**2. Materials and methods**

#### **2.1 Miniaturization concept**

It may not be out of place to look into the reasons behind the ongoing technological revolution based on miniaturization. A higher degree of intelligence can be achieved by drastically increasing the amount of sensory data (by many orders of magnitude) obtained from a large number of variable fermentation experiments. High-throughput screening of fermentations demands that the bioreactors, as well as the sensors, are miniaturized so that a large number of these can be accommodated in small areas and at the same time that neither the cost nor the energy consumption exceeds acceptable limits.

When all aspects of the bioreactor scale in a similar way, the geometric integrity is maintained with the downsizing. Such type of scaling is called "isomorphic" (or "isometric") scaling [22]. On the other hand, if different elements of a system with different functionalities do not scale similarly, the scaling is called "allometric" scaling [23, 24]. Scaling laws deal with the structural and functional consequences of changes in size or scale among otherwise similar structures/organisms; thus, only through the scaling laws a designer becomes aware of physical consequences of downscaling devices and systems. Scaling effects on problems of mechanics are significant and are essential to take into account while designing systems at mini scales [24].

#### **2.2 Miniature bioreactor design**

Computer-aided design (CAD) representations were created in AutoCAD Fusion, and drawings with the external and internal dimensions and design parameters are presented in **Figure 1**. The mini-bioreactor device was designed with four ports; each one corresponds to a different function (influent, effluent, gas exit, pH electrode).

Close attention was paid to the aspect ratio of the bioreactor. In general, the aspect ratio of a vessel (the ratio between its height and its diameter) should be 1:1 at the working volume for cell culture and 2.2:1 at the working volume for microbial systems [24].

**179**

**Figure 2.**

**Figure 1.**

*Development of an Anaerobic Digestion Screening System Using 3D-Printed Mini-Bioreactors*

After the baseline design, a 40-mL bioreactor was fabricated using a stereolithography (SLA)-based 3D printer as a proof of principle (see **Figure 2**). The CAD files were converted to the STL format, which is a file type that interfaces between CAD software and additive manufacturing platforms. The PREFORM software was used to print the bioreactor. The bioreactors were printed on a Formlabs Vat Polymerization platform (Form 1) using the commercially available Formlabs Clear FLGPCL02 proprietary resin. The lowest resolution available in the machine was employed (0.1 mm) for the printing. A sturdy and rigid device was created layer by layer using a laser which initiated polymerization in the photopolymer resin. The reactor was then extensively cleaned and flushed with isopropyl alcohol (IPA) to avoid after-curing of the resin on the walls and

A post-processing step of fine polishing shortly after fabrication with this resin produced clean and semitransparent bioreactors. This offers the possibility to observe the flow patterns and enables the application of visual techniques and

*The fabrication steps of an ultra-scale bioreactor. (a) A CAD model was designed to meet the requirements of a suitable bioreactor. (b) The reactor device was fabricated using a Form 1 SLA printer. (c) The reactor was* 

*polished, cleaned, and extensively flushed with IPA to remove the residual resin.*

*DOI: http://dx.doi.org/10.5772/intechopen.88623*

**2.3 Miniature bioreactor fabrication**

internal channels of the bioreactor.

in-line spectroscopy for process characterization.

*Internal and external dimensions of the mini-bioreactor.*

*Development of an Anaerobic Digestion Screening System Using 3D-Printed Mini-Bioreactors DOI: http://dx.doi.org/10.5772/intechopen.88623*

### **2.3 Miniature bioreactor fabrication**

*New Advances on Fermentation Processes*

through anaerobic digestion) [20, 21].

**2. Materials and methods**

**2.1 Miniaturization concept**

consumption exceeds acceptable limits.

