Biobased short chain fatty acid production - Exploring microbial community dynamics and metabolic networks through kinetic and microbial modeling approaches.

In recent years, there has been growing interest in harnessing anaerobic digestion technology for resource recovery from waste streams. This approach has evolved beyond its traditional role in energy generation to encompass the production of valuable carboxylic acids, especially volatile fatty acids (VFAs) like acetic acid, propionic acid, and butyric acid. VFAs hold great potential for various industries and biobased applications due to their versatile properties. Despite increasing global demand, over 90% of VFAs are currently produced synthetically from petrochemicals. Realizing the potential of large-scale biobased VFA production from waste streams offers significant eco-friendly opportunities but comes with several key challenges. These include low VFA production yields, unstable acid compositions, complex and expensive purification methods, and post-processing needs. Among these, production yield and acid composition stand out as the most critical obstacles impacting economic viability and competitiveness. This paper seeks to offer a comprehensive view of combining complementary modeling approaches, including kinetic and microbial modeling, to understand the workings of microbial communities and metabolic pathways in VFA production, enhance production efficiency, and regulate acid profiles through the integration of omics and bioreactor data.

Exploitation of microbial activities at low pH to enhance planetary health.

Awareness is growing that human health cannot be considered in isolation but is inextricably woven with the health of the environment in which we live. It is, however, under-recognized that the sustainability of human activities strongly relies on preserving the equilibrium of the microbial communities living in/on/around us. Microbial metabolic activities are instrumental for production, functionalization, processing, and preservation of food. For circular economy, microbial metabolism would be exploited to produce building blocks for the chemical industry, to achieve effective crop protection, agri-food waste revalorization, or biofuel production, as well as in bioremediation and bioaugmentation of contaminated areas. Low pH is undoubtedly a key physical-chemical parameter that needs to be considered for exploiting the powerful microbial metabolic arsenal. Deviation from optimal pH conditions has profound effects on shaping the microbial communities responsible for carrying out essential processes. Furthermore, novel strategies to combat contaminations and infections by pathogens rely on microbial-derived acidic molecules that suppress/inhibit their growth. Herein, we present the state-of-the-art of the knowledge on the impact of acidic pH in many applied areas and how this knowledge can guide us to use the immense arsenal of microbial metabolic activities for their more impactful exploitation in a Planetary Health perspective.

Impact of wastewater characteristics on the removal of organic micropollutants by Chlorella sorokiniana.

Microalgae-based technologies can be used for the removal of organic micropollutants (OMPs) from different types of wastewater. However, the effect of wastewater characteristics on the removal is still poorly understood. In this study, the removal of sixteen OMPs by Chlorella sorokiniana, cultivated in three types of wastewater (anaerobically digested black water (AnBW), municipal wastewater (MW), and secondary clarified effluent (SCE)), were assessed. During batch operational mode, eleven OMPs were removed from AnBW and MW. When switching from batch to continuous mode (0.8 d HRT), the removal of most OMPs from AnBW and MW decreased, suggesting that a longer retention time enhances the removal of some OMPs. Most OMPs were not removed from SCE since poor nutrient availability limited C. sorokiniana growth. Further correlation analyses between wastewater characteristics, biomass and OMPs removal indicated that the wastewater soluble COD and biomass concentration predominantly affected the removal of OMPs. Lastly, carbon uptake rate had a higher effect on the removal of OMPs than nitrogen and phosphate uptake rate. These data will give an insight on the implementation of microalgae-based technologies for the removal of OMPs in wastewater with varying strengths and nutrient availability.

Conceptual system for sustainable and next-generation wastewater resource recovery facilities.

Shifting the concept of municipal wastewater treatment to recover resources is one of the key factors contributing to a sustainable society. A novel concept based on research is proposed to recover four main bio-based products from municipal wastewater while reaching the necessary regulatory standards. The main resource recovery units of the proposed system include upflow anaerobic sludge blanket reactor for the recovery of biogas (as product 1) from mainstream municipal wastewater after primary sedimentation. Sewage sludge is co-fermented with external organic waste such as food waste for volatile fatty acids (VFAs) production as precursors for other bio-based production. A portion of the VFA mixture (product 2) is used as carbon sources in the denitrification step of the nitrification/denitrification process as an alternative for nitrogen removal. The other alternative for nitrogen removal is the partial nitrification/anammx process. The VFA mixture is separated with nanofiltration/reverse osmosis membrane technology into low-carbon VFAs and high-carbon VFAs. Polyhydroxyalkanoate (as product 3) is produced from the low-carbon VFAs. Using membrane contactor-based processes and ion-exchange techniques, high-carbon VFAs are recovered as one-type VFA (pure VFA) and in ester forms (product 4). The nutrient-rich fermented and dewatered biosolid is applied as a fertilizer. The proposed units are seen as individual resource recovery systems as well as a concept of an integrated system. A qualitative environmental assessment of the proposed resource recovery units confirms the positive environmental impacts of the proposed system.

