Field of the invention

The present invention relates to a method of producing polyhydroxyalkanoates (PHAs) in a cost-efficient way using organic acids resulting from hydrothermal treatment of biomass.

Technical Background

PHAs, polyhydroxyalkanoates is a family of renewable and biodegradable polymers produced and degraded by many naturally occurring bacteria [1], PHA can be biodegraded in compost, soil, marine and freshwater environment [1], In contrast to other bioplastics, PHA consists of about 150 molecular variants and has big potential to be processed into different biomaterials tuned to the specific application, from large volume consumables to low volume niche products [2], [3], In order for PHA to compete with fossilbased plastics and current bioplastics, the PHA production cost needs to be reduced in the currently available processes [4]— [6]. The use of underutilized streams and mixed microbial cultures have gained interest in research, as it enables cost reductions in comparison to single-substrate monocultures and homogenous feedstocks [7]— [9].

Many different industrial waste streams have been tested with varying degrees of PHA yield [10], Forest industry residual streams have been investigated for PHA production due to their large availibility, but often need to be pre-treated to make the material available for fermentation into volatile fatty acids (VFA) [11], [12], which are then used as substrates in the selection of biomass and PHA accumulation [13]— [15].

Woody biomass, or lignocellulosic material in general, consists of three main components in various ratios; cellulose, lignin and hemicellulose. The three components have different properties, degradation products and thermal stability. All three have decomposition pathways that leads to organic acids [29], that can serve as suitable feed for bacteria capable of PHA accumulation. The hemicellulose is the least thermally stable out of the three, and typically starts hydrolysing into degradation products during hydrothermal treatment already at temperatures above 150 degrees C [17], A significant portion of the degradation products created consist of acetic acid, which is a volatile fatty acid (VFA). Acetic acid is one of the most common VFA in PHA production and produces the homopolymer PHB [18], A water phase containing the water soluble organic acids (VFAs) resulting from hydrothermal treatment of woody biomass can be used directly as substrate in a PHA accumulation step [19], [20],

There are a number of ways to hydrothermally treat biomass. Below follows a list of potential treatments, and then a more detailed description for each:

• Acidic Hydrolysis

• Enzymatic Hydrolysis

• Hydrothermal Liquefaction (HTL)

• Hydrothermal Carbonisation (HTC)

• Biomass fractionation using ionic liquids (IL)

• Biomass fractionation using organic solvents (Organosolv)

Hydrolysis is a broad term that implies that water is involved in breaking chemical bonds of a substrate. The hydrolysis process can be assisted by adding acids, like sulfuric acid (acidic hydrolysis), or enzymes (enzymatic hydrolysis). Acidic hydrolysis is used within the forest industry to pretreat woody biomass. The pretreatment enables the hemicellulose to be broken down and dissolved, later on resulting in a purer cellulose product, like in the case of making dissolving pulp [31], The mechanics of acidic hydrolysis also occurs in the sulfite pulping process, similarly resulting in degradation products coming from hemicellulose, like organic acids [33], Water containing these organic acids (VFAs) can be fed directly to bacteria capable of using the feedstock to accumulate PHA.

Hydrothermal liquefaction (HTL), in some cases also called hydropyrolysis, and Hydrothermal carbonisation (HTC) are processes based on water acting as a catalyst for depolymerizing organic content at high temperature and pressure in an oxygen-free environment. For HTL, the temperature ranges from 260-360 degress C, with a pressure range of 50-250 bars. For HTC, the temperature ranges from 180-260 degrees C, with a pressure range of 10-80 bar. A liquid oil and a solid char-like material are typically formed in the process [17], The resulting proportion of oil obtained is typically higher at higher temperature and pressure [17], At lower pressure (20-50 bar) and temperature (180-260 °C), the main product is solid in the form of biochar [16], The process is well suited for handling feedstock with a significant water content (80-95 % and 70-90 %, respectively^ 6]), such as various industrial waste sludges. System and method for hydrothermal treatment of sludge are described in the patent applications with publication numbers WO 2017/003358 and WO 2010/112230. During the process, the various components of the material is decomposed, whereof a significant portion is dissolved and follows the process water. The VFA content of the process water can be fed to a reactor for PHA accumulation.

