Engineering and microbiology insights into co-fermentation of waste activated sludge and food waste

Abstract

[eng] According to the circular economy principles, wastewater treatment plants (WWTPs) should be transformed into water resource recovery facilities. Urban organic waste, including sewage sludge from WWTPs, can be converted into valuable byproducts, such as volatile fatty acids (VFA), through acidogenic fermentation (AF). AF involves hydrolysis, acidogenesis, and acetogenesis, while limiting methanogenesis, especially aceticlastic Archaea that consume acetic acid. One strategy to increase VFA yields from organic wastes is co-fermentation, which aims to ferment two or more complementary substrates simultaneously. The mixture of waste activated sludge (WAS) and food waste (FW) is one of the most studied combination for acidogenic co-fermentation, as WAS provides buffering capacity and indigenous microbial communities, while FW offers highly biodegradable organic matter. However, most studies (∼40 %) rely on batch tests, limiting insights into biomass migration and the effects of operational variables on the microbial community observed in continuous operation. Key operating variables, such as temperature, retention time, and organic loading rate (OLR) remain underexplored. This thesis addresses key challenges in acidogenic co-fermentation, including enhancing fermentation yields, optimising product profiles, and preventing methanogenesis. This research evaluates the long-term co-fermentation of WAS and FW under varying operational conditions, including the effects of OLR shock, microbial source variability, operational temperature, and WAS collection period on fermentation performance and microbial dynamics, with the goal of achieving a consistent and reproducible co-fermentation process. Three experimental studies on semi-continuous acidogenic co-fermentation have been conducted to evaluate different operational variables. In the first study, two key perturbations were evaluated in the acidogenic co-fermentation at 35 °C: an increase in OLR and changes in WAS sources (same WWTP, different collection periods). A control reactor was operated at 11 gVS/(L⋅d), while a test reactor experienced a sustained OLR increase to 18 gVS/(L⋅d). Despite the OLR increase, fermentation yields remained consistent (~300 mgCOD/gVS). However, the product profile shifted: at 11 gVS/(L⋅d), the fermentation liquid was enriched in acetic, butyric and propionic acids, while at 18 gVS/(L⋅d), acetic acid, ethanol, and caproic acid predominated. Reverting the OLR to 11 gVS/(L⋅d) restored the original profile. Changes in the collected WAS introduced acetic

acid-consuming methanogens, whose growth was temporarily suppressed by the higher OLR. This study demonstrated that microbial monitoring and post-fermentation tests are effective tools for the early detection of acetic acid consumption events. The second study investigated long-term temperature effects on co-fermentation yields and microbial dynamics. Semi-continuous fermenters were evaluated at operating temperatures of 25, 35, 45, and 55 °C. WAS batches collected at different periods were used to evaluate their microbiota's influence on the fermenters' microbial communities. Results showed and improvement of the solubilisation at higher temperatures and the highest fermentation yield was at 55 °C (425 ± 28 mgCOD/gVS). Temperature also had a crucial impact on the fermentation product profile. At 55 °C, acetic and butyric acids were the dominated products. Then, at 35 °C, acetic, butyric, and propionic acids predominated, and at 45 °C, caproic acid was accumulated. Distinct microbial communities emerged at each temperature, also influenced by the WAS microbiota. Methanogens introduced with WAS were probably responsible for the acetic acid consumption detected in one of the fermenters operating at 35 °C. In the third and last study, two long-term co-fermentation experiments evaluated the effects of temperature (35 °C vs. 55 °C), WAS origin, and collection periods. At 55 °C, solubilisation yields improved compared to 35 °C, although fermentation yields did not consistently higher. The product profile at 55 °C was dominated by acetic and butyric acids in 13 operated fermenters. Temperature had a greater impact on microbial community structure than WAS origin. Acetic acid consumption, observed only at 35 °C in fermenters fed with WAS_A, was attributed to Methanosarcina species (midas_s_9557), which were inhibited at 55 °C. Functional redundancy ensured stable metabolic functions among fermenters at 55 °C. These findings demonstrate the robustness and consistency of thermophilic co-fermentation as a biotechnological solution for managing WAS and FW.

