Biogas production from protein-rich biomass: fed-batch anaerobic fermentation of casein and of pig blood and associated changes in microbial community composition

Abstract

It is generally accepted as a fact in the biogas technology that protein-rich biomass substrates should be avoided due to inevitable process inhibition. Substrate compositions with a low C/N ratio are considered difficult to handle and may lead to process failure, though protein-rich industrial waste products have outstanding biogas generation potential. This common belief has been challenged by using protein-rich substrates, i.e. casein and precipitated pig blood protein in laboratory scale continuously stirred mesophilic fed-batch biogas fermenters. Both substrates proved suitable for sustained biogas production (0.447 L CH4/g protein oDM, i.e. organic total solids) in high yield without any additives, following a period of adaptation of the microbial community. The apparent key limiting factors in the anaerobic degradation of these proteinaceous materials were the accumulation of ammonia and hydrogen sulfide. Changes in time in the composition of the microbiological community were determined by next-generation sequencing-based metagenomic analyses. Characteristic rearrangements of the biogas-producing community upon protein feeding and specific differences due to the individual protein substrates were recognized. The results clearly demonstrate that sustained biogas production is readily achievable, provided the system is well-characterized, understood and controlled. Biogas yields (0.45 L CH4/g oDM) significantly exceeding those of the commonly used agricultural substrates (0.25-0.28 L CH4/g oDM) were routinely obtained. The results amply reveal that these high-energy-content waste products can be converted to biogas, a renewable energy carrier with flexible uses that can replace fossil natural gas in its applications. Process control, with appropriate acclimation of the microbial community to the unusual substrate, is necessary. Metagenomic analysis of the microbial community by next-generation sequencing allows a precise determination of the alterations in the community composition in the course of the process.

Figure 1. Protease activity changes during acclimation.

The biogas-producing community was cultivated on casein (blue line) or pig blood (red line) as protein-rich substrates. The weekly protein doses are indicated by the blue (casein) and red (pig blood) columns. The vertical black line denotes the end of the adaptation period, i.e. feeding with a constant, low protein dose.

Figure 2. Biogas production from casein.

The fed-batch reactors were fed with increasing amounts of protein at weekly intervals. The protein dosages are shown by red line. The columns indicate the average specific biogas yields generated each week. Dashed line indicates biogas productivity according to the batch test.

Figure 3. Biogas production from pig blood.

The fed-batch reactors were fed with increasing amounts of protein at weekly intervals. The protein dosages are shown by red line. The columns indicate the specific biogas yield generated during each week. Dashed line indicates biogas productivity according to the batch test.

Figure 4. Changes in the VOAs/TAC values during the AD of casein (blue line) and blood (red line).

The protein loads administered at weekly intervals are indicated by the blue (casein) and red (blood) columns. The vertical black line denotes the end of the adaptation period, i.e. feeding with a constant, low protein dose. The gray area indicates the VOAs/TAC range under optimal operational conditions.

Figure 5. Changes in the volatile fatty acid accumulation during the AD of casein.

The colors indicate the acetate (blue), propionate (red), isobutyrate (green), butyrate (violet) and isovalerate (light blue) concentrations. The gray area illustrates the acetate concentration range under optimal operational conditions. The vertical black line denotes the end of the adaptation period, i.e. feeding with a constant, low protein dose (see Figures 1 and 4).

Figure 6. Accumulation of ammonium during the AD of casein (blue line) and pig blood (red line).

The gray area indicates the ammonium-nitrogen concentration range under optimal operational conditions. The vertical black line denotes the end of the adaptation period, i.e. feeding with a constant, low protein dose.

Figure 7. Comparison of acetate accumulation during growth on casein (blue line) or on pig blood (red line) in fed-batch AD.

The vertical black line indicates the end of the adaptation period, i.e. feeding with a constant, low protein dose.

Figure 8. Scaling-up of AD of blood protein.

Weekly average specific biogas yields were determined in completely stirred tank reactors of 5 L (blue columns) or 50 L (red columns). The X-axis indicates the weekly substrate doses for the 5 L (first number) and 50 L (second number) reactors. Note that both reactors were fed with the same amount of substrate for 3 weeks and the weekly dose was raised afterwards.

Figure 9. Testing of sustainability of biogas production from pig blood.

The weekly average specific biogas production is indicated by the blue columns, and the blood protein dosing is shown by the red line.

Figure 10. Changes induced in the Bacteria microbial community composition by adaptation to casein as sole carbon source.

The relative abundances of the taxonomic groups were determined in week 0 (light-blue columns), week 5 (medium-blue columns), week 9 (dark-blue columns) and week 12 (purple columns).

Figure 11. Changes induced in the Archaea microbial community composition by adaptation to casein as sole carbon source.

The relative abundances of the taxonomic groups were determined in week 0 (lightblue columns), week 5 (medium-blue columns), week 9 (dark-blue columns) and week 12 (purple columns).

Figure 12. Changes induced in the Bacteria microbial community composition by adaptation to pig blood protein as sole carbon source.

The relative abundances of the taxonomic groups were determined in week 0 (light-brown columns), week 8 (medium-brown columns) and week 12 (dark-brown columns).

Figure 13. Changes induced in the Archaea microbial community composition by adaptation to pig blood as sole carbon source.

The relative abundances of the taxonomic groups were determined on week 0 (light-brown columns), week 8 (medium-brown columns) and week 12 (dark-brown columns).