Modular 3d-printed bioreactor systems

Abstract

Disclosed is a modular and reconfigurable solid-support bioreactor utilizing 3D printed media beds for the growth and maturation of biofilms of organisms of interest for biomanufacturing, bioremediation, and scientific study. By leveraging additive manufacturing, the bioreactors and all components can be produced entirely through 3D printing at the point of need, with dimensions and features optimized for diverse applications. The modular design permits the end caps, reactor body sections, and media bed segments to be interchanged or replaced independently.

Description

MODULAR 3D-PRINTED BIOREACTOR SYSTEMSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to U.S. Provisional Patent Application Serial Number 63 / 756,727, filed February 10, 2025; which is incorporated by reference herein in its entirety into this disclosure.FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

[0002] The United States Government has ownership rights in this subject disclosure. Licensing inquiries may be directed to Office of Technology Transfer, US Naval Research Laboratory, Code 1004, 4555 Overlook Avenue SW, Washington, DC 20375, USA; +1.202.767.7230; techtran@nrl.navy.mil, referencing NC 212544.BACKGROUND OF THE SUBJECT DISCLOSUREField of the Subject Disclosure

[0003] The present subject disclosure relates generally to bioreactor systems.More particularly, the present subject disclosure relates to modular 3D-printed bioreactor systems.Background of the Subject Disclosure

[0004] Bioreactors are used to provide controlled environments for biological processes, enabling targeted growth of cells and microorganisms. Traditional designs often rely on planktonic suspensions, achieving mixing through mechanical stirring or gas sparging. While effective at the industrial scale, these approaches can suffer from lower cell densities and may require large volumes to compensate.

[0005] In contrast, packed bed bioreactors (a type of solid-support bioreactor) offer high surface areas for cell attachment and biofilm formation, which can lead to increased productivity. These systems also reduce washout of biomass and can allow continuous operation. However, drawbacks include diffusion limitations and the challenge of accessing or replacing the internal support media. Cleaning or regenerating such reactors can be complex, particularly when the media is not designed for easy removal.

[0006] Additive manufacturing (3D printing) has disrupted conventional methods by enabling rapid prototyping of complex, geometrically precise scaffolds.Researchers can now design support media with engineered porosity, high surface-to-volume ratios, and tailored flow paths, drastically improving mass transfer and biofilm growth. Despite these improvements, existing 3D-printed solid-support bioreactors remain monolithic, limiting their reusability and complicating sampling or media bed replacement.

[0007] Fixed bed bioreactors produced by Yates et al. [1] demonstrate the use of selective laser sintering (SLS) 3D printing to fabricate nylon-based monolithicreactor structures with optimized porosity. Biofilms of Marinobacter atlanticus successfully produced wax esters from non-sugar feedstocks under continuous flow. While this design showcased the potential of 3D-printed bioreactors, it was limited by single-use constraints and required destructive sampling (cutting open the reactor to access the biofilm). Assembly challenges also arose from removing excess printing material inside the reactor chambers. A U.S. patent application has been filed for this approach and is incorporated by reference herein in its entirety into this disclosure [2],

[0008] A need exists for more versatile, reusable and environmentally friendly bioreactor systems.SUMMARY OF THE SUBJECT DISCLOSURE

[0009] The present subject disclosure relates generally to bioreactor systems.

[0010] In an improvement over conventional systems, the present subject disclosure provides modular, reconfigurable, and reusable bioreactor systems that mitigate the major drawbacks of existing monolithic designs. By enabling easy disassembly and individual replacement of components, the system prevents resource-intensive disposal of the entire bioreactor after each run.

[0011] Presented in this disclosure is a modular and reconfigurable solid-support bioreactor utilizing 3D printed media beds for the growth and maturation of biofilms of organisms of interest for biomanufacturing, bioremediation, and scientific study. By leveraging additive manufacturing, the bioreactors and all components can be produced entirely through 3D printing at the point of need,with dimensions and features optimized for diverse applications. The modular design permits the end caps, reactor body sections, and media bed segments to be interchanged or replaced independently. This modularity enables some of the advantages provided herein, and others as recognized by one having ordinary skill in the art;1. Pre-seeding of biofilms prior to assembly in bioreactors.2. Easy sampling or analysis, by removing the media bed for biofilm composition studies.3. Inexpensive replacement of used media beds to allow reuse of the other reactor components, thus reducing waste and lowering operational costs.

[0012] The bioreactors can be sterilized using steam, UV, or chemical sterilants, ensuring high reliability and reproducibility. Materials are selected for biocompatibility to support the growth of even the most sensitive organisms. The reusability of the main reactor body and end caps, coupled with 3D-printed media beds designed for high surface area and optimized fluid flow, address the need for a scalable and versatile solution in bioprocessing.