[cat] L’objectiu d’aquesta tesi doctoral és abordar els principals reptes de la co-fermentació, com la millora dels rendiments de fermentació, l’optimització del perfil de productes i evitar el creixement de microorganismes consumidors d’àcid acètic. La recerca es centra en la co-fermentació contínua de llots actius (LA) i residus alimentaris (RA) en diferents condicions d’operació. En aquesta tesi s’han realitzat 3 estudis de co-fermentació contínua de LA i RA sota diferents condicions operatives. En el primer estudi, es van avaluar dues pertorbacions clau en fermentadors a 35 °C: un augment de la càrrega orgànica (CO) i canvis en les fonts de LA (mateixa EDAR, diferents períodes de recol·lecció). Malgrat l’increment de CO de 11 a 18 gSV/(L·d), els rendiments de fermentació es van mantenir. El perfil de productes va canviar, dominat per àcids acètic, butíric i propiònic a 11 gSV/(L·d), a àcid acètic, etanol i àcid caproic a 18 gSV/(L·d). Restaurar la CO va recuperar el perfil original. Els canvis en LA van introduir metanògens aceticlàstics, el creixement dels quals va ser temporalment suprimit per l’augment de CO. El segon estudi va explorar els efectes de la temperatura (25, 35, 45 i 55 °C) sobre la co-fermentació i les comunitats microbianes. El major rendiment de solubilització es va observar a 45 °C, mentre que a 55 °C es va obtenir el major rendiment de fermentació. A 55 °C, van predominar els àcids acètic i butíric, mentre que a 45 °C es va detectar acumulació d’àcid caproic. Les comunitats microbianes van ser específiques de cada temperatura, influenciades pel microbiota del LA. En un dels fermentadors operats a 35 °C, es va observar un consum d’àcid acètic, que va ser inhibit a 55 °C. En el tercer estudi, es van comparar els efectes de temperatura (35 °C vs. 55 °C), origen de LA i períodes de recol·lecció. Tots els fermentadors operats a 55 °C van obtenir rendiments i perfil de productes similars. A 55 °C, no es va observar consum d’àcid acètic, atribuït a la inhibició de Methanosarcina. Aquests resultats demostren la robustesa de la co-fermentació de LA i RA termòfila com a tecnologia.

[spa] El objetivo de esta tesis es abordar los principales retos de la co-fermentación, como la mejora de los rendimientos de fermentación, optimizar el perfil de productos, y evitar el crecimiento de microorganismos consumidores de ácido acético. La investigación se centra en la co-fermentación en continuo de lodos activos (LA) y residuos alimentarios (RA) en diferentes condiciones de operación. En esta tesis se investigaron tres estudios de co-fermentación continua de LA y RA bajo distintas condiciones operativas. En el primer estudio, se evaluaron dos perturbaciones clave en fermentadores a 35 °C: un aumento de la CO y cambios en las fuentes de LA (misma EDAR, diferentes periodos de recolección). A pesar del incremento de CO de 11 a 18 gSV/(L·d), los rendimientos de fermentación se mantuvieron. El perfil de productos cambió, dominado por ácidos acético, butírico y propiónico a 11 gSV/(L·d) a ácido acético, etanol y ácido caproico a 18 gSV/(L·d). Restaurar la CO recuperó el perfil original. Los cambios en LA introdujeron metanógenos aceticlásticos, cuyo crecimiento fue temporalmente suprimido por el aumento de CO. El segundo estudio exploró los efectos de la temperatura (25, 35, 45 y 55 °C) sobre la co-fermentación y las comunidades microbianas. El mayor rendimiento de solubilización se observó a 45 °C, mientras que a 55 °C se obtuvo el mayor rendimiento de fermentación. A 55 °C, predominaron los ácidos acético y butírico, mientras que a 45 °C se detectó acumulación de ácido caproico. Las comunidades microbianas fueron específicas de cada temperatura, influenciadas por el microbiota del LA. En uno de los fermentadores operados a 35 °C, se observó un consumo de ácido acético, que fue inhibido a 55 °C. En el tercer estudio, se compararon los efectos de temperatura (35 °C vs. 55 °C), origen del LA y periodos de recolección. Todos los fermentadores operados a 55 °C (13) obtuvieron rendimientos y perfil de productos similares. A 55 °C, no se observó consumo de ácido acético, atribuido a la inhibición de Methanosarcina. Estos resultados demuestran la robustez de la co-fermentación de LA y RA termofílica como tecnología.