Statistical Analysis of SARS-CoV-2 Using Wastewater-Based Data of Stockholm, Sweden.

An approach based on wastewater epidemiology can be used to monitor the COVID-19 pandemic by assessing the gene copy number of SARS-CoV-2 in wastewater. In the present study, we statistically analyzed such data from six inlets of three wastewater treatment plants, covering six regions of Stockholm, Sweden, collected over an approximate year period (week 16 of 2020 to week 22 of 2021). SARS-CoV-2 gene copy number and population-based biomarker PMMoV, as well as clinical data, such as the number of positive cases, intensive care unit numbers, and deaths, were analyzed statistically using correlations and principal component analysis (PCA). Despite the population differences, the PCA for the Stockholm dataset showed that the case numbers are well grouped across wastewater treatment plants. Furthermore, when considering the data from the whole of Stockholm, the wastewater characteristics (flow rate m3/day, PMMoV Ct value, and SARS-CoV gene copy number) were significantly correlated with the public health agency's report of SARS-CoV-2 infection rates (0.419 to 0.95, p-value < 0.01). However, while the PCA results showed that the case numbers for each wastewater treatment plant were well grouped concerning PC1 (37.3%) and PC2 (19.67%), the results from the correlation analysis for the individual wastewater treatment plants showed varied trends. SARS-CoV-2 fluctuations can be accurately predicted through statistical analyses of wastewater-based epidemiology, as demonstrated in this study.

Long-term SARS-CoV-2 surveillance in the wastewater of Stockholm: What lessons can be learned from the Swedish perspective?

Wastewater-based epidemiology (WBE) can be used to track the spread of SARS-CoV-2 in a population. This study presents the learning outcomes from over two-year long monitoring of SARS-CoV-2 in Stockholm, Sweden. The three main wastewater treatment plants in Stockholm, with a total of six inlets, were monitored from April 2020 until June 2022 (in total 600 samples). This spans five major SARS-CoV-2 waves, where WBE data provided early warning signals for each wave. Further, the measured SARS-CoV-2 content in the wastewater correlated significantly with the level of positive COVID-19 tests (r = 0.86; p << 0.0001) measured by widespread testing of the population. Moreover, as a proof-of-concept, six SARS-CoV-2 variants of concern were monitored using hpPCR assay, demonstrating that variants can be traced through wastewater monitoring. During this long-term surveillance, two sampling protocols, two RNA concentration/extraction methods, two calculation approaches, and normalization to the RNA virus Pepper mild mottle virus (PMMoV) were evaluated. In addition, a study of storage conditions was performed, demonstrating that the decay of viral RNA was significantly reduced upon the addition of glycerol to the wastewater before storage at -80 °C. Our results provide valuable information that can facilitate the incorporation of WBE as a prediction tool for possible future outbreaks of SARS-CoV-2 and preparations for future pandemics.

The effects of pH on the production of volatile fatty acids and microbial dynamics in long-term reactor operation.

Volatile fatty acids, intermediate products of anaerobic digestion, are one of the most promising biobased products. In this study, the effects of acidic (pH 5), neutral (without pH adjustment) and alkali (pH 10) pH on production efficiency and composition of volatile fatty acids (VFAs) and bacterial community profile were analyzed. The anaerobic sequencing batch reactors were fed cheese production wastewater as substrate and inoculated by anaerobic granular seed sludge. The results showed that acidic pH improved VFA production yield (0.92 at pH 5; 0.42 at pH 10 and 0.21 gCOD/gVS at neutral pH). Furthermore, propionic acid was dominant under both pH 10 (64 ± 20%) and neutral pH (72 ± 8%), whereas, acetic acid (23 ± 20%4), propionic acid (22 ± 3%), butyric acid (21 ± 4%) and valeric acid (15 ± 8%) were almost equally distributed under pH 5. Adaptation of bacterial community to different pH conditions might steer the acid profile: Bacteroidetes (50.07 ± 2%) under pH 10, Proteobacteria (40.74 ± 7%) under neutral pH and Firmicutes (47.64 ± 9%) under pH 5 were the most dominant phylum, respectively. Results indicated pH plays a significant role in VFA production, acid composition, and bacterial community structure. However, in order to gain a concrete understanding effects of pH, characterization of intracellular and extracellular metabolites with dynamics of the microbial community is required.

Bioaugmented Mixed Culture by Clostridium aceticum to Manipulate Volatile Fatty Acids Composition From the Fermentation of Cheese Production Wastewater.

