**3.1 Biogas production**

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 as obtained in the commercial reactor after 3xHRT.

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.

### **3.2 pH**

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.

**Figure 4.** *Daily biogas production during the experimental period.*

Methanogenic archaea consume acetate, CO2, and H2 but do not perform very well at a pH below 6.5, and the acidification accelerates until all microorganisms are not able to grow anymore. At this point, the anaerobic digestion comes to a halt and is not able to recover unless the pH is actively increased to pH 7. Therefore, early signs of acidification of an anaerobic reactor that produces biogas are an indication to change the process operation parameters to maintain pH neutrality (**Figure 5**).

Until day 50, the pH showed higher stability for the commercial reactors, whereas the pH of the mini-bioreactors (MR1 and MR2) was a little less stable but varied within an acceptable range. After day 50, a small but steady decrease is observed in both commercial bioreactors and the mini-bioreactors. Milk powder is mainly composed of carbohydrates, proteins, and lipids and may not result in the optimal growth conditions for especially the methanogens. An imbalance in the different processes is likely and volatile fatty acids accumulate in the reactors, and the pH decreases [31]. The similar pH profile in all reactors indicates that the minibioreactors behave similarly as the commercial reactors and anaerobic digestion can be downscaled and performed in 3D-printed microreactors leading to the same pH profile as in anaerobic digestion performed in commercial reactors.

### **3.3 FOS/TAC**

The changes in VFA and TA in reactors were also monitored, and the results of the VFA/TA ratio are shown in **Figure 6**. With a ratio of less than 0.20, the microbes begin to "feel hungry," and the inoculum-to-substrate ratio must be decreased to obtain a stable process. A VFA/TA ratio greater than 0.3 indicates the beginning of "indigestion" [32, 33]. The content of the commercial bioreactors showed a significant higher buffer capacity, maintaining an optimal pH for the methanogenic bacteria. No extra alkalinity was added in the bioreactors, and the inoculum was considered as the only source of alkalinity. After 3xHRT, MR1 ranged between 0.21 and 0.28, whereas the MR2 was between 0.23 and 0.27. R1 and R2 showed lower ratios between 0.18 and 0.2, indicating better stability. It is notable that if the TVFA/TA ratio falls in the range between 0.20 and 0.3, the anaerobic digestion process is usually stable [34, 35].

**185**

**3.5 Redox**

**3.4 Ammonia**

**Figure 6.**

composed of ammonium ions (NH4

*FOS/TAC variation during the experimental period.*

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

Ammonia is formed from the decomposition of proteins and urea in milk. It is an essential nutrient that serves as a nitrogen source for the bacteria and archaea in the reactor. Without nitrogen, the microorganisms are unable to grow and will gradually wash out of the rectors. The total ammonia nitrogen is primarily

) and free ammonia (NH3) (i.e., free ammo-

shifts toward NH3.

+

+

or pH increases, the equilibrium between NH3 and NH4

commercial reactors and the 3D-printed mini-bioreactors.

required by the anaerobic methanogenic archaea [39].

maintain their optimal intracellular pH [37].

nia nitrogen (FAN)). The predominant form of these two components mainly depends on process temperature and pH [36]. To illustrate, if the temperature

Furthermore, the FAN is the most toxic species of the total ammonia nitrogen (TAN). FAN diffuses through the bacterial cell membrane and results in a proton imbalance in the cytosol. The intercellular pH increases and a rise in maintenance energy requirements inhibit the microorganisms because they will attempt to

In all reactors, the concentration of FAN increases with the same rate until day 50. After that, the rate decreases, and the level of FAN stabilized at 1.25 g/l (**Figure 7**). The degradation of the protein-rich substrate leads to the formation of FAN, and the microbial community needs to adapt to this substrate to effectively convert milk powder into biogas. The adaption seems to follow the same path in the

The reduction oxidizing potential (i.e., redox potential) has been shown as a successful monitoring parameter in many AD systems due to redox-reaction-catalyzed enzymes that degrade organic materials in the anaerobic environment [38]. The strictness of the anaerobic environment is well known, which is indicated by a redox potential of ≤ −200 mV [39]. Preferably, the redox potential is between −330 and −450 mV for an optimal AD process environment. The facultative anaerobic microorganisms consume the oxygen and other oxidizing components that are dissolved in the growth medium, resulting in a sufficiently low redox potential

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

**Figure 5.** *pH variation during the experimental period.*

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

**Figure 6.** *FOS/TAC variation during the experimental period.*

#### **3.4 Ammonia**

*New Advances on Fermentation Processes*

(**Figure 5**).