[0013] In one exemplary embodiment, the present subject disclosure is a modular biofilm bioreactor system. The system includes a reusable reactor body defining an internal chamber and an access opening at at least one end; at least one removable end cap configured to close the access opening and comprising a fluid connector providing an inlet or outlet fluid path communicating with the internal chamber; and a removable media-bed assembly disposed within the internal chamber, the media-bed assembly comprising a plurality of discretemedia-bed segments arranged end-to-end along a longitudinal axis. Each mediabed segment comprises a three-dimensionally printed lattice scaffold defining interconnected voids forming a through-flow path for fluid contacting an attached biofilm. Adjacent media-bed segments include complementary mating features that align the segments and define a bypass-resistant interface inhibiting fluid from flowing around the lattice scaffolds, and alignment and / or indexing features configured to align adjacent segments and to permit maintaining a selected axial order and / or orientation. At least one media-bed segment includes a frangible plane, score, or separable seam configured to permit post-operation splitting of the segment into at least two portions for different analyses. At least one of the end cap(s) and the media-bed assembly are removable and replaceable independently, and the components are produced by 3D printing.

[0014] In another exemplary embodiment, the present subject disclosure includes a set of interchangeable end caps for a bioreactor body. Each end cap is produced by 3D printing and defines an external mating interface configured to removably couple to the bioreactor body and at least one external fluid connector configured to communicate with the internal chamber. The end cap may optionally include an integral dispersion plate, flow distributor, and / or static mixing structure.

[0015] In yet another exemplary embodiment, the present subject disclosure includes a method of assembling and operating a modular bioreactor system, including printing components, assembling a removable media-bed assembly from multiple media-bed segments, coupling end caps to establish a closed flowpath, operating the bioreactor, retrieving at least one media-bed segment, and splitting the retrieved segment along a separable seam for multiple analyses.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The patent or application file may contain at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0017] FIGS. 1A-1H show a modular, packed bed bioreactor of 10 mL internal volume and associated end cap, according to an exemplary embodiment of the present subject disclosure.

[0018] FIG. 1 A shows a perspective view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0019] FIG. 1 B shows a first end view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0020] FIG. 1 C shows a second end view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0021] FIG. 1 D shows a side view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0022] FIG. 1 E shows an internal side view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0023] FIG. 1 F shows a perspective view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0024] FIG. 1G shows a side view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0025] FIG. 1 H shows an internal side view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0026] FIGS. 2A-2I show a modular, packed bed bioreactor of 100 mL internal volume and associated end cap, according to another exemplary embodiment of the present subject disclosure.

[0027] FIG. 2A shows a perspective view of a bioreactor and end cap, according to an exemplary embodiment of the present subject disclosure.

[0028] FIG. 2B shows a first end view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0029] FIG. 2C shows a side view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0030] FIG. 2D shows an internal side view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0031] FIG. 2E shows a first end view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0032] FIG. 2F shows a side view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0033] FIG. 2G shows a perspective view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0034] FIG. 2H shows a second perspective view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0035] FIG. 2I shows an internal side view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0036] FIGS. 3A-3C show a threaded bioreactor body containing porous media beds with varying periodic structures, according to an exemplary embodiment of the present subject disclosure.

[0037] FIG. 3A shows a threaded bioreactor body containing porous media beds with internal solid octahedron supports, according to an exemplary embodiment of the present subject disclosure.

[0038] FIG. 3B shows a threaded bioreactor body containing porous media beds with internal hollow octahedron supports, according to an exemplary embodiment of the present subject disclosure.

[0039] FIG. 3C shows a threaded bioreactor body containing porous media beds with hexagonal mesh stacks, according to an exemplary embodiment of the present subject disclosure.

[0040] FIGS. 4A-4O show a modular, packed bed bioreactor of 10 mL internal volume with multiple modular components, according to an exemplary embodiment of the present subject disclosure.

[0041] FIG. 4A shows a first end view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0042] FIG. 4B shows a second end view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0043] FIG. 4C shows a first perspective view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0044] FIG. 4D shows a second perspective view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0045] FIG. 4E shows a side surface view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0046] FIG. 4F shows a side view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0047] FIG. 4G shows a perspective view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0048] FIG. 4H shows a side view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0049] FIG. 41 shows a side internal view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0050] FIG. 4J shows a first end view of an internal solid support, according to an exemplary embodiment of the present subject disclosure.

[0051] FIG. 4K shows a side vertical view of an internal solid support, according to an exemplary embodiment of the present subject disclosure.

[0052] FIG. 4L shows a perspective view of an internal solid support, according to an exemplary embodiment of the present subject disclosure.

[0053] FIG. 4M shows a second end view of an internal solid support, according to an exemplary embodiment of the present subject disclosure.

[0054] FIG. 4N shows a side horizontal view of an internal solid support,according to an exemplary embodiment of the present subject disclosure.

[0055] FIG. 40 shows a side internal view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0056] FIGS. 5A-5M show a modular, packed bed bioreactor of 100 ml_ internal volume with multiple modular components, according to an exemplary embodiment of the present subject disclosure.

[0057] FIG. 5A shows an end view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0058] FIG. 5B shows a perspective view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0059] FIG. 5C shows a side surface view of a bioreactor, according to an exemplary embodiment of the present subject disclosure.

