Performance Of Bioreactor Research Articles

A perspective-driven and technical evaluation of machine learning in bioreactor scale-up: A case-study for potential model developments.

Bioreactor scale-up and scale-down have always been a topical issue for the biopharmaceutical industry and despite considerable effort, the identification of a fail-safe strategy for bioprocess development across scales remains a challenge. With the ubiquitous growth of digital transformation technologies, new scaling methods based on computer models may enable more effective scaling. This study aimed to evaluate the potential application of machine learning (ML) algorithms for bioreactor scale-up, with a specific focus on the prediction of scaling parameters. Factors critical to the development of such models were identified and data for bioreactor scale-up studies involving CHO cell-generated mAb products collated from the literature and public sources for the development of unsupervised and supervised ML models. Comparison of bioreactor performance across scales identified similarities between the different processes and primary differences between small- and large-scale bioreactors. A series of three case studies were developed to assess the relationship between cell growth and scale-sensitive bioreactor features. An embedding layer improved the capability of artificial neural network models to predict cell growth at a large-scale, as this approach captured similarities between the processes. Further models constructed to predict scaling parameters demonstrated how ML models may be applied to assist the scaling process. The development of data sets that include more characterization data with greater variability under different gassing and agitation regimes will also assist the future development of ML tools for bioreactor scaling.

How high salt shock affects performance and membrane fouling characteristics of a halophilic membrane bioreactor used for treating hypersaline wastewater

In the present study, the effect of short-term salt shocks (13% and 20%) on the performance of a halophilic MBR bioreactor used to treat a hypersaline (5% salt) synthetic wastewater was considered. 13% and 20% salt shocks resulted in a transient and permanent decrease in chemical oxygen demand removal efficiency, respectively which could be correlated with soluble microbial products (SMP) concentration and specific oxygen uptake rate values of the halophilic population. DNA leakage tests suggested that both 13% and 20% short-term salt shocks resulted in some cell structural damage. During both 13% and 20% salt shocks mixed liquor SMP, extracellular polymeric substances (EPS), zeta potential and endogenous respiration increased while relative hydrophobicity, EPSp/EPSc and exogenous respiration decreased; in both cases, however, the pre-shock values for these parameters were restored after the removal of the salt shock. 13% salt shock resulted in a transient increase in the membrane fouling rate and a permanent rise in total membrane resistance (Rt). On the other hand, both membrane fouling rate and Rt increased during 20% salt shock. Membrane fouling rate initially reduced after the 20% salt shock removal but after 5 days a “TMP jump” occurred. The latter was caused by the higher steady state SMPc and SMPp concentrations after removal of 20% salt shock compared to pre-shock values. This might have either resulted in a decrease in critical flux or an increase in local flux above critical flux in some parts of the membrane. The contribution of cake layer resistance to overall membrane resistance increased after the 13% and 20% salt shocks. The findings of the present study reveal the robustness of halophilic MBRs against salt shocks in the treatment of hypersaline wastewater. However, in cases of very high salt shocks, appropriate membrane fouling reduction strategies should be carried out during its operation.

Performance evaluation and metagenomic analysis of sequencing batch reactor under transient 2,4,6-trichlorophenol shock

The transient chlorophenol shock under some emergency conditions might directly affect the pollutant removal of bioreactor. Therefore, the recovery of bioreactor performance after transient chlorophenol shock is a noteworthy issue. In the present research, the performance, antioxidant response, microbial succession and functional genes of sequencing batch reactor (SBR) were evaluated under transient 2,4,6-trichlorophenol (2,4,6-TCP) shock. The chemical oxygen demand (COD) and ammonia nitrogen (NH4+-N) removal efficiencies decreased sharply in the first 4 days after 60 mg/L 2,4,6-TCP shock for 24 h and gradually recovered to normal in the subsequent 8 days. The nitrogen removal rates and their corresponding enzymatic activities rapidly decreased after transient 2,4,6-TCP shock and then gradually increased to normal. The increase of antioxidant enzymatic activity, Cu-Zn SOD genes and Fe-Mn SOD genes contributed to the recovery of SBR performance. The abundance of genes encoding ammonia monooxygenase and hydroxylamine dehydrogenase decreased after transient 2,4,6-TCP shock, including amoA, amoC and nxrA. Thauera, Dechloromonas and Candidatus_Competibacter played key roles in the restorative process, which provided stable abundances of narG, norB , norC and nosZ. The results will deeply understand into the effect of transient 2,4,6-TCP shock on bioreactor performance and provide theoretical basis to build promising recoveries strategy of bioreactor performance.