Several methods have been developed to fractionate lignocellulosic biomass into it’s main components cellulose, lignin and hemicellulose. One proposed method is to dissolve the lignin and hemicellulose fraction using organic solvents to isolate the cellulose. The method was first developed during the 1960s as an alternative to the kraft pulping process, and a number of branded processes has been developed since then, like the Organocell and Alcell processes. They are collectively referred to as organosolv processes. The organosolv core process typically works in a temperature range close to 200 degrees Celsius and in a pressure range of 20-30 bar, which implies that part of the lignocellulosic material is broken down into various degradation products, like organic acids, that dissolves in the solvent/water phase of the process.

Progress has been made in fractionating biomass using Ionic Liquids (ILs). Ionic Liquids are defined as salts in liquid state at an arbitrary temperature, usually below 100 degree Celsius. Ionic Liquids can selectively dissolve part of the biomass, like lignin and hemicellulose, for isolation of cellulose pulp. ILs selected for this purpose typically exhibits very low vapour pressure and high thermal stability, and stays intact for easy recovery. Process conditions can range between 150-200 degrees C. The high production cost of ILs have been hindering the commercialization of IL based fractionation, but recent development has resulted in low cost ILs that still exhibits the same functionality [32], As in previous cases, the conditions promotes degradation of part of the lignocellulosic material into organic acids that can be fed directly to a PHA accumualtion process.

Woody biomass is a suitable feedstock for the above mentioned processes, and beneficial to the purpose of this innovation because of its high hemicellulose content. Hemicellulose is partly broken down into organic acids at relatively mild conditions, and is, in the case of high purity cellulose production, an unwanted component that is broken down and removed in commercially available processes.

Other side streams that are readily available can serve as suitable starting material for the above mentioned processes. Fiber sludge coming from a pulping process has a low nitrogen content (0.1 -0.5 % dry substance (DS) for primary pulp and paper sludge, 3.3-7.0 % DS for pulp and paper biosludge [21]) and a high proportion of hemicellulose (4.9-14.2 % DS for primary pulp and paper sludge and 12.0-15.0 % DS for pulp and paper biosludge[22]), which favors the creation of organic acids in a hydrothermal treatment process, and especially the conversion to acetic acid [16],

In most installations of PHA production, a separate acidogenic and anaerobic fermentation step is used to digest the starting material into organic acids suitable for the PHA accumulation step. The anaerobic fermentation demands closed reactors and is a cost-driving step in the overall PHA production process [23], [24], If the starting material is lignocellulosic, the material might also need to be pretreated to make the cellulose and hemicellulose available for the degrading organisms [21], Many microorganisms can degrade cellulose and hemicellulose, but untreated lignocellulosic material is however very resistant to microbial activity, which results in very long process retention times [25], Cellulose and hemicellulose can be depolymerized into its monomer components, sugars, which are the most easily fermented materials, but depolymerizing cellulose to monomers is an energy-intensive process [22], The fermentation retention time depends on how accessible and easily fermented the lignocellulosic material is [21], A trade off must be made between pre-treatment of the material and the length of retention time in the fermentation step [21], Sewage sludge is also considered a promising raw material for PHA production, and is also an attractive material for producing biochar in an HTC process. Sewage sludge does however not contain significant amounts of VFA for direct use, thus the sewage sludge also needs to be fermented [26], Sewage sludge typically also contain significant amounts of nitrogen, which acts as an inhibitor in the PHA accumulation process and needs to be decreased [20],

In addition to pre-treatment and fermentation, the raw material itself accounts for a large part of the cost [27], Most commercial processes are based on pure vegetable oils or sugars as a raw material [28], There are however commercial ambitions to use forest based raw materials as well. In the patent WO2019CA51797, a process is described using forest industry residues, a fermentation step and a monoculture, which, however, results in a costly process.

One aim of the present invention is to provide a cost-efficient process to produce PHA by using an already existing infrastructure to retrieve organic acids originating from low-nitrogen forest industrial waste streams with a significant proportion of hemicellulose that without fermentation can be used directly as a substrate in a mixed microbial culture PHA accumulation step.