**2.2 Miniature bioreactor design**

stability.

throughput [16]. This highlights the need for automated multi-parallel mini-

bioreactor systems [17–19]. The pilot-scale reactors are often considered impractical since they require more feedstock, space, and energy than the mini-scale reactors. This makes the commercial bioreactors expensive, unrealistic, and inefficient as a process screening method. Additionally, the current state-of-the-art minibioreactors do not apply to complex microbial systems (e.g., the biogas production

A downsized approach of anaerobic digestion using mini-digesters is presented. In this study, 40 mL bioreactors were designed, fabricated, and operated to evaluate the anaerobic digestion performance and stability at a small scale. The start-up and operation of the mini-bioreactors were investigated. The results demonstrated that AD in low working volumes was feasible and efficient in terms of biogas quantity and quality. The results also established links between scale-down and process

It may not be out of place to look into the reasons behind the ongoing technological revolution based on miniaturization. A higher degree of intelligence can be achieved by drastically increasing the amount of sensory data (by many orders of magnitude) obtained from a large number of variable fermentation experiments. High-throughput screening of fermentations demands that the bioreactors, as well as the sensors, are miniaturized so that a large number of these can be accommodated in small areas and at the same time that neither the cost nor the energy

When all aspects of the bioreactor scale in a similar way, the geometric integrity is maintained with the downsizing. Such type of scaling is called "isomorphic" (or "isometric") scaling [22]. On the other hand, if different elements of a system with different functionalities do not scale similarly, the scaling is called "allometric" scaling [23, 24]. Scaling laws deal with the structural and functional consequences of changes in size or scale among otherwise similar structures/organisms; thus, only through the scaling laws a designer becomes aware of physical consequences of downscaling devices and systems. Scaling effects on problems of mechanics are significant and are essential to take into account while designing systems at mini

Computer-aided design (CAD) representations were created in AutoCAD Fusion, and drawings with the external and internal dimensions and design parameters are presented in **Figure 1**. The mini-bioreactor device was designed with four ports; each one corresponds to a different function (influent, effluent, gas exit, pH

Close attention was paid to the aspect ratio of the bioreactor. In general, the aspect ratio of a vessel (the ratio between its height and its diameter) should be 1:1 at the working volume for cell culture and 2.2:1 at the working volume for microbial

**178**

scales [24].

electrode).

systems [24].

After the baseline design, a 40-mL bioreactor was fabricated using a stereolithography (SLA)-based 3D printer as a proof of principle (see **Figure 2**). The CAD files were converted to the STL format, which is a file type that interfaces between CAD software and additive manufacturing platforms. The PREFORM software was used to print the bioreactor. The bioreactors were printed on a Formlabs Vat Polymerization platform (Form 1) using the commercially available Formlabs Clear FLGPCL02 proprietary resin. The lowest resolution available in the machine was employed (0.1 mm) for the printing. A sturdy and rigid device was created layer by layer using a laser which initiated polymerization in the photopolymer resin. The reactor was then extensively cleaned and flushed with isopropyl alcohol (IPA) to avoid after-curing of the resin on the walls and internal channels of the bioreactor.

A post-processing step of fine polishing shortly after fabrication with this resin produced clean and semitransparent bioreactors. This offers the possibility to observe the flow patterns and enables the application of visual techniques and in-line spectroscopy for process characterization.

**Figure 1.** *Internal and external dimensions of the mini-bioreactor.*

#### **Figure 2.**

*The fabrication steps of an ultra-scale bioreactor. (a) A CAD model was designed to meet the requirements of a suitable bioreactor. (b) The reactor device was fabricated using a Form 1 SLA printer. (c) The reactor was polished, cleaned, and extensively flushed with IPA to remove the residual resin.*

In terms of fabrication, the 40-mL reactor, including support structures, required 63 mL of resin and just over 8 h to complete (**Table 1**). The large build platform of the Form 1 printer allows that both the vessel and lid are printed simultaneously reducing manufacturing time.

#### **2.4 Inoculum and substrate**

The microbial inoculum for this study was obtained from the wastewater treatment plant in Garmerwolde (Groningen, Netherlands). Anaerobic sludge was collected from an anaerobic digester degrading municipal waste and stored at 6°C. The inoculum was gently homogenized to reduce the size of big particles somewhat. The characteristics of the inoculum and substrate are shown in **Table 2**. Dried milk was used as a constant complex substrate that consists of a mixture of carbohydrates, lipids, proteins, and minerals. Dried milk powder was purchased from the local grocery market. The components of dried milk are carbohydrates (lactose) 39%, butter fat 28.2%, proteins 25.1%, moisture 3%, calcium 930 mg, phosphorus 75 mg, other minerals 3.88 g, vitamin A 636.3 μg, vitamin D3 8.8 μg, vitamin E 0.8 mg, vitamin B2 1.4 mg, and vitamin B12 1.8 μg.

