BIO-SPEC: An open-source bench-top parallel bioreactor system

Abstract

The BIO-SPEC is an open-source, cost-effective, and modular bench-top bioreactor system designed for batch, sequencing batch, and chemostat cultivation. Featuring thermoelectric condensers to eliminate the need for a chiller, it ensures stable long-term operation. Controlled by a Raspberry Pi, the BIO-SPEC offers flexibility in headplate design, gas supply, and feeding strategies, making it a versatile alternative to high-cost commercial systems. This paper details the design, construction, and validation of the BIO-SPEC system, demonstrating its potential to advance microbiology and bioprocessing research through accessible and reliable hardware at a fraction of the cost of commercial systems.

Keywords: Bioreactor; Chemostat; Microbiology; Open-source; Raspberry Pi; Sequencing batch.

Graphical abstract

Fig. 1

Overview of the BIO-SPEC system. (a) shows a picture of two bioreactors operated in sequencing batch mode, and (b) indicates a schematic diagram of the relevant connections and components.

Fig. 2

Pictures of the reactor vessel and its headplate design. (a) shows the assembly of the vessel with the headplate (and fittings), (b) the headplate with internal tubes, and (c) a top and bottom view of the headplate.

Fig. 3

Pictures of the Peltier condenser. (a) shows the condenser in operation, (b) the pipe freezing up without temperature control (red circle), and (c) the slot where the off-gas pipe connects to the condenser components.

Fig. 4

Schematic diagram of a tube-in-tube off-gas condenser based on standard Swagelok parts and their technical datasheets. The required fittings shown here are bored-through reducers (A: 2x SS-12M0-1-8BT), adapters (B: 2x SS-12-TA-7-8), and tees (C: 2x SS-1210-3). The design needs the extra tube adapters (orange, B) to work around the limited bored-through fitting offer for metric pipes (since the off-gas pipe (blue) is 12 mm). If the fittings are bored through by the user, there are not only more size options available, but the adapters can be omitted as well. The outer pipe should have a diameter of 3/4 inch in this example.

Fig. 5

(a,b) pictures of the electrical cabinet, and (c) a render of the component layout. The stacked power supplies shown in (a) power the 12 V thermoelectric condensers. A possible button layout on the side of the cabinet is indicated in (b). The Raspberry Pi can be mounted on the blue DIN rail relay board visible in (c).

Fig. 6

(a) side and (b) top view of the FAST pump as replicated from the design of Jönsson et al. .

Fig. 7

Pictures of the manufactured headplates. (a) shows the 3D printed version in Formlabs BioMed Amber Resin ${}^{\text{TM}}$ , while (b) and (c) show the stainless steel version with appropriate O rings in the blind holes.

Fig. 8

Assembly of (a) the reactor lid, (b) a fitting, and (c) the aeration stone.

Fig. 9

Reactor level calibration using tube heights (a-b) and the final reactor headplate assembly (c).

Fig. 10

Heat sink assembly. (a) indicates where to drill the holes in the heat sink, (b) the assembly at the hot side of the Peltier elements, and (c) the assembly at the cold side.

Fig. 11

Thermoelectric condenser assembly. (a) shows the insulation sleeve, (b) the assembly of the Peltier elements, aluminium plate, and heat sink, and (c) the final assembly on the off-gas pipe.

Fig. 12

Electrical cabinet assembly. (a) shows the bare cabinet with the power supply rack, while (b) and (c) show the cabinet with all components mounted. A 3D render of the cabinet layout is provided in Fig. 14.

Fig. 13

Example close-up pictures of electrical connections using terminal blocks. Arrays of live, neutral and ground terminal blocks are labelled with red, blue and green-yellow electrical tape at the end brackets. (a) shows the top part of the cabinet with the power supplies and circuit breaker, while (b) shows the connections to the relay board.

Fig. 14

3D render of the proposed layout of the electrical cabinet as assembled in Fig. 12. The annotations correspond to the part identifiers of the bill of materials.

Fig. 15

(a) top and (b) side view of an example layout of the circuit board for the low-voltage electronics. The annotations reference the part identifiers of the bill of materials.

Fig. 16

Pictures and diagrams of how to set up the gas supply for two sets of three reactors. (a) shows the solenoid valve connections, (b) the rotameter and pressure regulator box, and (c) the plexi box for the gas valve array.

Fig. 17

Screenshots of the GUI during an anaerobic feeding (a) and a withdrawal (b) phase. The buttons change their colour (BLUE/ORANGE) and text (ON/OFF) according to the current phase. The Peltier elements are all turned off here, since this was run without any probes connected.

Fig. 18

(a) reactors are connected to feed and waste bottles using luer locks in a laminar flow cabinet. In the setup shown in (b), every reactor is connected to an individual 5 L feed bottle, and the waste lines are interconnected after the peristaltic pump tubing to two 10 L waste bottles in series.

Fig. 19

Inoculation and feeding options for the sampling valve. The blunt needle applied in (a) keeps the valve intact to allow the use of the same port for the manual addition of spiking solutions through a 0.2 µm filter in (b).

Fig. 20

(a) nitrogen and (b) carbon metabolite concentrations during anoxic/oxic cycles in a coculture experiment for two replicates. Denitrification (Eq. (3)), fermentation (Eq. (1)), and respiration (Eq. (2)) are observed. Notice the accumulation and removal of nitrite as an intermediary product during the anoxic phase, and the immediate consumption of acetate in aerobic conditions.

Fig. 21

Nitrate profiles for one reactor of a coculture experiment in SBR operation. A similar decline was observed in all four replicates, indicating the loss of nitrifiers over time.