Summary of the invention

The purpose stated above is achieved by a method comprising the production of polyhydroxyalkanoates (PHAs) from an organic material, wherein the organic material has undergone hydrothermal treatment. In a most general aspect, the present invention is directed to a method comprising providing a hydrothermally treated organic material; and producing polyhydroxyalkanoates (PHAs) from the hydrothermally treated organic material. The method is based on using organic material as a feedstock for intracellular PHA accumulation, where the organic material has been made suitable for the microbial process by first undergoing hydrothermal treatment. The present invention has the advantage of being able to use the process water from a hydrothermal treatment process, which in many cases is considered a waste stream, directly as a feedstock for PHA production.

According to the present invention, there is no need to use an independent pre-treatment of lignocellulosic biomass to make the material available for digestion in a fermentation step, which in its turn can result in organic acids suitable for PHA production. By using an underutilized waste stream from already existing installations, significant cost reductions can be achieved.

Embodiments of the invention

In one embodiment of the invention, the organic material are fibre residues resulting from a pulping process and are collected and treated in a hydrothermal carbonisation (HTC) plant, with the main purpose of the plant to produce biochar. The HTC process generates large amounts of process water, where up to 40% of the organic material going into the process is dissolved or suspended in the water phase. The organic material in the water phase adds cost to the HTC biochar production process by demanding a wastewater treatment solution to release the water to a recipient. In this embodiment, the temperature of the hydrothermal treatment is kept at 200 °C and the pressure at 20 bar, which results in the hemicellulose of the starting material decomposing into organic acids with various carbon chain length. Due to using fibre residues from a pulping process in this embodiment, which have a low nitrogen content, means that only limited amounts of ammonia are formed. Further in this embodiment, a mixed microbial culture (MMC), originating from the wastewater treatment plant of a pulp and paper mill, is fed with a portion of the HTC process wastewater, and exposed to feast and famine cycles to selectively promote a culture that is capable of significant PHA accumulation. After the selection process, the biomass produced is fed with another portion of the HTC process water for a final PHA accumulation. The PHA rich biomass is then treated in another process to extract the PHA from the biomass for further usage.

Below some embodiments of the present invention are disclosed and discussed further.

According to one embodiment, the hydrothermal treatment provides decomposition of at least a fraction of the organic material into organic acids, said organic acids preferably being suitable as a carbon source for PHA accumulating bacteria. In this regard it should be noted that the hydrothermal treatment may be seen as step occurring before the method according to the present invention, or may be part of the method as such. In line with this, according to one embodiment of the present invention, the method comprises a step of hydrothermal treatment of a starting organic material and which step provides decomposition of the starting organic material into organic acids, said organic acids being suitable as a carbon source for PHA accumulating bacteria.

Furthermore, according to one embodiment of the present invention, the hydrothermally treated organic material is separated in a solid phase and an aqueous phase. See fig. 1 where one example is presented showing this possible separation. Moreover, according to yet another embodiment, the aqueous phase of the hydrothermally treated organic material has a total acetic acid concentration of at least 1 g per kg of water. The acid concentration is of relevance for the possible PHA accumulating in one or more subsequent steps according to the present invention.

According to yet another embodiment, the temperature of the hydrothermal treatment is kept in a range of 150 - 400 degrees C, more preferably in a range of 170 - 300 degrees Celsius. Furthermore, according to one specific embodiment of the present invention, the pressure of the hydrothermal treatment is kept in a range of 10 - 250 bars, more preferably in a range of 10 - 50 bars.

According to yet another embodiment, wherein the starting organic material has a nitrogen content < 20% by dry weight, preferably < 10% by dry weight, more preferably < 5% by dry weight, before entering the hydrothermal treatment. In this regarding, the starting organic material should be understood to be the material provided before the hydrothermal treatment.

Moreover, according to one embodiment, the starting organic material has a hemicellulose content > 3% by dry weight, preferably > 5% by dry weight, before entering the hydrothermal treatment.

According to one specific embodiment, the starting organic material originates from a pulping process.

Furthermore, in accordance with yet another embodiment, the step of producing PHAs are performed by means of a mixed microbial culture (MMC). Moreover, according to yet another embodiment, the mixed microbial culture (MMC) originates from a wastewater treatment plant of a pulp and/or paper mill.

According to yet another embodiment of the present invention, the step of producing PHAs are performed free from an intermediate fermentation step between the hydrothermal treatment and the PHA accumulation.