Performance of Fe-dosing membrane bioreactor and evaluation of phosphorus recovery as vivianite after long-term operation

In this study, the influence of high concentration of iron on the nutrient removal, membrane fouling and microbial diversity of membrane bioreactor for wastewater treatment were systematically investigated. Moreover, the potential of phosphorus recovery in the form of vivianite was assessed after the long-term operation. The results show that TP concentration in effluent was decreased to 1.07 ± 0.47 mg/L (without Fe addition), 0.41 ± 0.23 mg/L (Fe/P = 2), 0.20 ± 0.12 mg/L (Fe/P = 4). Compared with control group, the TN concentration in effluent was higher at the high concentration of iron, which was due to the decreased nitrite-oxidizing bacteria (NOB) by microbial population analysis. Membrane fouling was alleviated by enlarging floc size and reducing supernatant COD via adding iron. Besides, it was detected that the amorphous ferric oxide and Fe(III) bound phosphorus were the main Fe and P speciation and vivanite (ca. 29 %). The anaerobic environment was conducive to the formation of vivianite and the phosphorus recovery as vivanite was recommended based on the results of current study.

Mg2+ addition: Unlocking optimized treatment performance and anti-fouling property in microalgal-bacterial membrane bioreactors

While microalgal-bacterial membrane bioreactors (microalgal-bacterial MBRs) have risen as an important technique in the realm of sustainable wastewater treatment, the membrane fouling caused by free microalgae is still a significant challenge to cost-effective operation of the microalgal-bacterial MBRs. Addressing this imperative, the current study investigated the influence of magnesium ion (Mg2+) addition on the biological dynamics and membrane fouling characteristics of the laboratory-scale submerged microalgal-bacterial MBRs. The results showed that Mg2+, important in augmenting photosynthetic process, yielded a biomass concentration of 2.92 ± 0.06 g/L and chlorophyll-a/MLSS (mixed liquor suspended solids) of 33.95 ± 1.44 mg/g in the RMg (Mg2+ addition test group). Such augmentation culminated in elevated total nitrogen and phosphorus removal efficiencies, clocking 81.73 % and 80.98 % respectively in RMg. Remarkably, despite the enhanced microalgae activity and concentration in RMg, the TMP growth rate declined by a significant 46.8 % compared to R0. Detailed characterizations attributed reduced membrane fouling of RMg to a synergy of enlarged floc size and reduced EPS contents. This transformation is intrinsically linked to the bridging action of Mg2+ and its role in creating a non-stressed ecological environment for the microalgal-bacterial MBR. In conclusion, the addition of Mg2+ in the microalgal-bacterial MBR appears an efficient approach, improving the efficiency of pollutant treatment and mitigating fouling, which potentially revolutionizes cost-effective applications and propels the broader acceptance of microalgal-bacterial MBRs. It also of great importance to promote the development and application of microalgal-bacterial wastewater treatment technology.

Dissolved organic matter characteristics linked to bacterial community succession and nitrogen removal performance in woodchip bioreactors

Woodchip bioreactors are an eco-friendly technology for removing nitrogen (N) pollution. However, there needs to be more clarity regarding the dissolved organic matter (DOM) characteristics and bacterial community succession mechanisms and their association with the N removal performance of bioreactors. The laboratory woodchip bioreactors were continuously operated for 360 days under three influent N level treatments, and the results showed that the average removal rate of TN was 45.80 g N/(m3·day) when the influent N level was 100 mg N/L, which was better than 10 mg N/L and 50 mg N/L. Dynamic succession of bacterial communities in response to influent N levels and DOM characteristics was an important driver of TN removal rates. Medium to high N levels enriched a copiotroph bacterial module (Module 1) detected by network analysis, including Phenylobacterium, Xanthobacteraceae, Burkholderiaceae, Pseudomonas, and Magnetospirillaceae, carrying N-cycle related genes for denitrification and ammonia assimilation by the rapid consumption of DOM. Such a process can increase carbon limitation to stimulate local organic carbon decomposition to enrich oligotrophs with fewer N-cycle potentials (Module 2). Together, this study reveals that the compositional change of DOM and bacterial community succession are closely related to N removal performance, providing an ecological basis for developing techniques for N-rich effluent treatment.

