Abstract/Summary

The increasing demand for whey protein products has led to significant production of whey permeate as the byproduct. Whey permeate has a high organic load, mainly due to high lactose concentration with small amount of other organic compounds, which limits its potential applications. The high organic load of whey permeate poses environmental risks if directly discharged without prior treatments. Thus, effective treatment strategies for whey permeate become important to support the growth and circularity within the dairy industry. The current applications of whey permeate have been facing various challenges, such as limited consumer acceptance when used as consumable products — especially for consumers with lactose intolerance, handling difficulties due to lactose crystallization, as well as secondary waste generation and needing complex processes that further adds to the cost of treating the byproduct. By considering these challenges, anaerobic digestion (AD) emerges as a promising solution for whey permeate treatment. The AD technology offers practicality and versatility in processing diverse feedstocks to produce renewable energy in the form of methane (CH4) gas and valuable byproducts like volatile fatty acids (VFAs). Furthermore, the AD process simultaneously reduce organic load of the digested feedstock with minimum secondary waste generation. This study investigates optimization strategies for AD of whey permeate to enhance CH4 production by initially observed whey permeate potential as an AD feedstock under batch reactors. Subsequently, optimization strategies for whey permeate AD were explored under semi-continuous stirred tank reactor (s-CSTR) system, which include the application of different organic loading rate (OLR), digestate recirculation, activated carbon (AC) supplementation, as well as organic (urea) and inorganic (ammonium sulfate) nitrogen supplementation. Initial investigations into AD of whey permeate were conducted through biochemical methane potential (BMP) tests by evaluating the effect of inoculum-to-substrate ratios (ISRs), pH levels, and temperatures under batch reactors. The highest CH4 production has iii shown to be achieved at different temperatures when different source of whey permeate were used. In our first BMP test using liquid whey permeate, optimal conditions were achieved at an ISR of 2, pH 8, and 30 °C. This operational condition resulted with a cumulative CH4 yield of 466.29 ± 13.71 NLCH4.kgVS⁻¹. However, when powdered whey permeate from a different source was tested, the optimal operation shifted to ISR of 2, pH 7.5, and 37 °C, which produced a higher cumulative CH4 production of 653.64 ± 12.16 NLCH4.kgVS⁻¹. Furthermore, directly applying kinetic parameters as determined from the BMP tests data into s-CSTR system revealed operational challenges. Despite achieving a high CH4 yield in batch systems under ISR 2 with technical digestion time (T80-90) of 14 d, applying these parameters to determine the OLR and hydraulic retention time (HRT) values for s-CSTR system resulted with operational instability and ultimately system failure. In response to this, kinetic parameters from BMP tests were only used as rough guidelines. The observation of whey permeate AD under s-CSTR system was then operated at OLR of 2.5 and 4 gVSL -1d -1 with HRT of 30 d for 150 days. In this operational set up, CH4 production rate was declining for the first three HRTs before stabilizing for the rest of the observation. This study revealed that OLR of 2.5 gVSL -1d -1 produced higher cumulative CH4, with the average daily CH4 production of 38.20 ± 5.04 NLCH4kgVS⁻¹d -1 during steady period. The decline in CH4 production during the first three HRTs, as well as different AD performance between each observed reactors, were shown to be correlated with volatile solids degradation, VFA accumulation, and shifts in microbial communities. Initially, acetogenic bacteria (e.g., Trichococcus and Sedimentibacter) dominated the digestate, but over time, propionic acid-producing bacteria (e.g., Actinomyces and Acidipropionibacterium) became prevalent. Concurrently, the dominant archaea in the digestate shifted from Methanosarcina to Methanobacterium, indicating a transition in methanogenesis pathways that directly impacted AD performance. iv Following results from different OLRs experiment, AC supplementation and digestate recirculation alongside trace elements addition were investigated at OLR of 2.5 gVSL -1d -1 to further explore strategies for AD optimization. In this experiment, stable CH4 productions were observed in all reactors since the first HRT. This indicates that trace elements addition at the concentration of 0.3 ppm could stabilize CH4 production in whey permeate AD. Moreover, combining one-time AC supplementation with digestate recirculation significantly improved CH4 yields, with the daily average of 60.53 ± 13.72 NLCH4kgVS⁻¹d -1 . This CH4 yield outperforms reactors with daily AC supplementation or digestate recirculation alone. These interventions also influenced microbial communities, where Methanosarcina and Methanoculleus populations were enriched. Separately, this study also investigates nitrogen and AC supplementation strategies towards AD of whey permeate. Two different nitrogen sources, urea (organic nitrogen source) at the concentration of 0.07 g.L-1 and ammonium sulfate (inorganic nitrogen source) at the concentration of 0.16 g.L-1 are supplemented separately to fix the C/N ratio of the system into 25. The effects of the nitrogen alongside AC supplementation on AD performance of whey permeate are then evaluated. This investigation reveals that nitrogen alongside AC supplementation increased CH4 production, with the average increase at 7–42% as compared to reactor without nitrogen supplementation. Furthermore, urea supplementation alone achieved the highest CH4 yield at the average daily production of 81.73 ± 20.32 NLCH4kgVS⁻¹d -1 . Similar to the different OLRs experiment, microbial analysis indicated a transition from Methanosarcina to hydrogenotrophic methanogens like Methanobacterium and Methanoculleus, suggesting that digestate recirculation had an effect to maintain acetoclastic methanogens population, while nitrogen supplementation promotes hydrogenotrophic methanogens, regardless of AC supplementation. In summary, while AD presents a viable pathway for whey permeate valorization, achieving stable and efficient operations requires careful optimization of parameters such as ISR, pH, temperature, and nutrient balance. Strategies like AC supplementation, digestate recirculation, and nitrogen management offer promising avenues for enhancing AD performance and microbial stability. Further research is needed to address scalability, and long-term operational challenges, paving the way for sustainable and economically feasible whey permeate utilization.