#### **2.5 Experimental setup**

In this study, the development process of the 3D-printed mini-bioreactor consists of the vessel design and fabrication, operation test, and the baseline study. In addition to the manufacturing of the 3D-printed bioreactors, baseline studies involving commercial stirred bioreactors were carried out to examine the process in parallel with the mini-bioreactors. The two setups are schematically described in **Figure 3**.

The daily biogas production rate was determined to evaluate the behavior of the anaerobic digestion process and the stability of the miniature bioreactor. The experimental conditions and the content of the reactors are shown in **Table 3**. Biogas composition, pH, and COD reduction have also been employed as valuable parameters for further understanding and evaluation of the microreactor performance. A single-stage semicontinuous process was performed in two 400-mL BioBLU single-use vessels (Eppendorf, USA) with a working volume of 300 mL. The vessel was placed in a temperature-controlled water bath (36°C) and fed once a day. The milk powder suspension was impelled with a syringe pump (AL-1000HP, World Precision Instruments, USA) equipped with a 30-mL syringe (Terumo, inner diameter 23.1 mm) and Teflon tubing (1.37 × 1.07 mm).

Before use, the inoculum (anaerobic sludge) was first incubated anaerobically until no methane production was observed anymore (37°C, 6–7 days). For


**181**

matrix.

**Figure 3.**

**Table 2.**

mL biogas per g VSadded per day.

*the conceptual research with the 3D-printed mini-bioreactors.*

water bath and kept at 36 ± 1°C.

*Development of an Anaerobic Digestion Screening System Using 3D-Printed Mini-Bioreactors*

*Physicochemical characteristics of the inoculum and substrate (influent) used in the experiments.*

**Parameter Unit Anaerobic sludge Milk powder** pH 7.36 — TS g · kg−1 39.5 ± 1.7 968.4 ± 3.5 VS g · kg−1 27.3 ± 0.4 924.9 ± 2.9 COD g · kg−1 40.7 ± 1.9 1147.6 TVFA mg acetic acid · L−1 716 1400 TA mg CaCO3 · L−1 5884 3000

both the mini-bioreactors and the commercial reactors, no additional external nutrients/trace elements were added to the influent as it was assumed that they are sufficiently present in the inoculum and the milk powder. The reactors were mixed twice a day (2 × 5 min) by integrated magnetic stirrers (miniature bioreactors) or by propellers (commercial bioreactors) to achieve a homogenized

*The validation step of the miniature system consists of a baseline study operating commercial bioreactors and* 

The experiments were carried out in a semicontinuous mode using the water displacement method to measure the biogas production for 95 days. The biogas production rate was based on the volume of biogas produced daily and is defined as

The bioreactors were filled with sieved anaerobic sludge to provide sufficient consortia of microbes to degrade organic material in the influent. The bioreactors were flushed with N2-gas for 2 min to achieve anaerobic conditions, placed in a

*DOI: http://dx.doi.org/10.5772/intechopen.88623*

**Table 1.**

*Technical data from bioreactors used in the experiments.*

*Development of an Anaerobic Digestion Screening System Using 3D-Printed Mini-Bioreactors DOI: http://dx.doi.org/10.5772/intechopen.88623*


**Table 2.**

*New Advances on Fermentation Processes*

taneously reducing manufacturing time.

**2.4 Inoculum and substrate**

B12 1.8 μg.

**Figure 3**.

(1.37 × 1.07 mm).