[0060] FIG. 5D shows a first end view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0061] FIG. 5E shows a perspective view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0062] FIG. 5F shows a side surface view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0063] FIG. 5G shows a side internal view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0064] FIG. 5H shows a first end view of a periodic solid support, according to an exemplary embodiment of the present subject disclosure.

[0065] FIG. 5I shows a perspective view of a periodic solid support, according to an exemplary embodiment of the present subject disclosure.

[0066] FIG. 5J shows a side vertical surface view of a periodic solid support, according to an exemplary embodiment of the present subject disclosure.

[0067] FIG. 5K shows a second end view of a periodic solid support, according to an exemplary embodiment of the present subject disclosure.

[0068] FIG. 5L shows a side horizontal surface view of a periodic solid support, according to an exemplary embodiment of the present subject disclosure.

[0069] FIG. 5M shows a side internal view of a periodic solid support, according to an exemplary embodiment of the present subject disclosure.

[0070] FIGS. 6A-6D show periodic solid supports with internal frangible planes, according to an exemplary embodiment of the present subject disclosure.

[0071] FIG. 6A shows a first periodic solid support with attached internal frangible planes, according to an exemplary embodiment of the present subject disclosure.

[0072] FIG. 6B shows a first periodic solid support with separated internal frangible planes, according to an exemplary embodiment of the present subject disclosure.

[0073] FIG. 6C shows a second periodic solid support with attached internal frangible planes, according to an exemplary embodiment of the present subject disclosure.

[0074] FIG. 6D shows a second periodic solid support with separated internal frangible planes, according to an exemplary embodiment of the present subject disclosure.

[0075] FIGS. 7A-7C show an end cap, according to an exemplary embodiment of the present subject disclosure.

[0076] FIG. 7A shows a perspective view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0077] FIG. 7B shows a side view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0078] FIG. 7C shows an internal side view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0079] FIGS. 8A-8D show an end cap, according to an exemplary embodiment of the present subject disclosure.

[0080] FIG. 8A shows an end view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0081] FIG. 8B shows a perspective view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0082] FIG. 8C shows a side view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0083] FIG. 8D shows an internal side view of an end cap, according to an exemplary embodiment of the present subject disclosure.

[0084] FIG. 9A show various size and shape packed bed bioreactors, according to an exemplary embodiment of the present subject disclosure.

[0085] FIG. 9B show various size and shape packed bed bioreactors, according to an exemplary embodiment of the present subject disclosure.

[0086] FIG. 10 shows a conventional BIOPACS (Biological Point-of-need Advanced Chemical Synthesis) 3-D printed continuous flow fixed bed bioreactor.

[0087] FIG. 11A shows that by tracking the oxygen in the media going in (influent) and coming out (effluent) of the bioreactor, biofilm growth as a function of time in terms of oxygen utilization may be monitored.

[0088] FIG. 11 B shows fastest to slower growth of various cells.

[0089] FIG. 12A shows that changing the resin of the bioreactor from nylon to a more bio-compatible polymer encourages biofilm formation.

[0090] FIG. 12B shows time required for biofilm growth.

[0091] FIG. 12C shows M. atlanticus recovery on different resins.

[0092] FIG. 13A shows a modular design of a bioreactor with removable,perforated interior sections that enable the application of multiple sample analysis techniques from one bioreactor, even if they are destructive.

[0093] FIG. 13B shows biomass by bioreactor segment.

[0094] FIG. 14 shows % 02 over time for different samples.DETAILED DESCRIPTION OF THE SUBJECT DISCLOSURE

[0095] The present subject disclosure addresses the shortcomings of conventional bioreactors, as discussed above.

[0096] Definitions

[0097] Before describing the present subject disclosure in detail, it is to be understood that the terminology used in the specification is for the purpose of describing particular embodiments, and is not necessarily intended to be limiting. Although many methods, structures and materials similar, modified, or equivalent to those described herein can be used in the practice of the present subject disclosure without undue experimentation, the preferred methods, structures and materials are described herein. In describing and claiming the present subject disclosure, the following terminology will be used in accordance with thedefinitions set out below.

[0098] As used herein, the singular forms “a”, “an,” and “the” do not preclude plural referents, unless the content clearly dictates otherwise.

[0099] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0100] As used herein, the term “about” when used in conjunction with a stated numerical value or range denotes somewhat more or somewhat less than the stated value or range, to within a range of ±10% of that stated.

[0101] As used herein, the term “end cap” includes a lid, cap, or manifold configured to couple to an end of a reactor body and to provide at least one fluid connector in fluid communication with an internal chamber of the reactor body.

[0102] As used herein, the term "interchangeable end caps" refers to a plurality of end caps that are each configured to removably couple to a common reactor body.

[0103] As used herein, the term "sampling port" refers to a fluid connector or port configured to permit withdrawal of fluid from the internal chamber without disassembling the reactor body.

[0104] As used herein, the term "dispersion plate" refers to a plate, screen, or manifold region configured to distribute influent across a cross-section of an internal chamber and / or a media-bed assembly.

[0105] As used herein, the term "alignment feature" refers to a mating structure on a media-bed segment configured to align the segment relative to an adjacent segment and / or to the reactor body during assembly.