Impact of agitation and non-agitation on microbiota and reactor performance in anaerobic digestion

Optimizing process conditions in anaerobic digestion could enhance the utilization of organic matter for renewable energy generation. Thus, initial upset substrates with elevated volatile fatty acids were investigated under agitation and non-agitation conditions for optimal bioreactor performance. There were two continuous agitation scenarios for the liquid-state (40 and 100 rpm) with a non-agitated scenario. Similarly, a non-agitated and 40 rpm scenario for the solid-state. The result indicated that the non-agitated liquid-state reactor had the highest methane yield (193 L/kgVS) and lowest retention (51 days) despite delayed microbial adaptation. Of the prominent microbes, the relative abundance of Firmicutes and Archaea_unclassified negatively correlated with VFA at 100 rpm. Contrarily at 40 rpm, Firmicutes correlated positively with VFA, an indication that Firmicutes could withstand acid production at agitation speed ≤40 rpm suggesting that agitation associated with VFA might reduce microbial diversity in an initial upset liquid-state bioreactor. Thus, upset influent could be utilized for energy generation with a non-agitated liquid-state bioreactor.

Unsteady state municipal wastewater treatment: MBR response to organic and hydraulic shock-loads

ABSTRACT Ensuring the effluent quality despite fluctuations in influent flow rate and organic load to municipal wastewater treatment plants is critical. This study investigated the performance of a membrane bioreactor (MBR) in handling hydraulic and organic loading shocks during wastewater treatment. The ability of the MBR to remove COD, BOD5, and turbidity from synthetic wastewater was evaluated at steady-state and after applying five levels of organic shock loads (1.5×-4×) and four levels of hydraulic shocks (1.2×-4.7×). Due to the short-term shock-loadings no discernible effect was observed on the mixed liquor suspended solids implying biological stability. The average COD removal in MBR exposed to organic and hydraulic shock loads was 93.8% and 94%, respectively, similar to average BOD5 removals of 94.4% and 93.6%. The membrane retained suspended particles well throughout the process. However, it became quickly fouled only by applying a strong hydraulic shock. The MBR guaranteed the quality of the effluent.

Replacing rice bran with low-molecular-weight substrates affected the performance and metabolic feature of sulfate-reducing bioreactors treating acid mine drainage

The sulfate-reducing bioreactor presents an auspicious approach for eradicating toxic metals from acid mine drainage (AMD). In earlier studies, the incorporation of rice bran as a substrate in bioreactors was observed to foster the proliferation of a diverse range of microorganisms, consequently leading to the undesirable development of biofilms atop the bioreactor. To selectively stimulate sulfate-reducing bacteria (SRB) within the bioreactor, we examined the consequences of introducing three low-molecular-weight substrates in lieu of rice bran under 12 varying conditions. The sulfate reduction performance was marginally impacted by the incorporation of lactate with corn steep liquor (CSL) compared to ethanol or glucose with CSL, and the microbial composition in the presence of lactate differed significantly from those containing ethanol or glucose. The integration of ethanol and glucose with/without CSL efficiently lowered oxidation-reduction potential and promoted the proliferation of Desulfosporosinus-related SRB (relative abundance: ∼26.6%). Given that the employment of glucose initially activated fermentative bacteria such as Clostridium spp., ethanol emerged as an efficacious alternative substrate to rice bran, which directly fosters the accumulation of Desulfosporosinus sp. without encouraging the growth of other microorganisms that contribute to the unwanted formation of biofilms within the sulfate-reducing bioreactors employed in AMD treatment.

Harnessing Fermentation May Enhance the Performance of Biological Sulfate-Reducing Bioreactors.