Furthermore, according to yet another embodiment, the aqueous phase of the hydrothermally treated material is divided into two streams, wherein one stream is fed to a reactor for producing biomass capable of accumulating PHA, and the other stream is fed to a reactor containing biomass that accumulates PHA.

The organic acids included in the material being processed according to the present invention may originate from different pre-processes. According to one embodiment, the organic acids suitable for PHA production originates from an acid hydrolysis of organic material in a sulfite pulping process. According to yet another embodiment, the organic acids suitable for PHA production originates from an acidic hydrolysis pretreatment step of organic material in a pulping process. Moreover, according to yet another embodiment of the present invention, the organic acids suitable for PHA production originates from an enzymatic hydrolysis pretreatment step of organic material in a pulping process. Furthermore, according to one other embodiment, the organic acids suitable for PHA production originates from an organosolv process of organic material. According to yet another embodiment, the organic acids suitable for PHA production originates from an Ionic Liquid based process of organic material.

It should be noted that all types of combinations of different embodiments mentioned above are possible according to the present invention.

Description of the drawing

In fig. 1 one embodiment of the present invention is shown.

Initially, organic material of fibre- and biosludge (1.), that is a starting organic material as defined in the claims of the present invention, is hydrothermally treated (A.) to form organic acids prior to microbial conversion. The hydrothermal water phase containing formed organic acids is separated from residual organic material (2.), the resulting organic acid-rich aqueous streams (3.) is sent to aerobic biomass production (B.) and (4.) to aerobic PHA accumulation (C.), respectively. In the aerobic biomass production (B.), the organic acids (3.) are used to select and enrich a mixed microbial culture (MCC) of PHA-accumulating biomass under feast and famine conditions. The enriched PHA-accumulating biomass (6.) is separated from residual water (5.) and sent to the PHA accumulation step (C.). In the PHA accumulation (C.), the organic acid-rich aqueous stream (4.) is used as carbon source for the microorganisms in the PHA-accumulating biomass (6.) for the organic acid- PHA conversion. From the PHA accumulation (C.), PHA-rich biomass (8.) is separated from residual water (7.) and sent for PHA extraction (D.). Extracted PHA is separated from residual organic material (9.) and a pure extract of PHA (10.) is obtained.

In relation to above it should be noted that different types of separation technologies are possible to implement in the method according to the present invention. Moreover, to combine different separation technologies for different separation steps are also fully possible. References

[1] E. Bugnicourt, P. Cinelli, A. Lazzeri, and V. Alvarez, “Polyhydroxyalkanoate (PHA): Review of synthesis, characteristics, processing and potential applications in packaging,” Express Polymer Letters, vol. 8, no. 11 , pp. 791- 808, 2014, doi: 10.3144/expresspolymlett.2014.82.

[2] Bauchmuller Verena et al., “BioSinn Products for which biodegradation makes sense,” Hurth, May 2021. [Online], Available: www. renewable- carbon. eu/publications

[3] C. Consultants, “Polyhydroxyalkanoates: plastic the way nature intended?,” pp. 1-12, 2018, [Online], Available: https://www.cambridgeconsultants.com/sites/default/files/upl oaded-pdfs/PHA - plastic the way nature intended_1 .pdf

[4] Y. K. Leong, P. L. Show, J. C. W. Lan, H. S. Loh, H. L. Lam, and T. C. Ling, “Economic and environmental analysis of PHAs production process,” Clean Technologies and Environmental Policy, vol. 19, no. 7, pp. 1941-1953, 2017, doi: 10.1007/sl 0098-017-1377-2.

[5] C. Fernandez-Dacosta, J. A. Posada, R. Kleerebezem, M. C. Cuellar, and A. Ramirez, “Microbial community-based polyhydroxyalkanoates (PHAs) production from wastewater: Techno-economic analysis and ex-ante environmental assessment,” Bioresource Technology, vol. 185, pp. 368-377, Jun. 2015, doi: 10.1016/j.biortech.2015.03.025.

[6] Y. K. Leong et al., “Preliminary integrated economic and environmental analysis of polyhydroxyalkanoates (PHAs) biosynthesis,” Bioresources and Bioprocessing, vol. 3, no. 1 , Dec. 2016, doi: 10.1186/s40643-016-0120-x.