[0106] The following component label parts will be used consistently throughout the disclosure and drawings:101 bioreactor body (e.g., 15 mm)102A hose barb on bioreactor body102B hose barb on end cap103 periodic solid support104 male threaded element105 mesh filter106 end cap (e.g., 15 mm)107 dispersion plate108 O-ring retainer109 female threaded element110 reactor body (e.g., 45 mm)111 end cap (e.g., 45 mm)112A Luer lock on bioreactor body112B Luer lock on end cap112C Luer lock on sample port113 solid octahedron supports114 hollow octahedron supports115 hexagonal mesh stack116 hollow interior117 alignment feature118 insert (e.g., 15 mm)119 frangible plane120 sampling ports121 insert (e.g., 45 mm)123 bisected insert124 flow distributor125 sampling chamber

[0107] Overview

[0108] In a first exemplary embodiment, depicted in FIGS. 1A-1 H, a modular, packed bed bioreactor 100 (e.g., 10 mL internal volume) comprises two pieces, a threaded end cap 106 with a hose barb connector 102B, and a threaded reactor body 101 containing a porous media bed 103, fluid dispersion plate, and terminating in a barbed hose connector 102A. A gasket, such as an O-ring, is used to seal the threaded end cap 106 to the reactor body 101 upon connection of the two halves.

[0109] As shown in FIGS. 1A-1H, bioreactor 100 has a reusable reactor body 101 defining an elongated internal chamber about a longitudinal axis and having an access opening at at least one end (see FIG. 1A). At least one removable end cap 106 configured to close the access opening and including a fluid connector 102B provides an inlet or outlet fluid path communicating with the internal chamber, where a removable media-bed assembly 103 is disposed within. The media-bed assembly 103 may include a plurality of discrete media-bed segments (see FIG. 3C) arranged end-to-end along the longitudinal axis. Each media-bed segment 103 comprises a three-dimensionally printed, unitary lattice scaffolddefining a plurality of interconnected voids forming a through-flow path for fluid contacting an attached biofilm. Adjacent media-bed segments all include mating features that align the segments coaxially and define a bypass-resistant interface that inhibits fluid from flowing around, rather than through, the lattice scaffolds. The media-bed segments 103 include alignment and / or indexing features 117 configured to facilitate assembly and to indicate and / or maintain a selected axial order and / or orientation of the segments in the reactor body. At least one of the media-bed segments includes a frangible plane, score, or separable seam 119 configured to permit post-operation splitting of the segment into at least two portions for performing different analyses on material recovered from the segment (see FIGS. 6A-6D), wherein the media-bed assembly is removable from the reactor body without destroying the reactor body. Alignment feature 117 (body) and frangible plane 119 (segment) are mated features that constrain the orientation of segments relative to the body, ensuring alignment of segments with one another so that flow channels are aligned between segments. The feature on the media bed segments is part of the frangible plane feature, particularly the notch / seam forming the outer edge of each frangible plane feature.

[0110] As best shown in FIGS. 1 A-1 E, bioreactor 100 has a central reactor body 101 and a hose barb 102A on one end. The other end has a wide opening with a male threaded element 104. The wide opening is large enough to allow a solid support body 103 to be inserted and withdrawn within a central chamber therein. An internal mesh filter 105 may be positioned between the solid support body 103 and the barb 102A and serves to filter out components as desired.

[0111] As best shown in FIGS. 1F-1H, end cap 106 has a hose barb 102B (similar to hose barb 102A in bioreactor 101) and an internal concave portion with a female thread element 109 which is complementary to the male thread element 104 of bioreactor body 101. An O-ring retainer 108 positions an O-ring (not shown) to seal the connection between end cap 106 and bioreactor body 101. A dispersion plate 107 serves to distribute the flow evenly across the body of the solid support 103.

[0112] The various exemplary embodiments shown and discussed in the present subject disclosure have similar and shared components and, thus, each embodiment will not be described by each of its individual features when such features are also shown, identified and described in other embodiments. The features and functions identified by the same label are similar throughout all embodiments.

[0113] In a second exemplary embodiment, depicted in FIGS. 2A-2I, a modular, packed bed bioreactor contains an internal volume of 100 ml_, but, otherwise similar to the embodiment shown in FIG. 1. The structure and function of the components in FIGS. 2A-2I are the same as shown and described for FIGS. 1A- 1 H, other than the volume of the internal chamber of reactor body 101 and, thus, will not be repeated here.

[0114] FIGS. 3A, 3B, and 3C depict another exemplary embodiment of a modular bioreactor having a threaded bioreactor body 101 contains porous media beds with varying periodic structures 113, 114, 115, each possessing varied surface areas and flow characteristics. The reactor bodies additionally terminate in Luerlock connectors 112A. As shown in FIG. 3A, a solid octahedron support 113 spans the interior chamber of the bioreactor body 101. FIG. 3B shows a hollow octahedron support 114 spanning the interior chamber of the bioreactor body 101. FIG. 3C shows a series of hexagonal mesh discs stacked as a long tubular structure within the interior chamber of the bioreactor body 101. A combination of one or more of the structures 113, 114, 115 and others may also be possible.