Biological sulfate reduction (BSR) represents a promising strategy for bioremediation of sulfate-rich waste streams, yet the impact of metabolic interactions on performance is largely unexplored. Here, genome-resolved metagenomics was used to characterize 17 microbial communities in reactors treating synthetic sulfate-contaminated solutions. Reactors were supplemented with lactate or acetate and a small amount of fermentable substrate. Of the 163 genomes representing all the abundant bacteria, 130 encode 321 NiFe and FeFe hydrogenases and all genomes of the 22 sulfate-reducing microorganisms (SRM) encode genes for H2 uptake. We observed lactate oxidation solely in the first packed bed reactor zone, with propionate and acetate oxidation in the middle and predominantly acetate oxidation in the effluent zone. The energetics of these reactions are very different, yet sulfate reduction kinetics were unaffected by the type of electron donor available. We hypothesize that the comparable rates, despite the typically slow growth of SRM on acetate, are a result of the consumption of H2 generated by fermentation. This is supported by the sustained performance of a predominantly acetate-supplemented stirred tank reactor dominated by diverse fermentative bacteria encoding FeFe hydrogenase genes and SRM capable of acetate and hydrogen consumption and CO2 assimilation. Thus, addition of fermentable substrates to stimulate syntrophic relationships may improve the performance of BSR reactors supplemented with inexpensive acetate.

Development of bioreactor scale-down model using orthogonal projections to latent structures method and CO2 supplementation.

Scale-down model qualification is an important step for developing a large-scale cell culture process to enhance process understanding and support process characterization studies. Traditionally, only harvest data are used to show consistency between small-scale and large-scale bioreactor performance, allowing attributes that are dynamic over the cell culture period to be overlooked. A novel statistical method, orthogonal projections to latent structures (OPLS) analysis, can be utilized to compare time-course cell culture data across scales. Here we describe an example where OPLS is used to identify gaps between small-scale and large-scale bioreactor performances. In this case, differences in the partial pressure of carbon dioxide (pCO2) and lactate profiles were observed between small- and large-scale bioreactors, which were linked to differences in the product-quality attributes fragments and galactosylation. An improved small-scale model was developed, leading to improved consistency in the process performance and product qualities across scales and qualification of the scale-down model for regulatory submissions. This new statistical approach can provide valuable insights into process understanding and process scale-up.

Predicting Anaerobic Membrane Bioreactor Performance Using Flow-Cytometry-Derived High and Low Nucleic Acid Content Cells.

Having a tool to monitor the microbial abundances rapidly and to utilize the data to predict the reactor performance would facilitate the operation of an anaerobic membrane bioreactor (AnMBR). This study aims to achieve the aforementioned scenario by developing a linear regression model that incorporates a time-lagging mode. The model uses low nucleic acid (LNA) cell numbers and the ratio of high nucleic acid (HNA) to LNA cells as an input data set. First, the model was trained using data sets obtained from a 35 L pilot-scale AnMBR. The model was able to predict the chemical oxygen demand (COD) removal efficiency and methane production 3.5 days in advance. Subsequent validation of the model using flow cytometry (FCM)-derived data (at time t - 3.5 days) obtained from another biologically independent reactor did not exhibit any substantial difference between predicted and actual measurements of reactor performance at time t. Further cell sorting, 16S rRNA gene sequencing, and correlation analysis partly attributed this accurate prediction to HNA genera (e.g., Anaerovibrio and unclassified Bacteroidales) and LNA genera (e.g., Achromobacter, Ochrobactrum, and unclassified Anaerolineae). In summary, our findings suggest that HNA and LNA cell routine enumeration, along with the trained model, can derive a fast approach to predict the AnMBR performance.

Studying the Effect of HRT, SRT, and MLSS on Membrane Bioreactor Performance for Wastewater Treatment

Studying the Effect of HRT, SRT, and MLSS on Membrane Bioreactor Performance for Wastewater Treatment

Harnessing diurnal dynamics: Deciphering the interplay of light cycles on algal-bacterial membrane bioreactors

Light profoundly modulates the algal-bacterial membrane bioreactor (algal-bacterial MBR) performance. Yet, its outdoor deployment grapples with the inherent diurnal cycle of sunlight, engendering suboptimal light conditions. The adaptability of such systems to these fluctuating light conditions and their implications for practical outdoor applications remained an under-explored frontier. In response, this study meticulously scrutinized two laboratory-scale algal-bacterial MBRs under varying light regimes: a 24-h continuous and a 12-h cyclic illumination. Over 70 days, continuous illumination was observed to yield superior biomass production and total nitrogen and total phosphorus removal efficiencies compared to its cyclic counterpart. Contrarily, when focusing on membrane fouling, the 12-h cyclic illumination exhibited lower membrane fouling. The spectral analyses coupled with adhesion ability evaluation, traced the enhanced membrane fouling under continuous illumination to the elevated organics and heightened adhesive properties of the flocs. Given the tangible benefits of reduced membrane fouling and the potential harnessing of solar radiation, the 12-h cyclic illumination emerges as an economically astute operational paradigm for algal-bacterial MBRs. The significance of this study is to promote the application of algal-bacterial MBR in sewage treatment and provide robust support for the development of green technology in the future.

