. Due to the phototaxis of PPB, they grow on the reactor walls; therefore, the attached biomass was periodically removed by wiping the inner surface once per HRT cycle. In the first few days of operation, the soluble chemical oxygen demand (SCOD) of the effluent fluctuated but gradually stabilized. After 100 days, the SCOD remained stable at approximately 100 mg / L. Figure 2 As shown in b, the ratio of COD consumption to ammonium is approximately 30:1. Figure 2 As shown in c, *Rhodopseudomonas palustris* was the dominant species in the reactor, reaching a relative abundance of over 80%. In summary, the pilot-scale reactor demonstrated stable operation, effective PPB enrichment, and stable biomass production.

[0059] Example 3 - Plant Cultivation

[0060] PPB is increasingly used in various fields, such as soil remediation, wastewater treatment, aquaculture, and energy recovery. However, research on the role of PPB in promoting plant growth is limited. To fill this gap, it is hypothesized that PPB can function as a plant growth-promoting rhizosphere bacterium (PGPR), providing nutrients, secreting growth hormones, and enhancing stress resistance.

[0061] Plant hormone contents were detected using MetWare (http: / / www.metware.cn / ) on the AB Sciex QTRAP6500 LC-MS / MS platform. As shown in Table 1, the main plant hormones found in dried PPB biomass and bacterial cultures included 2-hydroxyindole-3-acetic acid, indole-3-acetic acid (IAA), L-tryptophan, L-phenylalanine, and indole-3-carboxaldehyde. The presence of these hormones indicates that PPB has the potential to significantly enhance plant growth.

[0062] Table 1. Major plant hormones in dried biomass and bacterial solutions.

[0063]

[0064] Several experiments were conducted using PPB in plant culture, including lettuce and amaranth. three-colored amaranth For soil culture, add 20 mL of PPB solution to 1 L of soil, or apply 1.1 L of PPB solution to 1 hectare of farmland. For hydroponic culture, add PPB solution to achieve a 5% volume ratio.

[0065] Regarding the promotion of lettuce growth: Two different operational methods were employed. In one method, PPB was dispersed on the soil, while in the other, the microorganisms were sprayed onto the leaves and roots. Results showed that the addition of PPB improved the overall growth performance of the edible plant, including the yield and quality of edible plant biomass. Analysis of the microbial community in the plant roots revealed that plant pathogens such as Pantotheca pineapple (…) Pantoea ananatis ) can be effectively inhibited, and some probiotics such as Pseudomonas mournikosa ( Pseudomonas mosselii ) and Bacillus belesi ( Bacillus velezensis The relative abundance of PPB increases. Compared with the control group, the method of the present invention shows an overwhelming advantage in terms of cost and edible plant growth performance. That is, the biomass not only provides common nutrients that are slowly released and fertilize the soil, but also generates added value by containing substances that promote plant growth, which are not found in conventional fertilizers. This indicates that PPB biomass cultured using the method of the present invention has the potential to be developed into probiotics.

[0066] Regarding the promotion of amaranth and rice growth: Compared with treatments using chemical fertilizers, PPB inoculation showed a significant promoting effect on amaranth growth. This result highlights that PPB application increases amaranth yield and shortens its growth cycle (see [link to relevant documentation]). Figure 3 (a) Furthermore, PPB inoculation promoted the growth of rice with higher chlorophyll content ( Figure 3 (b) and 3(c)).

[0067] in conclusion

[0068] This method can transform low-quality wastewater into high-quality value-added products (in the form of probiotics) instead of sludge requiring additional treatment costs. This method not only treats wastewater but also generates commercially valuable products, making it an economically viable solution. The method disclosed herein enables the production of new and reliable products that are aligned with and support Singapore's Sustainable Development Goals. It should be noted that the application of this technology as probiotics is still in its early stages, and further exploration of each specific application is an iterative process requiring close collaboration between research and industry.

Claims

1. A method for culturing purple phototrophic bacteria (PPB), the method comprising the following steps: (a) Feeding a mixed microbial community containing PPB and wastewater into a tubular reactor; (b) Under near-infrared (NIR) irradiation, the microbial community and wastewater in the tubular reactor are circulated for a period of time, and a portion of the resulting biomass is harvested; and (c) Repeat step (b).

2. The method according to claim 1, wherein the tubular reactor, which is in its initial state prior to step (a), has not been sterilized.

3. The method according to claim 1 or claim 2, wherein the surface area to volume ratio of the tubular reactor is 20 m². 2 / m 3 up to 70 m 2 / m 3 For example, 30 m 2 / m 3 up to 50 m 2 / m 3 For example, 33.3 m 2 / m 3 Up to 40 m 2 / m 3 .

4. The method according to any one of the preceding claims, wherein the NIR optical density in the tubular reactor is 10 W / m². 2 Up to 40 W / m 2 For example, 15 W / m 2 Up to 30 W / m 2 For example, approximately 21 W / m 2 .