[0115] FIGS. 4A-4O depict another exemplary embodiment of a modular packed bed bioreactor of 10 mL internal volume with multiple modular components. A threaded end cap 106 is shown with a fluid dispersion plate 107, O-ring retainer 108, female threaded element 109, and Luer lock connector 112B. A threaded reactor body 101 has Luer lock connector 112A. Porous media bed sections 118 may be inserted into the reactor body 101 prior to attachment of the end cap 106. One or more alignment features 117 may be used to mate an exterior portion of an insert 118 (e.g., 15 mm) with the hollow interior 116 of the bioreactor body 101. As shown best in FIGS. 4K, 4L, 4N, and 40, insert 118 has a frangible plane 119 that spans the entire longitudinal length of the insert 118, and serves to separate the insert into two or more components, as will be described in more detail.

[0116] FIGS. 5A-5M depict a modular, packed bed bioreactor of 100 mL internal volume with multiple modular components. In this embodiment, two threaded end caps 111 (e.g., 45 mm) with fluid dispersion plates 107, a reactor body threaded on both ends 104 (see FIG. 5C), and one or more porous media bed sections 121 are inserted into the reactor body 101 prior to attachment of the end caps106. One or more alignment features 117 and frangible planes 119 may also be included. One or more sampling ports 120, which may be in the configuration of a Luer lock 112C, may also be used to sample, introduce or withdraw fluids from within the reactor body 110 interior. In some embodiments, the alignment feature 117 and frangible plane 119 may coincide.

[0117] FIGS. 6A-6D show a periodic support 103 in various configurations. In FIGS. 6A-6B, a smaller (e.g., 15 mm) insert 118 is shown having a frangible plane 119, which is unseparated in FIG. 6A and partially separated in FIG. 6B as a bisected insert 123. Similarly, FIGS. 6C-6D shows a larger (e.g., 45 mm) insert 121 having a frangible plane 119, which is unseparated in FIG. 6C and partially separated in FIG. 6D as a bisected insert 123. In both sizes and shapes shown in FIGS, 6A-6D, the inserts 118, 121 are modular and easily insertable and removable from a bioreactor body, and once removed, the insert 118, 121 may be separated at frangible plane 119 to allow further and detailed examination of the surface of the cells within the interior passages of the solid support 103.

[0118] FIGS. 7A-7C show an exemplary embodiment of end cap 106 (e.g., 15 mm) having a dispersion plate 107 and flow distributor 124 (which can be mesh 105), which work together to promote an even distribution of fluid flow within the interior of the end cap 106 before the fluid passes through the mesh 105.

[0119] FIGS. 8A-8D show another exemplary embodiment of end cap 106 (e.g.,15 mm) having a sampling port 125 in the shape of a Luer lock 112C, projecting at a tangential position from its Luer lock 112B on the end cap 106. In this embodiment, an internal sampling chamber 125 allows the mixing of one or morefluids together before its flow through the interior of the bioreactor body. As shown best in FIG. 8D, one or more fluids from each of Luer lock ports 112B and 112C allow for a desired mixing of fluids before their flow through the bioreactor.

[0120] In exemplary embodiments, one or more end caps 106 are produced as monolithically 3D-printed manifolds that provide one or more external fluid connectors in fluid communication with an internal chamber 125. The end caps 106 may include an integral dispersion plate 107, flow distributor 124, and / or static mixing structure to promote uniform flowthrough the media-bed assembly. Different end caps 106 may provide different connector types and / or internal flow features and may be interchanged on a common reactor body.

[0121] FIGS 9A-9B show various styles, shapes, sizes and configurations of modular bioreactors 100 according to the present subject disclosure. It should be noted that the descriptions of components provided herein for a specific embodiment are not specific to (or limited to) any particular embodiment, and may be added to or removed from any bioreactor to produce a bioreactor of desired properties, as appreciated by one having ordinary skill in the art. Further, specific components (e.g., Luer locks or barb locks) are mere examples used for simplicity, and any suitable substitute (e.g., press fit, clips, etc.) may be used, as appreciated by one having ordinary skill in the art.

[0122] FIGS. 9A-9B show samples of a new bioreactor design according to the present subject disclosure. The primary improvements to the conventional bioreactor design include, for example:New 3D-printing resin to improve biocompatibility;New bead shape to improve biofilm formation;Screw top design (no more assembly / epoxy);Luer lock ports for convenient assembly; andIndividual, removable interior bead blocks to improve ease of analysis.

[0123] EXAMPLE 1

[0124] Packed bed bioreactors in the formats described herein were made from proprietary Formlabs Inc. resins using Formlabs Form 3 and Form 4 UV stereolithography 3D printers. The bioreactor components were made with a variety of resins, including Clear V4 and V5, BioMed Amber V1 , Biomed Durable V1.1 , and Biomed Black V1. Several examples were also made from a polyamide, Nylon 6,6, using a selective laser sintering (SLS) 3D printer. The reactors varied in length from 120 to 140 mm and possessed diameters ranging from 25 to 65 mm. Internal volumes ranged from 10 to 100 mL, with porous media beds possessing varying geometries:- Repeating octahedron in a square lattice with center to center spacing of 1.7 mm (FIG. 3A), possessing relative surface areas of approximately 700 m2 / m3,- Wireframe octahedron in a square lattice with center to center spacing of 2 mm (FIG. 3B), possessing relative surface area of approximately 870 m2 / m3,- Stacked hexagonal mesh disks of 2 mm thickness and 3 mm center to center spacing (FIG. 3C), with relative surface area of approximately 1800 m2 / m3.