Production of targeted volatile fatty acid (VFA) composition by fermentation is a promising approach for upstream and post-stream VFA applications. In the current study, the bioaugmented mixed microbial culture by Clostridium aceticum was used to produce an acetic acid dominant VFA mixture. For this purpose, anaerobic sequencing batch reactors (bioaugmented and control) were operated under pH 10 and fed by cheese processing wastewater. The efficiency and stability of the bioaugmentation strategy were monitored using the production and composition of VFA, the quantity of C. aceticum (by qPCR), and bacterial community profile (16S rRNA Illumina Sequencing). The bioaugmented mixed culture significantly increased acetic acid concentration in the VFA mixture (from 1170 ± 18 to 122 ± 9 mgCOD/L) compared to the control reactor. Furthermore, the total VFA production (from 1254 ± 11 to 5493 ± 36 mgCOD/L) was also enhanced. Nevertheless, the bioaugmentation could not shift the propionic acid dominancy in the VFA mixture. The most significant effect of bioaugmentation on the bacterial community profile was seen in the relative abundance of the Thermoanaerobacterales Family III. Incertae sedis, its relative abundance increased simultaneously with the gene copy number of C. aceticum during bioaugmentation. These results suggest that there might be a syntropy between species of Thermoanaerobacterales Family III. Incertae sedis and C. aceticum. The cycle analysis showed that 6 h (instead of 24 h) was adequate retention time to achieve the same acetic acid and total VFA production efficiency. Biobased acetic acid production is widely applicable and economically competitive with petroleum-based production, and this study has the potential to enable a new approach as produced acetic acid dominant VFA can replace external carbon sources for different processes (such as denitrification) in WWTPs. In this way, the higher treatment efficiency for WWTPs can be obtained by recovered substrate from the waste streams that promote a circular economy approach.

Bioaugmentation as a strategy for tailor-made volatile fatty acid production.

This study aimed to develop a novel strategy for tailor-made volatile fatty acid (VFA) composition. For this purpose, the mixed microbial culture was bioaugmented by Propionibacterium acidipropionici. Anaerobic sequencing batch reactors were operated with cheese wastewater under alkali pH. While the maximum propionic acid production almost four times increased (3779 ± 201 mgCODeq propionic acid/L in the bioaugmented reactor and 942 ± 172 mgCODeq propionic acid/L in the control reactor), there was no significant difference in VFA composition. The gene copy number of P.acidipropionici increased 20 times after the bioaugmentation. Furthermore, the gene copy number of P.acidipropionici was positively correlated with total VFA and isovaleric acid concentration. The relative abundance of family Flavobacteriaceae increased in the bioaugmented reactor, which might be caused by the syntrophic relation between Flavobacteriaceae and P. acidipropionici. The cycle analysis results showed that the shorter cycle (6h) could ensure the same efficiency.

Bioconversion of food waste to volatile fatty acids: Impact of microbial community, pH and retention time.

Bio-based production of materials from waste streams is a pivotal aspect in a circular economy. This study aimed to investigate the influence of inoculum (three different sludge taken from anaerobic digestors), pH (5 & 10) and retention time on production of total volatile fatty acids (VFAs), VFA composition as well as the microbial community during anaerobic digestion of food waste. The highest VFA production was ∼22000 ± 1036 mg COD/L and 12927 ± 1029 mg COD/L on day 15 using the inoculum acclimated to food waste at pH 10 and pH 5, respectively. Acetic acid was the dominant VFA in the batch reactors with initial alkaline conditions, whereas both propionic and acetic acids were the dominant products in the acidic condition. Firmicutes, Chloroflexi and Bacteroidetes had the highest relative abundance in the reactors. VFA generation was positively correlated to the relative abundance of Firmicutes.

Bio-based conversion of volatile fatty acids from waste streams to polyhydroxyalkanoates using mixed microbial cultures.

Production of polyhydroxyalkanoates is an important field in the biorefinery as bio-alternative to conventional plastics. However, its commercialization is still limited by high production cost. In this study, a process with the potential to reduce the production cost of polyhydroxyalkanoates was proposed. Mixed cultures accumulated polyhydroxyalkanoates using volatile fatty acid-rich effluents from waste streams, without pH and temperature control. In addition, the impact of two types of carbon sources was investigated by analyzing the microbial community as well as the polyhydroxyalkanoate accumulation capacity. Mixed cultures successfully adapted to different substrates, consuming the volatile fatty acids in their totality. The phyla Proteobacteria, Bacteroidetes and Firmicutes dominated the bacterial community. The highest polyhydroxyalkanoate content was 43.5% w/w, which is comparable to contents reported from mixed cultures using synthetic carbon sources. The biopolymer consisted of (R)-3-hydroxybutyrate 94.8 ± 1.7% w/w and (R)-3-hydroxyvaletare 5.2 ± 1.7% w/w.

Benchmarking virus concentration methods for quantification of SARS-CoV-2 in raw wastewater.