5. The method according to any one of the preceding claims, wherein the purple phototrophic bacteria (PPB) comprises Rhodopseudomonas palustris (… Rhodopseudomonas palustris ), among which, After 5 to 7 days of operation of the method, the relative abundance of Rhodopseudomonas palustris is 50% to 90% of the total bacterial community, for example 70% to 85%, for example about 80%.

6. The method according to any one of the preceding claims, wherein the wastewater is food processing wastewater, and optionally the food processing wastewater includes soybean wastewater.

7. The method according to any one of the preceding claims, wherein one or more of the following are applicable: (i) The tubular reactor comprises 5 to 30 tubular vessels, such as 10 to 14 horizontal tubular vessels, wherein the tubular vessels are optionally connected in series. (ii) The diameter of each tubular container is from 10 mm to 100 mm, for example from 20 mm to 60 mm, for example from 28 mm to 60 mm; and (iii) The length of each tubular container is from 500 mm to 5,000 mm, for example from 1,000 mm to 2,800 mm.

8. The method according to any one of the preceding claims, wherein the wavelength of the near-infrared (NIR) irradiation is from 800 nm to 1,000 nm, for example, about 850 nm.

9. The method according to any one of the preceding claims, wherein the time period is 5 to 7 days.

10. The method according to any one of the preceding claims, wherein the method is operated in a semi-continuous mode with a hydraulic retention time (HRT) of 10 to 40 days, for example 15 to 30 days, for example about 20 days, optionally wherein one to three cleaning operations are performed per HRT cycle to remove bacteria adhering to the inner wall of the tubular reactor.

11. The method according to any one of the preceding claims, wherein one or more of the following are applicable: (ai) Total chemical oxygen demand (TCOD) is 5 g / L to 20 g / L, for example 6 g / L to 15 g / L, for example about 7 g / L, for example 7.04 g / L; (aii) Soluble chemical oxygen demand (SCOD) is 0.01 g / L to 10 g / L, for example 0.05 g / L to 7 g / L, for example 4.2 g / L to 5 g / L, for example about 4.28 g / L, for example 0.1 g / L to 0.5 g / L, for example about 0.1 g / L; (aiii) The organic loading rate in step (b) is from 0.1 g COD / L / d to 5 g COD / L / d, for example from 0.5 g COD / L / d to 2 g COD / L / d, for example about 0.56 g COD / L / d; (aiv) from ammonia (NH4) + The nitrogen concentration provided by (-N) is from 10 mg / L to 200 mg / L, for example 20 mg / L to 100 mg / L, for example 25 mg / L to 50 mg / L, for example about 28 mg / L; (av) is composed of phosphate (PO4) 3- -P) provides phosphorus concentrations from 50 mg / L to 200 mg / L, for example from 70 mg / L to 100 mg / L, for example about 72.97 mg / L; (avi) The biomass concentration before harvest is at least 2.0 g total suspended solids (TSS) / L; (avii) Biomass production ranging from 0.5 g total solids / g COD consumed to 1 g total solids / g COD consumed; and (aviii) The ratio of COD consumption to ammonia is 10:1 to 100:1, for example 20:1 to 40:1, for example about 30:

1.

12. The method according to any one of the preceding claims, wherein the biomass obtained by step (b) comprises one or more of 2-hydroxyindole-3-acetic acid, indole-3-acetic acid (IAA), L-tryptophan, L-phenylalanine, and indole-3-carboxaldehyde, and more particularly, comprises 5-aminolevulinic acid, optionally wherein the biomass obtained by step (b) comprises 2-hydroxyindole-3-acetic acid, indole-3-acetic acid (IAA), L-tryptophan, L-phenylalanine, and indole-3-carboxaldehyde, and 5-aminolevulinic acid.

13. A biomass obtained by the method according to any one of claims 1 to 12, wherein the biomass comprises one or more of 2-hydroxyindole-3-acetic acid, indole-3-acetic acid (IAA), L-tryptophan, L-phenylalanine, and indole-3-carboxaldehyde, and more particularly, comprises 5-aminolevulinic acid, optionally wherein the biomass obtained by step (b) comprises 2-hydroxyindole-3-acetic acid, indole-3-acetic acid (IAA), L-tryptophan, L-phenylalanine, and indole-3-carboxaldehyde, and 5-aminolevulinic acid.

14. The biomass of claim 13, wherein the biomass is provided in dried form or as a bacterial solution.

15. A method for cultivating plants, the method comprising: (bi) Provide biomass obtained according to any one of claims 1 to 12 or biomass according to claim 13 or claim 14, and plants in soil or liquid culture medium; as well as (bii) Adding the biomass to one or both of the plant and soil, or to one or both of the plant and liquid culture medium, to increase the growth rate of the plant.

16. Use of biomass obtained according to any one of claims 1 to 12 or biomass according to claim 13 or 14 for increasing the growth rate of plants.

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