[0125] EXAMPLE 2

[0126] A modified biofilm bioreactor for use with marine chassis organisms has been created. This study presented the design, optimization, and implementation of a 3D-printed packed bed bioreactor for biomanufacturing efforts, focusing on applications with biofilm-forming chassis organisms. Biofilm bioreactors exhibit numerous advantages over traditional bioreactors, primarily through increased cell density and greater cell robustness, enabling increased production rates per volume. Here, the previously developed biofilm bioreactor, called BIOPACS (Biological Point-of-need Advanced Chemical Synthesis), was modified to increase the applicability and the ease of post-run processing to facilitate potential future deployment of the system. The new design, incorporating materials selected for biocompatibility and a novel modular design, improves the scalability and versatility of the previous system.

[0127] FIG. 10 shows BIOPACS (Biological Point-of-need Advanced Chemical Synthesis) 3-D printed continuous flow fixed bed bioreactor. In this configuration, bioreactors are printed out of nylon and are comprised of a cylindrical outer body filled with fixed octahedral beads. At the inlet is a dispersion plate designed to help evenly distribute media flow throughout the reactor. Various media sizes are possible. Total void volume is 10 mL. Media is flowed through the bioreactor at a consistent rate with a pump. Media oxygen is measured before and after running through the bioreactor, allowing time to monitor biofilm growth over time.

[0128] FIG. 11A shows that by tracking the oxygen in the media going in (influent) and coming out (effluent) of the bioreactor, biofilm growth as a functionof time in terms of oxygen utilization may be monitored. Influent and effluent oxygen are recorded every 10 seconds for 100 hours. “Recovery” is defined as the oxygen % in the media after the overnight culture is pumped out. This value provides a relative idea of how much of the bacteria “stuck” to the bioreactor. Oxygen utilization performance for various cells are show in FIG. 11B, and including a chart of fastest to slowest growth.

[0129] FIGS. 12A, 12B, and 12C show that changing the resin of the bioreactor from nylon to a more bio-compatible polymer encouraged biofilm formation. Oxygen concentration in the bioreactor effluent, used as a proxy for metabolic activity of the biofilm, indicated increased cell density with a shorter growth period. In addition, the new resin can be sterilized using steam, UV, or chemical sterilants, ensuring high reproducibility by decreasing the risk of contamination.

[0130] FIG. 13A shows a modular design of a bioreactor with removable,perforated interior sections that enable the application of multiple sample analysis techniques from one bioreactor. The interior bead sections have a center, easily-breakable seam so that each piece may be split to allow multiple analyses. Individual, removable bead sections also allow for more spatially refined analysis of the bioreactor (e.g., protein concentration on each section as an idea of relative biofilm growth and how it changes across the length of the bioreactor). FIG. 13B shows a comparison of biomass protein by bioreactor segment position for M. atlanticus and M. nauticus.

[0131] FIG. 14 shows comparison result charges for four samples, such that the resilient resin and modular design of the bioreactor allows the system to be easilyadaptable for use with alternative feedstocks (e.g., hydrocarbons).

[0132] Some alterations may be made to adapt the system to hydrocarbons, include:1. Rubber bioreactor tubing was replaced with hydrocarbon- resistant PVDF tubing.2. Surfactant was incorporated into the media bottles to solubilize the hydrocarbon feedstock.3. Bioreactor pieces were chemically sterilized rather than steam- sterilized post-run.

[0133] Some conclusions from this study are:1. 3D-printed packed bed bioreactors exhibit promise for application in biomanufacturing efforts, with current designs being further optimized to improve versatility, ease of operation, and scalability.2. Switching from Nylon to a biocompatible resin for the bioreactor resulted in quicker biofilm formation of Marinobacter atlanticus (measured by oxygen utilization).3. Removable interior bead segments allow for simple, spatially refined sample analysis while also introducing versatility through enabling reusability.4. The novel design is easily adaptable for applications involving alternative feedstocks such as hydrocarbons.

[0134] ADVANTAGES

[0135] The devices, systems, and methods presented in the current subject disclosure have a number of advantages, as appreciated by one having ordinary skill in the art after consideration of the present disclosure. Some of these advantages include, but are not limited to:

[0136] 1. Modular Assembly and Reusability

[0137] By separating the reactor body, end caps, and media bed sections, users can replace or upgrade individual parts without discarding the entire system. This significantly reduces costs, simplifies maintenance, and promotes sustainable bioprocessing.