**2.5 Experimental setup**

In terms of fabrication, the 40-mL reactor, including support structures, required 63 mL of resin and just over 8 h to complete (**Table 1**). The large build platform of the Form 1 printer allows that both the vessel and lid are printed simul-

The microbial inoculum for this study was obtained from the wastewater treatment plant in Garmerwolde (Groningen, Netherlands). Anaerobic sludge was collected from an anaerobic digester degrading municipal waste and stored at 6°C. The inoculum was gently homogenized to reduce the size of big particles somewhat. The characteristics of the inoculum and substrate are shown in **Table 2**. Dried milk was used as a constant complex substrate that consists of a mixture of carbohydrates, lipids, proteins, and minerals. Dried milk powder was purchased from the local grocery market. The components of dried milk are carbohydrates (lactose) 39%, butter fat 28.2%, proteins 25.1%, moisture 3%, calcium 930 mg, phosphorus 75 mg, other minerals 3.88 g, vitamin A 636.3 μg, vitamin D3 8.8 μg, vitamin E 0.8 mg, vitamin B2 1.4 mg, and vitamin

In this study, the development process of the 3D-printed mini-bioreactor consists of the vessel design and fabrication, operation test, and the baseline study. In addition to the manufacturing of the 3D-printed bioreactors, baseline studies involving commercial stirred bioreactors were carried out to examine the process in parallel with the mini-bioreactors. The two setups are schematically described in

The daily biogas production rate was determined to evaluate the behavior of the anaerobic digestion process and the stability of the miniature bioreactor. The experimental conditions and the content of the reactors are shown in **Table 3**. Biogas composition, pH, and COD reduction have also been employed as valuable parameters for further understanding and evaluation of the microreactor performance. A single-stage semicontinuous process was performed in two 400-mL BioBLU single-use vessels (Eppendorf, USA) with a working volume of 300 mL. The vessel was placed in a temperature-controlled water bath (36°C) and fed once a day. The milk powder suspension was impelled with a syringe pump (AL-1000HP, World Precision Instruments, USA) equipped with a 30-mL syringe (Terumo, inner diameter 23.1 mm) and Teflon tubing

Before use, the inoculum (anaerobic sludge) was first incubated anaerobically until no methane production was observed anymore (37°C, 6–7 days). For

**Parameter Units 3D-printed reactor Commercial reactor** Reactor volume mL 40 mL 400 mL Inner diameter mm 28 mm 62 mm Inner height mm 56 mm 124 mm Resin volume mL 63 mL — Fabrication time h min (8 h 11 min) —

**180**

**Table 1.**

*Technical data from bioreactors used in the experiments.*

*Physicochemical characteristics of the inoculum and substrate (influent) used in the experiments.*

#### **Figure 3.**

*The validation step of the miniature system consists of a baseline study operating commercial bioreactors and the conceptual research with the 3D-printed mini-bioreactors.*

both the mini-bioreactors and the commercial reactors, no additional external nutrients/trace elements were added to the influent as it was assumed that they are sufficiently present in the inoculum and the milk powder. The reactors were mixed twice a day (2 × 5 min) by integrated magnetic stirrers (miniature bioreactors) or by propellers (commercial bioreactors) to achieve a homogenized matrix.

The experiments were carried out in a semicontinuous mode using the water displacement method to measure the biogas production for 95 days. The biogas production rate was based on the volume of biogas produced daily and is defined as mL biogas per g VSadded per day.

The bioreactors were filled with sieved anaerobic sludge to provide sufficient consortia of microbes to degrade organic material in the influent. The bioreactors were flushed with N2-gas for 2 min to achieve anaerobic conditions, placed in a water bath and kept at 36 ± 1°C.


**Table 3.**

*Process conditions and masses of organic materials in experimental tests.*

#### **2.6 Analytical methods**

Total solid (TS) and volatile solid (VS) contents were determined according to the standard method 1684 (EPA) [25]. The total volatile fatty acids (TVFA) were measured using the test kit LCK 365 (Hach Lange GmbH). The samples were centrifuged (10 min, 6000 rpm), and the supernatant was filtered. The time from the sampling up to the execution of the analytical procedure was identical for each sample to ensure the best possible quality of the results. A pH meter (HI991001, Hanna Instruments) was used to measure the pH in commercial reactors, and a mini pH meter (VWR, USA) was used to measure the pH in the miniature reactors.