Wastewater-based epidemiology offers a cost-effective alternative to testing large populations for SARS-CoV-2 virus, and may potentially be used as an early warning system for SARS-CoV-2 pandemic spread. However, viruses are highly diluted in wastewater, and a validated method for their concentration and further processing, and suitable reference viruses, are the main needs to be established for reliable SARS-CoV-2 municipal wastewater detection. For this purpose, we collected wastewater from two European cities during the Covid-19 pandemic and evaluated the sensitivity of RT-qPCR detection of viral RNA after four concentration methods (two variants of ultrafiltration-based method and two adsorption and extraction-based methods). Further, we evaluated one external (bovine corona virus) and one internal (pepper mild mottle virus) reference virus. We found a consistently higher recovery of spiked virus using the modified ultrafiltration-based method. This method also had a significantly higher efficiency (p-value <0.01) for wastewater SARS-CoV-2 detection. The ultracentrifugation method was the only method that detected SARS-CoV-2 in the wastewater of both cities. The pepper mild mottle virus was found to function as a potentially suitable internal reference standard.

A comprehensive study of volatile fatty acids production from batch reactor to anaerobic sequencing batch reactor by using cheese processing wastewater.

Volatile fatty acids (VFAs) has great potential for closed-loop production in dairy industries via resource recovery from waste-streams. In the current study, the transition of VFA production from batch reactor to anaerobic sequencing batch reactor (ASBR) by using cheese industry wastewater under alkali pH was evaluated with respect to seed sludge structure, microbial diversity and reactor type. The transition from the batch reactor to the ASBR demonstrated that the maximum VFA production yield (g COD/g SCOD) was comparable in two reactors (batch: 0.97; ASBR: 0.94), whereas, the dominant acid type was different (batch: 49% lactic acid; ASBR: 80% propionic acid). There was a significant correlation between the productions of butyric acid with Gracilibacteraceae and Desulfovibrionaceae; propionic acid with Desulfovibrionaceae and Synergistaceae; lactic acid with Pseudomonadaceae and Rhodocyclaceae. The high VFA production efficiency can be achieved by long term reactor operation, which enables the shift from industrial waste-streams to biorefineries.

Volatile fatty acid production from semi-synthetic milk processing wastewater under alkali pH: The pearls and pitfalls of microbial culture.

Volatile fatty acids (VFA) are one of the most promising sustainable and environmentally friendly bioproduct owing to their wide usage area and high market demand. For this reason, in this study, the evaluation of VFA production from pure and mixed bacterial cultures was aimed. Three different mixed cultures with C. aceticum, C. butyricum and P. acidipropionici as pure cultures were used for inoculation of milk processing wastewater fermentation under pH 10 for 15 days. The mixed culture fermentation had the highest VFA production efficiency whereas the highest amount of acetic, butyric and propionic acid productions were obtained by C. aceticum, C. butyricum and P. acidipropionici, respectively. Also, the mixed cultures demonstrated faster pH regulation and acclimation than the pure cultures tested. Therefore, development of synthetic cultures may offer a useful approach to produce VFA mixtures with one-dominant acid type and with high production efficiency.

Volatile fatty acids production via mixed culture fermentation: Revealing the link between pH, inoculum type and bacterial composition.

The aim of the study was to investigate the effects of operational parameters, inoculum type and bacterial community on mixed culture fermentation to produce one dominant acid type in the mixture of volatile fatty acids (VFA). The study was performed using three different inocula (large&small granular and slurry) with glucose under various initial pH. The VFA production efficiency reached to 0,97 (gCOD/gSCOD) by granular sludge. VFA composition was changed by initial pH: in neutral conditions, acetic acid; in acidic conditions, acetic and butyric acids, in alkali conditions butyric acid were dominated, respectively. The VFA production was positively affected by the high relative abundance of Firmicutes. On the contrary, a negative correlation was seen between VFA production and the relative abundance of Chloroflexi. The results revealed the physical sludge structure of inoculum was the key factor for production efficiency, whereas, pH was the most important parameter to affect VFA composition.

Bio-based volatile fatty acid production and recovery from waste streams: Current status and future challenges.

Bio-based volatile fatty acid (VFA) production from waste-stream is getting attention due to increasing market demand and wide range usage area as well as its cost-effective and environmentally friendly approach. The aim of this paper is to give a comprehensive review of bio-based VFA production and recovery methods and to give an opinion on future research outlook. Effects of operation conditions including pH, temperature, retention time, type of substrate and mixed microbial cultures on VFA production and composition were reviewed. The recovery methods in terms of gas stripping with absorption, adsorption, solvent extraction, electrodialysis, reverse osmosis, nanofiltration, and membrane contractor of VFA were evaluated. Furthermore, strategies to enhance bio-based VFA production and recovery from waste streams, specifically, in-line VFA recovery and bioaugmentation, which are currently not used in common practice, are seen as some of the approaches to enhance bio-based VFA production.