**3.3 FOS/TAC**

process is usually stable [34, 35].

*pH variation during the experimental period.*

Methanogenic archaea consume acetate, CO2, and H2 but do not perform very well at a pH below 6.5, and the acidification accelerates until all microorganisms are not able to grow anymore. At this point, the anaerobic digestion comes to a halt and is not able to recover unless the pH is actively increased to pH 7. Therefore, early signs of acidification of an anaerobic reactor that produces biogas are an indication to change the process operation parameters to maintain pH neutrality

Until day 50, the pH showed higher stability for the commercial reactors, whereas the pH of the mini-bioreactors (MR1 and MR2) was a little less stable but varied within an acceptable range. After day 50, a small but steady decrease is observed in both commercial bioreactors and the mini-bioreactors. Milk powder is mainly composed of carbohydrates, proteins, and lipids and may not result in the optimal growth conditions for especially the methanogens. An imbalance in the different processes is likely and volatile fatty acids accumulate in the reactors, and the pH decreases [31]. The similar pH profile in all reactors indicates that the minibioreactors behave similarly as the commercial reactors and anaerobic digestion can be downscaled and performed in 3D-printed microreactors leading to the same pH

The changes in VFA and TA in reactors were also monitored, and the results of the VFA/TA ratio are shown in **Figure 6**. With a ratio of less than 0.20, the microbes begin to "feel hungry," and the inoculum-to-substrate ratio must be decreased to obtain a stable process. A VFA/TA ratio greater than 0.3 indicates the beginning of "indigestion" [32, 33]. The content of the commercial bioreactors showed a significant higher buffer capacity, maintaining an optimal pH for the methanogenic bacteria. No extra alkalinity was added in the bioreactors, and the inoculum was considered as the only source of alkalinity. After 3xHRT, MR1 ranged between 0.21 and 0.28, whereas the MR2 was between 0.23 and 0.27. R1 and R2 showed lower ratios between 0.18 and 0.2, indicating better stability. It is notable that if the TVFA/TA ratio falls in the range between 0.20 and 0.3, the anaerobic digestion

profile as in anaerobic digestion performed in commercial reactors.

**184**

**Figure 5.**

Ammonia is formed from the decomposition of proteins and urea in milk. It is an essential nutrient that serves as a nitrogen source for the bacteria and archaea in the reactor. Without nitrogen, the microorganisms are unable to grow and will gradually wash out of the rectors. The total ammonia nitrogen is primarily composed of ammonium ions (NH4 + ) and free ammonia (NH3) (i.e., free ammonia nitrogen (FAN)). The predominant form of these two components mainly depends on process temperature and pH [36]. To illustrate, if the temperature or pH increases, the equilibrium between NH3 and NH4 + shifts toward NH3. Furthermore, the FAN is the most toxic species of the total ammonia nitrogen (TAN). FAN diffuses through the bacterial cell membrane and results in a proton imbalance in the cytosol. The intercellular pH increases and a rise in maintenance energy requirements inhibit the microorganisms because they will attempt to maintain their optimal intracellular pH [37].

In all reactors, the concentration of FAN increases with the same rate until day 50. After that, the rate decreases, and the level of FAN stabilized at 1.25 g/l (**Figure 7**). The degradation of the protein-rich substrate leads to the formation of FAN, and the microbial community needs to adapt to this substrate to effectively convert milk powder into biogas. The adaption seems to follow the same path in the commercial reactors and the 3D-printed mini-bioreactors.