Modeling large-scale bioreactors with diffusion equations. Part II: Characterizing substrate, oxygen, temperature, carbon dioxide, and pH profiles.

Large-scale fermentation processes involve complex dynamic interactions between mixing, reaction, mass transfer, and the suspended biomass. Empirical correlations or case-specific computational simulations are usually used to predict and estimate the performance of large-scale bioreactors based on data acquired at bench scale. In this two-part-study, one-dimensional axial diffusion equations were studied as a general and predictive model of large-scale bioreactors. This second part focused on typical fed-batch operations where substrate gradients are known to occur, and characterized the profiles of substrate, pH, oxygen, carbon dioxide, and temperature. The physically grounded steady-state axial diffusion equations with first- and zeroth-order kinetics yielded analytical solutions to the relevant variables. The results were compared with large-scale Escherichia coli and Saccharomyces cerevisiae experiments and simulations from the literature, and good agreement was found in substrate profiles. The analytical profiles obtained for dissolved oxygen, temperature, pH, and were also consistent with the available data. Distribution functions for the substrate were defined, and efficiency factors for biomass growth and oxygen uptake rate were derived. In conclusion, this study demonstrated that axial diffusion equations can be used to model the effects of mixing and reaction on the relevant variables of typical large-scale fed-batch fermentations.

Syntrophy between bacteria and archaea enhances methane production in an EGSB bioreactor fed by cheese whey wastewater

The cheese-making process generates large amounts of cheese whey wastewater (CWW), which is abundant in nutrients but difficult to dispose of, contributing to the eutrophication of natural environments due to inadequate waste management. Here we show the anaerobic digestion of CCW by syntrophy between bacteria and archaea in an expanded granular sludge bed (EGSB) bioreactor as a low-cost alternative for bioremediation and biofuel production. The performance of the EGSB bioreactor and the composition of the natural microbial community were evaluated. During the operation of the EGSB bioreactor, physicochemical parameters such as alkalinity ratio (0.25), pH (7.5), and temperature (26°C) were attained and maintained, as well as light- and oxygen-free conditions, which favored the metabolism of oxygen-sensitive bacteria and methane-producing archaea (methanogens). Under these conditions, the chemical oxygen demand (COD) removal rate was highly efficient (> 89%). Methane (CH4) was produced from organic matter degradation by a few methanogens, mainly from Methanosaeta spp., and was enhanced by the metabolic interaction between bacteria and archaea. The biochemical methane potential (BMP) was >335 mL CH4/gCOD, indicating that the syntrophic microbial community is very efficient in removing organic matter and CH4 produced from CWW. Our results suggest that CWW could be treated in EGSB bioreactors and used as a sustainable alternative to CH4 production and also provide insights for the design of synthetic microbial communities (SynComs) for bioremediation, biogas production, and other biotechnological processes.

Evaluating the performance and membrane fouling of a submerged membrane bioreactor (MBR) treating plywood industry wastewater.

This study evaluates the performance of a membrane bioreactor (MBR) for treating wastewater from the laminated plywood industry. To this end, a pilot-scale MBR was operated for 60 days with a hydraulic retention time of 20 h and a solid retention time of 20 days. The reactor's performance was assessed based on the removal of chemical oxygen demand (COD), phenol, turbidity, and apparent color. Furthermore, we monitored the solids content, dissolved oxygen concentration, and pH of the mixed liquor, as well as the progression of the transmembrane pressure (TMP). The wastewater exhibited a COD/biochemical oxygen demand (BOD) ratio of 5.5, suggesting low biodegradability, usually when this ratio is higher than 4.0. Nevertheless, it was observed that the MBR's performance was stable and satisfactory, with average removal efficiencies of 98% for COD, 70% for phenol, 99% for turbidity, and 93% for true color. The evolution of TMP indicated gradual membrane fouling; however, the operational limit of 0.6 bar was not reached during the study period. In conclusion, the utilization of MBR presents a promising approach to mitigate the environmental impacts associated with wastewater from the laminated plywood industry.