[0138] 2. Tailored Media Bed Designs

[0139] The use of 3D printing for producing highly complex, porous growth media gives users precise control over flow paths and surface area. The customizable geometry leads to improved nutrient delivery, enhanced mass transfer, and high cell densities within biofilms.

[0140] 3. Scalability and Versatility

[0141] By adjusting the length or diameter of the reactor body (or linking multiple reactor bodies in series), the system can be adapted from small-scale laboratory settings to larger pilot-scale or industrial scenarios. This flexibility is critical for applications ranging from academic research to full-scale biomanufacturing.

[0142] 4. Enhanced Observability and Sampling

[0143] Removable media beds allow researchers to directly observe and analyze the biofilm without destructive methods. Sampling is facilitated by modular sections that can be examined independently, thereby accelerating R&D andreducing trial-and-error in process optimization.

[0144] 5. Compatibility with Multiple Sterilization Methods

[0145] Materials compatible with steam, UV, or chemical sterilization ensure robust operation across different laboratory and industrial environments. This enables repeat usage without compromising sterility or performance.

[0146] 6. Customization for Different Cell Types

[0147] Users can configure multiple distinct media bed sections for different cell lines or microbial species, enabling controlled co-cultures or layered bioprocessing within the same reactor footprint. This unlocks advanced biotechnology applications, such as sequential or multi-step conversions under continuous flow.

[0148] 7. Interchangeable Functional End Caps

[0149] The modular architecture can further include a set of interchangeable, monolithically 3D-printed end caps that are each compatible with a common reactor body. By swapping end caps, users can change connector type (e.g., hose barb or Luer lock) and / or internal flow features such as a dispersion plate, flow distributor, and / or static mixing structure, and can optionally provide a sampling port for withdrawing fluid while the reactor body remains assembled, thereby increasing versatility and reducing external plumbing complexity.

[0150] Overall, these features address many of the shortcomings of existing solid-support bioreactors by combining the adaptability of 3D printing with the inherent advantages of a modular design. The inventive system thus provides a powerful platform for efficient, reproducible, and cost-effective biofilm-basedprocesses in both research and industrial contexts.

[0151] Concluding Remarks

[0152] Although the present subject disclosure has been described in connection with preferred embodiments thereof, it will be appreciated by those skilled in the art that additions, deletions, modifications, and substitutions not specifically described may be made without departing from the spirit and scope of the subject disclosure. Terminology used herein should not be construed as being “means-plus-function” language unless the term “means” is expressly used in association therewith.

[0153] The foregoing disclosure of the exemplary embodiments of the present subject disclosure has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the subject disclosure to the precise forms disclosed. Many variations and modifications of the embodiments described herein will be apparent to one of ordinary skill in the art in light of the above disclosure. The scope of the subject disclosure is to be defined only by the claims appended hereto, and by their equivalents.

[0154] Further, in describing representative embodiments of the present subject disclosure, the specification may have presented the method and / or process of the present subject disclosure as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particularorder of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and / or process of the present subject disclosure should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the present subject disclosure.

[0155] The following references, cited within this disclosure, are incorporated by reference herein in their entirety into this disclosure:[1] Yates, M.D., Mickol, R.L., Tolsma, J.S. etal. Lipid production from biofilms of Marinobacter atlanticus in a fixed bed bioreactor. Microb Cell Fact 23, 336[2] US20230313105A1, 2023-10-05, Additive Manufacturing of a Packed Bed Bioreactor.

[0156] The following patents and publications, related to this disclosure, are also incorporated by reference herein in their entirety into this disclosure:CN111068592A Slurry bed reactor with multi-stage perforated structure distribution plateUS20230313105A1 Additive Manufacturing of a Packed Bed Bioreactor CA1265080A Porous inorganic carriers bearing a growth of microorganismsEP1292672A1 Immobilizing carrier comprising a porous medium US5266476A Fibrous matrix for in vitro cell cultivationCA2941910C An aerated biofilm reactor fibre membrane CN102639448A Fluidized membrane bioreactorCN102127505B Immobilized cell bioreactorW02022010982A1 Microscale bioreactor system for and method of providing cell culture growthUS20210024868A1 Packed-bed bioreactor systems and methods of using the sameUS20230348834A1 Fixed bed bioreactor vessel and methods of using the same US12116556B2 Fixed bed bioreactor and methods of using the sameUS20200248120A1 Woven cell culture substrates, bioreactor systems using the same, and relatedW02023009373A1 Uniform cell culture substrate for fixed bed bioreactor WO2022115305A1 Packed bed bioreactors with controlled zonal porosity CN112955532A Three-dimensional bioreactorUS20170321178A1 Three-dimensional bioreactor for cell expansion and related applicationsUS20180016533A1 Substrates for high-density cell growth and metabolite exchangeJP2024529490A Uniform cell culture substrate for fixed-bed bioreactors WO2024102296A1 Adherent cell culture systems with removable witness substrates for cell samplingCN113144919A 3D printing multifunctional filter membrane for sewage treatmentUS9677038B2 Device and method for industrial cultivation of cells US9228579B2 Method and device for industrial biolayer cultivation US8222026B2 Stacked array bioreactor for conversion of syngas components to liquid productsUS11987782B2 Biofilm bioreactorUS20240117286A1 Methods of Feedstock Conversion Using a Biofilm Bioreactor