[7] K. Khatami, M. Perez-Zabaleta, I. Owusu-Agyeman, and Z. Cetecioglu, “Waste to bioplastics: How close are we to sustainable polyhydroxyalkanoates production?,” Waste Management, vol. 119. Elsevier Ltd, pp. 374-388, Jan. 01 , 2021. doi: 10.1016/j.wasman.2020.10.008.

[8] C. Kourmentza et al., “Recent advances and challenges towards sustainable polyhydroxyalkanoate (PHA) production,” Bioengineering, vol. 4, no. 2, pp. 1- 43, 2017, doi: 10.3390/bioengineering4020055.

[9] R. Ganesh Saratale et al., “A comprehensive overview and recent advances on polyhydroxyalkanoates (PHA) production using various organic waste streams,” Bioresource Technology, vol. 325. Elsevier Ltd, Apr. 01 , 2021. doi: 10.1016/j.biortech.2021 .124685.

[10] M. Li and M. R. Wilkins, “Recent advances in polyhydroxyalkanoate production: Feedstocks, strains and process developments,” International Journal of Biological Macromolecules, vol. 156, pp. 691-703, Aug. 2020, doi: 10.1016/j. ijbiomac.2020.04.082.

[11] C. Veluchamy and A. S. Kalamdhad, “Influence of pretreatment techniques on anaerobic digestion of pulp and paper mill sludge: A review,” Bioresource Technology, vol. 245. Elsevier Ltd, pp. 1206-1219, 2017. doi:

10.1016/j.biortech.2017.08.179.

[12] N. Ali, Q. Zhang, Z. Y. Liu, F. L. Li, M. Lu, and X. C. Fang, “Emerging technologies for the pretreatment of lignocellulosic materials for bio-based products,” Applied Microbiology and Biotechnology, vol. 104, no. 2. Springer, pp. 455-473, Jan. 01 , 2020. doi: 10.1007/s00253-019-10158-w. [13] M. G. E. Albuquerque, V. Martino, E. Pollet, L. Averous, and M. A. M. Reis, “Mixed culture polyhydroxyalkanoate (PHA) production from volatile fatty acid (VFA)-rich streams: Effect of substrate composition and feeding regime on PHA productivity, composition and properties,” Journal of Biotechnology, vol. 151 , no. 1 , pp. 66-76, Jan. 2011 , doi: 10.1016/j.jbiotec.2010.10.070.

[14] S. Bengtsson and A. Werker, “The production of biopolymers for bioplastics using pulp and paper mill wastewater and residual fibre streams,” Lomma, Sverige, Dec. 2020.

[15] C. S. S. Oliveira, C. E. Silva, G. Carvalho, and M. A. Reis, “Strategies for efficiently selecting PHA producing mixed microbial cultures using complex feedstocks: Feast and famine regime and uncoupled carbon and nitrogen availabilities,” New Biotechnology, vol. 37, pp. 69-79, Jul. 2017, doi: 10.1016/j.nbt.2016.10.008.

[16] L. Leng et al., “Valorization of the aqueous phase produced from wet and dry thermochemical processing biomass: A review,” Journal of Cleaner Production, vol. 294. Elsevier Ltd, Apr. 20, 2021. doi:

10.1016/j.jclepro.2021 .126238.

[17] D. Lachos-Perez, P. Cesar Torres-Mayanga, E. R. Abaide, G. L. Zabot, and F. de Castilhos, “Hydrothermal carbonization and Liquefaction: differences, progress, challenges, and opportunities,” Bioresource Technology, vol. 343, p. 126084, Jan. 2022, doi: 10.1016/j.biortech.2021 .126084.

[18] M. Singh, P. Kumar, S. Ray, and V. C. Kalia, “Challenges and Opportunities for Customizing Polyhydroxyalkanoates,” Indian Journal of Microbiology, vol. 55, no. 3. Springer India, pp. 235-249, Sep. 06, 2015. doi: 10.1007/s12088- 015-0528-6.

[19] R. Moita and P. C. Lemos, “Biopolymers production from mixed cultures and pyrolysis by-products,” Journal of Biotechnology, vol. 157, no. 4, pp. 578-583, Feb. 2012, doi: 10.1016/j.jbiotec.2011 .09.021 .