#### **3.5 Redox**

The reduction oxidizing potential (i.e., redox potential) has been shown as a successful monitoring parameter in many AD systems due to redox-reaction-catalyzed enzymes that degrade organic materials in the anaerobic environment [38]. The strictness of the anaerobic environment is well known, which is indicated by a redox potential of ≤ −200 mV [39]. Preferably, the redox potential is between −330 and −450 mV for an optimal AD process environment. The facultative anaerobic microorganisms consume the oxygen and other oxidizing components that are dissolved in the growth medium, resulting in a sufficiently low redox potential required by the anaerobic methanogenic archaea [39].

**Figure 7.** *FAN variation during the experimental period.*

**Figure 8.** *Redox potential variation during the experimental period.*

In **Figure 8**, a constant redox potential in the reactors CR1 and CR2 was obtained throughout the whole experimental procedure. After 50 days, the redox potential in MR1 and MR2 slowly starts to increase. The increase may be due to small oxygen leakages in, e.g., the tubing connections. Small air leakages in the commercial bioreactors can be better handled by the system because more biomass is available (the same concentration but larger volume) to consume the oxygen. In the small microbioreactors, a similarly sized leakage causes considerable more disturbances to the strictly anaerobic methanogenic archaea. The smaller amount of biomass is not able to metabolize all of the intruded oxygen, and the redox potential will increase.

## **4. Conclusion**

Although there are several opportunities in the biogas sector, new challenges and barriers cannot be ignored and have to be overcome by using new process

**187**

wastewater.

**Conflict of interest**

**Author details**

Groningen, Netherlands

The authors declare no conflict of interest.

Spyridon Achinas and Gerrit Jan Willem Euverink\*

\*Address all correspondence to: g.j.w.euverink@rug.nl

provided the original work is properly cited.

Engineering and Technology institute Groningen, University of Groningen,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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

parameters and optimizing existing ones. The possibility of manufacturing bioreactors employing 3D printing has been demonstrated in this work. In this way, miniaturized semicontinuous bioreactors have been manufactured using low-cost SLA machines for the first time. The high resolution of the printer, coupled with the satisfactory solvent compatibility of the photopolymers employed, enabled the development of reactors with the possibility to easily add advanced features, such as regularly spaced and geometry-controlled baffles, sample ports, sensor inlets, or other internal structures by simple CAD design. Furthermore, the direct printing of high-quality threads allowed working under controlled back pressure. Indeed, the micro-bioreactor manufactured here showed a similar performance as the commer-

cial bioreactor in biogas production from the anaerobic digestion of milk.

The scale of the reactors demonstrated in this work adds an important step to the laboratory scale and the industrial scale, speeding up the research to obtain optimal fermentation conditions. The simplicity, low cost, and rapid uptake of 3D printing technology will enable the development of numerous applications of advanced reactor engineering in continuous-flow chemical manufacturing. The conclusions of this work justify the use of mini AD systems for high-throughput process screening to improve AD systems further. The excessive amounts of biowaste and wastewater produced in our society need to be taken care of properly. Better performing AD reactors contribute considerably to the sustainable treatment of biowaste and

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

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

parameters and optimizing existing ones. The possibility of manufacturing bioreactors employing 3D printing has been demonstrated in this work. In this way, miniaturized semicontinuous bioreactors have been manufactured using low-cost SLA machines for the first time. The high resolution of the printer, coupled with the satisfactory solvent compatibility of the photopolymers employed, enabled the development of reactors with the possibility to easily add advanced features, such as regularly spaced and geometry-controlled baffles, sample ports, sensor inlets, or other internal structures by simple CAD design. Furthermore, the direct printing of high-quality threads allowed working under controlled back pressure. Indeed, the micro-bioreactor manufactured here showed a similar performance as the commercial bioreactor in biogas production from the anaerobic digestion of milk.

The scale of the reactors demonstrated in this work adds an important step to the laboratory scale and the industrial scale, speeding up the research to obtain optimal fermentation conditions. The simplicity, low cost, and rapid uptake of 3D printing technology will enable the development of numerous applications of advanced reactor engineering in continuous-flow chemical manufacturing. The conclusions of this work justify the use of mini AD systems for high-throughput process screening to improve AD systems further. The excessive amounts of biowaste and wastewater produced in our society need to be taken care of properly. Better performing AD reactors contribute considerably to the sustainable treatment of biowaste and wastewater.