Improved performance of single-stage partial nitritation-anammox membrane bioreactor (PN/A MBR) by adding biofilm carriers: start-up, membrane operation and microbial structure

Serious sludge washout is a bottleneck in the partial nitritation-anammox (PN/A) process that needs to be addressed urgently. A membrane bioreactor (MBR) is suitable for the PN/A process because of its 100 % biomass retention ability. Therefore, this study establishes a single-stage PN/A MBR and a PN/A moving bed membrane reactor (PN/A MB-MBR) for investigating the operation performance, membrane fouling behavior, and effect of biofilm carrier addition. The results demonstrate that the PN/A MB-MBR achieves a significantly shorter start-up time and substantially higher maximum nitrogen removal rate (NRR) of 17 days and 1.02 g N/L/d, respectively, compared to that of PN/A MBR (61 days and 0.19 g N/L/d, respectively). Further, the PN/A MB-MBR exhibits a lower membrane fouling of 0.023 kPa/L compared to that of 0.050 kPa/L in PN/A MBR because of the integration of the biofilm carrier. The biofilm in PN/A MB-MBR shows a higher relative abundance of Candidatus Kuenenia (18.90–25.10 %), which is a major AnAOB, compared to that observed in the suspended sludge of PN/A MB-MBR (10.30–14.32 %). Furthermore, the relative abundance of Candidatus Kuenenia absorbed on the membrane surface of PN/A MB-MBR (8.6 %) was lower than that on PN/A MBR (15.5 %). This study presented a promising method for enhancing the performance of PN/A MBR, which can serve as a valuable reference for its extensive implementation.

Evaluation of the Effect of Microalgae Chlorella vulgaris on the Performance of Membrane Bioreactor for Industrial Wastewater Treatment

Water pollution, especially from industry, is a problem that continues to emerge and even get worse. Rising sludge events often occur in industrial wastewater treatment processes. Process engineering with the addition of microalgae is proposed in this study to overcome the occurrence of rising sludge and improve wastewater quality based on dissolved oxygen levels. The control variables used were MLSS 1000 mg/L, microalgae Chlorella vulgaris 106 L, and urea and TSP 1,75 kg each. The dependent variable is rising sludge and DO levels while the independent variable is time. This research went through several stages, namely seeding microalgae, seeding microalgae into activated sludge in MBBR, experimenting with combining microalgae and activated sludge in MBBR, and installing ultrafiltration membranes with a WWTP capacity of 150 m3/day. The addition of microalgae in the MBBR process has a positive impact in improving the quality of industrial wastewater, which is characterized by the absence of rising sludge and DO levels exceeding the minimum threshold.

Effects of MgCl2 on sludge anaerobic digestion: Hydrolysis, acidogenesis, methanogenesis, and microbial characteristics

Back diffused draw salt affects the anaerobic digestion performance in a forward osmosis membrane bioreactor. Previous studies have established the influence of MgCl2 on sludge anaerobic digestion; however, its effects on individual phases, such as hydrolysis, acidogenesis, and methanogenesis remain unclear. This study investigates the influence of different concentrations of MgCl2 on these stages of anaerobic digestion and compares variations in methane production under different substrates. The findings demonstrate that adding MgCl2 significantly enhances sludge hydrolysis. At a concentration of 50 g/L, the soluble chemical oxygen demand increases by 265.2 %, although with unstable reactions. MgCl2 significantly affects the production of fatty acids. Specifically, the production of acids was enhanced when the concentration of MgCl2 was below 20 g/L, while it was inhibited when the concentration exceeded 50 g/L. The highest concentration of acetic acid recorded is 1991.28 mg/L, which represents an 83.5 % increment. In contrast, methane production shows a higher susceptibility to MgCl2, experiencing inhibition at concentrations exceeding 1 g/L. The inhibitory concentration for methane production is lower than for acid production. The study also observes significant differences in microbial structure, particularly in Methanogen. When using different substrates, the predominant orders of methanogen are Methanobacteriales for tests with acid solution as a feed and Methanosarcinales for tests with waste sludge as a feed, respectively. Analyses of metabolic pathways reveal that carbon dioxide and acetic acid are the most prevalent pathways in terms of abundance. In summary, MgCl2 has varying effects on various stages of anaerobic digestion of sludge.