[20] S. Wijeyekoon, C. R. Carere, M. West, S. Nath, and D. Gapes, “Mixed culture polyhydroxyalkanoate (PHA) synthesis from nutrient rich wet oxidation liquors,” Water Research, vol. 140, pp. 1-11 , Sep. 2018, doi:

10.1016/j.watres.2O18.04.017.

[21] T. Meyer and E. A. Edwards, “Anaerobic digestion of pulp and paper mill wastewater and sludge,” Water Research, vol. 65. Elsevier Ltd, pp. 321-349, Nov. 15, 2014. doi: 10.1016/j.watres.2O14.07.022.

[22] R. Kaur, R. D. Tyagi, and X. Zhang, “Review on pulp and paper activated sludge pretreatment, inhibitory effects and detoxification strategies for biovalorization,” Environmental Research, vol. 182. Academic Press Inc., Mar. 01 , 2020. doi: 10.1016/j.envres.2O19.109094.

[23] W. S. Lee, A. S. M. Chua, H. K. Yeoh, and G. C. Ngoh, “A review of the production and applications of waste-derived volatile fatty acids,” Chemical Engineering Journal, vol. 235. pp. 83-99, Jan. 01 , 2014. doi:

10.1016/j.cej.2O13.09.002.

[24] M. Atasoy, I. Owusu-Agyeman, E. Plaza, and Z. Cetecioglu, “Bio-based volatile fatty acid production and recovery from waste streams: Current status and future challenges,” Bioresource Technology, vol. 268. Elsevier Ltd, pp. 773-786, Nov. 01 , 2018. doi: 10.1016/j.biortech.2018.07.042.

[25] K. Kucharska, P. Rybarczyk, I. Holowacz, R. Lukajtis, M. Glinka, and M. Kaminski, “Pretreatment of lignocellulosic materials as substrates for fermentation processes,” Molecules, vol. 23, no. 11. MDPI AG, Nov. 10, 2018. doi: 10.3390/molecules23112937.

[26] C. Samori, A. Kiwan, C. Torri, R. Conti, P. Galletti, and E. Tagliavini, “Polyhydroxyalkanoates and Crotonic Acid from Anaerobically Digested Sewage Sludge,” ACS Sustainable Chemistry and Engineering, vol. 7, no. 12, pp. 10266-10273, Jun. 2019, doi: 10.1021/acssuschemeng.8b06615.

[27] S. N. Mudliar, A. N. Vaidya, M. Suresh Kumar, S. Dahikar, and T.

Chakrabarti, “Techno-economic evaluation of PHB production from activated sludge,” Clean Technologies and Environmental Policy, vol. 10, no. 3, pp. 255-262, 2008, doi: 10.1007/s10098-007-0100-0.

[28] R. Muthuraj, O. Valerio, and T. H. Mekonnen, “Recent developments in short- and medium-chain- length Polyhydroxyalkanoates: Production, properties, and applications,” International Journal of Biological Macromolecules, vol.

187. Elsevier B.V., pp. 422-440, Sep. 30, 2021. doi: 10.1016/j.ijbiomac.2021.07.143.

[29] D. Chena, K. Cenb, X. Zhuang et. al, ’’Insight into biomass pyrolysis mechanism based on cellulose, hemicellulose, and lignin: Evolution of volatiles and kinetics, elucidation of reaction pathways, and characterization of gas, biochar and bio-oil”

[30] Fiorella P. Cardenas-Toro, Sylvia C. Alcazar-Alay, Tania Forster-Carneiro, M. Angela A. Meireles, “Obtaining Oligo-and Monosaccharides from Agroindustrial and Agricultural Residues Using Hydrothermal Treatments”

[31] Vena, P. F. et al. “Dilute sulphuric acid extraction of hemicelluloses from Eucalyptus Grandis and its effect on kraft and soda-AQ pulp and handsheet properties.” Cellulose Chemistry and Technology, vol. 49, nr 9-10, ss. 819- 832.

[32] Florence J. V. Gschwend, Agnieszka Brandt-Talbot, Clementine L. Chambon, and Jason P. Hallett, “Ultra-Low Cost Ionic Liquids for the Delignification of Biomass”

[33] Martin G. Wolfinger, Herbert Sixta, “Modeling of the acid sulfite pulping process”, Lenzinger Berichte, 83 (2004) 35-45