The volume of biogas that was produced from the 3D-printed microreactors and the 300-mL reactors was estimated by the water displacement method, and the measuring devices were standard serum bottles with a volume of 10 and 100 mL, respectively. Chemical oxygen demand (COD; g∙kg<sup>−</sup><sup>1</sup> ) and ammonium (NH4 + -N; g∙kg<sup>−</sup><sup>1</sup> ) were determined using commercial assay kits (Hach Lange GmbH, Germany) according to the manufacturer's instructions and were quantified by a spectrophotometer (DR3900, Hach, USA). Free ammonia nitrogen (FAN; g∙kg<sup>−</sup><sup>1</sup> ) was calculated based on equation 1 [26]:

$$N \to \infty \quad \text{or} \quad \omega = \frac{\omega\_1}{\omega\_2} \frac{\omega\_1}{\omega\_2} = \frac{\omega\_1}{\omega\_2} \frac{\omega\_1}{\omega\_2} = \omega\_1 \omega\_2$$

$$N - N H\_3 = \frac{\tan \pi \times 10^{pH}}{e^{\left(\frac{6444}{(27315 + P)}\right)} + 10^{pH}} \tag{1}$$

The biogas volume (mL∙g VSsubstrate<sup>−</sup><sup>1</sup> ∙day<sup>−</sup><sup>1</sup> ) was measured with the water displacement method and was standardized according to DIN 1343 (standard conditions: temperature (T) = 0°C and pressure (P) = 1.013 bar) [27]. The biogas volume was normalized according to equation 2 [28]:

$$\begin{array}{l}\text{ling to equation 2 [28]:}\\\text{V}\_{N} = \frac{V \times 273 \times (760 - p\_w)}{T \times 760} \end{array} \tag{2}$$

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**Figure 4.**

*Daily biogas production during the experimental period.*

*Development of an Anaerobic Digestion Screening System Using 3D-Printed Mini-Bioreactors*

In this study, 3D-printed mini-bioreactors of 40 mL (MR1 and MR2) and commercial bioreactors of 400 mL (R1 and R2) were operated for 95 days (4.75 × HRT). Dried milk powder was used as a substrate, and the OLR was set to 0.5 g VS/day. The rate of biogas production has the potential to be a valid online process condition indicator that determines the stability of a reactor. **Figure 4** clearly shows the stable production rate in the last 60 days of operation (3xHRT). In the first 20 days, the commercial reactors (R1 and R2) started with a fast production, reaching a constant rate within the range of 820–850 mL/g VSadded. MR1 and MR2 showed an increased biogas production for the first 60 days, reaching a similar production rate

The OLR was set at 0.5 g VS/day to avoid clogging problems during the operation system. Gou et al. [29] proposed that an OLR less than 5000 mg/L is necessary to ensure stable biogas production at mesophilic conditions. Similarly, Sun et al. [30] reported that an OLR in the range of 3000–5000 mg/L is more desirable for digester operation.

The pH is a very useful indicator for the behavior of anaerobic digestion and the overall process stability. When the pH in an anaerobic reactor decreases, it is usually the first signal that the process starts to become unstable. The acidification is caused by the accumulation of short-chain fatty acids that are not efficiently converted into biogas. Typically, the pH is kept constant by the process itself. Organic substrates are hydrolyzed and converted into short-chain fatty acids and further converted into acetate, H2, and CO2. Specific microorganisms, archaea, convert H2 plus CO2 or acetate into CH4 or CH4 and CO2, respectively. Different groups of microorganisms (bacteria) are responsible for the hydrolysis, acidogenesis, and acetogenesis phases. An imbalance in the ratio and activity of the bacteria and archaea may increase the concentration of acids in the reactor.

*DOI: http://dx.doi.org/10.5772/intechopen.88623*

as obtained in the commercial reactor after 3xHRT.

**3. Results**

**3.2 pH**

**3.1 Biogas production**

where VN is the volume of the dry biogas at standard temperature and pressure (mLN), V is the recorded volume of the biogas (mL), pw is the water vapor pressure as a function of ambient temperature (mmHg), and T is the ambient temperature (K).

All the experiments were carried out in duplicate (two bioreactors for the commercial reactor and two micro-bioreactors, and the experimental data from each reactor was plotted in the corresponding graphs), and the data analysis was conducted using Microsoft Excel.

*Development of an Anaerobic Digestion Screening System Using 3D-Printed Mini-Bioreactors DOI: http://dx.doi.org/10.5772/intechopen.88623*