Claims

WHAT IS CLAIMED IS:

1. A modular biofilm bioreactor system, comprising:a reusable reactor body defining an elongated internal chamber about a longitudinal axis and having an access opening at one end; an end cap configured to reversibly mate with the access opening and comprising a fluid connector providing a fluid path to the internal chamber; anda removable media-bed assembly disposed within the internal chamber, the media-bed assembly comprising a plurality of discrete media-bed segments arranged end-to-end along the longitudinal axis; wherein:each media-bed segment comprises a three-dimensionally printed, unitary lattice scaffold defining a plurality of interconnected voids forming a through-flow path for fluid;the media-bed segments include an alignment feature configured to facilitate assembly and maintain a selected orientation of the segments in the reactor body;at least one of the media-bed segments includes a frangible plane configured to divide the segment into two or more portions wherein the portions are detachable from each other without destruction of the segments; andwherein the media-bed assembly is removable from the reactor body without destruction of the reactor body.

2. The bioreactor system of claim 1 , wherein, upon removal of the end cap, each media-bed segment is individually retrievable from the reactor body.

3. The bioreactor system of claim 1 , wherein the alignment features comprise profiled protrusions and complementary grooves configured to align adjacent segments.

4. The bioreactor system of claim 1 , wherein the plurality of discrete media-bed segments define a plurality of discrete axial sampling positions enabling spatially resolved profiling of biofilm growth or metabolic activity along the reactor length.

5. The bioreactor system of claim 1 , wherein at least two media-bed segments are configured for different pre-plated microorganisms or cell types, and the alignment and / or indexing features are configured to permit maintaining a selected axial order during assembly to establish a staged or co-culture sequence.

6. The bioreactor system of claim 1 , wherein the reactor body and the end cap are reusable across runs and the media-bed segments are configured as replaceable consumables.

7. The bioreactor system of claim 1 , wherein each media-bed segment is produced by stereolithography or selective laser sintering and comprises a biocompatible polymer.

8. The bioreactor system of claim 1 , wherein the media-bed segments are configured to withstand chemical sterilization, ultraviolet sterilization, and / or steam sterilization.

9. The bioreactor system of claim 1 , wherein the end cap comprises an integral flow distributor positioned to distribute influent across a crosssection of the media-bed assembly and / or an integral static mixing structure upstream of the media-bed assembly.

10. The bioreactor system of claim 1 , wherein the reactor body is adjustable in length to accommodate varying numbers of media bed sections, thereby increasing the total capacity for culturing cells or other biological materials.11.A set of interchangeable three-dimensionally printed end caps for a bioreactor body defining an internal chamber, the set comprising a plurality of different end caps, wherein each end cap comprises:an external mating interface configured to removably couple to the bioreactor body;at least one external fluid connector configured to provide fluid communication with the internal chamber when the end cap is coupled to the bioreactor body; andone or more internal flow features selected from a mesh filter, a dispersion plate, a flow distributor, and a static mixing structure; wherein different end caps of the set provide different combinations of external fluid connectors and / or internal flow features while remaining compatible with the bioreactor body.

12. The set of claim 11 , wherein at least one end cap comprises a sampling port configured to permit withdrawal of a fluid sample from the internal chamber while the bioreactor body remains assembled.

13. The set of claim 11 , wherein at least one end cap comprises an integral dispersion plate or flow distributor positioned to distribute influent across a cross-section of the internal chamber.

14. The set of claim 11 , wherein at least one end cap comprises an integral static mixing structure upstream of the internal chamber.

15. The set of claim 11 , wherein at least one end cap comprises a hose barb connector.

16. The set of claim 11 , wherein at least one end cap comprises a gasket seat configured to receive an O-ring to seal the end cap to the bioreactor body.

17. The set of claim 11 , wherein the external mating interface comprises a threaded coupling.

18. A method of assembling and operating a modular bioreactor, comprising:three-dimensionally printing a reactor body, a plurality of interchangeable end caps, and a plurality of media-bed segments as separate components;assembling the media-bed segments in a selected axial order to form a removable media-bed assembly and placing the removable media-bed assembly within the reactor body;coupling an inlet end cap and an outlet end cap to the reactor body to establish a closed flow path through the internal chamber andflowing media through the removable media-bed assembly to grow and / or maintain a biofilm;optionally withdrawing a fluid sample from the internal chamber via a sampling port of at least one end cap without opening the reactor body;optionally decoupling a first end cap and coupling a second, different end cap to change a connector type and / or an internal flow feature; andafter operation, retrieving at least one media-bed segment and splitting the retrieved media-bed segment along a frangible plane to perform two or more different analyses on material obtained from the retrieved media-bed segment.

19. The method of claim 18, comprising pre-plating at least two different media-bed segments with different microorganisms or cell types prior to assembling the removable media-bed assembly.

20. The method of claim 18, comprising replacing one or more media-bed segments while reusing the reactor body and at least one end cap for a subsequent run.

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