Microbial Sludge Research Articles

The Relationship between Ribosomal RNA Operon Copy Number and Ecological Characteristics of Activated Sludge Microbial Communities across China

The Relationship between Ribosomal RNA Operon Copy Number and Ecological Characteristics of Activated Sludge Microbial Communities across China

Simultaneous organic wastewater treatment and bioelectricity production in a dual chamber microbial fuel cell with Scenedesmus obliquus biocathode

Algal biocathode MFCs (ABMs) are recently being heavily studied due to their advantage over abiotic cathode MFCs (ACMs) as dissolved oxygen (DO) generated by algal growth in the cathodic chamber is directly utilized in cathodic oxidant reduction. In this work growth of pure algae species Scenedesmus obliquus used in the cathode chamber has been studied under a photoperiod of 24 h illumination to supplement oxygen for continual MFC operation without any external supply of air or carbon dioxide and treatment of synthetic wastewater having chemical oxygen demand (COD) equivalent to medium strength domestic wastewater (5.35 g/L COD). Results of ABM showed the highest continuous current density generation of 23.93 mA/m2 and the highest continuous volumetric power density generation of 13.24 mW/m3 were around 120 h with COD removal efficiency of 92.52 % and coulombic efficiency (CE) of 41.88 %. Initial ohmic (Roh) and charge transfer (Rct) resistance values of ABM were found to be 8.3 Ω and Rct of 22.5 Ω which after 216 h of algal growth got reduced to 1 Ω and 15.1 Ω respectively. 16S microbiome profiling of the mixed microbial sludge inoculum used in the anodic chamber showed dominance of Xanthomonadaceae stenotrophomonas (9.7 %), Chitinophaga flexibacter (8.65 %), Sphingomonadaceae sphingomonas (6.82 %), Sphingobacterium multivorum (6.55 %), Dyadobacter fermentans (6.05 %), and Alcaligenaceae achromobacter (5.51 %). Elemental investigation of the bioanode’s and biocathode’s surface carried out using EDS analysis also shows that oxygen was generated in the cathodic chamber by pure Scenedesmus obliquus species.

Impact of Doxycycline Addition on Activated Sludge Microflora and Microbial Communities

Municipal wastewater treatment plants (WWTPs) are exposed to high concentrations of micropollutants that can impact conventional activated sludge treatment. The consequences of this include failure to meet discharge standards and the disintegration of flocs, leading to poor sludge settleability. This lab-scale study focuses on the influence of doxycycline, an antibiotic widely used against human and animal diseases, on protozoa, metazoa, and bacterial communities under sludge growing conditions. Doxycycline was added to the mixed liquor of a communal WWTP up to 0, 100, 200, and 400 mg of doxycycline L−1 and incubated in batch conditions for 23 days. The regular addition of nutrient and carbon sources was preformed every 2 days to prevent sludge starvation. Sludge growth, conductivity, and settleability were measured and compared to sludge microbial community structure, determined by microscopic observations and high-throughput 16S rDNA sequencing. The high doxycycline concentration negatively impacted settleability and correlated with a decrease in bacterial diversity and floc disintegration. The addition of doxycycline promoted the enrichment of Proteobacteria Brevundimonas sp., Luteibacter anthropi, and the Bacteroidetes Chryseobacterium massoliae. These species are known to be resistant to a wide spectrum of antibiotics, including tetracyclines. A study of a larger scale may be conducted based on this study’ results.

Identification of Activated Sludge Microbial Based on Improving YOLOv8

Identification of Activated Sludge Microbial Based on Improving YOLOv8

Effect of antibiotics addition on nutrient removal stability and microbial community change of the solar-powered oxidation ditch-membrane bioreactor in treating building wastewater

The solar-powered oxidation ditch-membrane bioreactors (SOD-MBR) system was developed and operated with long solid retention times (SRTs) of 80 and 160 days. The aim was to investigate the effects of using a long SRT and antibiotics in building wastewater on the stability of nutrient removal, as well as membrane fouling. An increase in the SRT from 80 days to 160 days did not significantly affect the performance of the SOD-MBR system. Ciprofloxacin and Sulfamethoxazole removal efficiencies were 94.47 ± 1.54% and 87.54 ± 24.7%. However, the presence of antibiotics resulted in lower removal efficiencies for NH4+-nitrogen and phosphorus and stimulated the production of extracellular polymeric substances (EPS), particularly proteins in L-EPS and T-EPS of the foulant. FTIR and FEEM analysis revealed that the microbial sludge primarily consisted of proteins, carbohydrates, and lipids. Furthermore, the relative abundance analysis of microbial communities identified bacteria associated with nitrogen removal in the SOD-MBR system, including Anammox, AOB (ammonia oxidizing bacteria), DNB (denitrifying bacteria), and NOB (nitrite oxidizing bacteria), with a total of 25 genera. The majority of these bacteria were stimulated by the presence of antibiotics, resulting in higher relative abundance. Finally, the SOD-MBR system achieved energy savings of 97.38% by utilizing photovoltaic (PV) technology.

Suppression of Hydrogen Sulfide Generation via the Coexistence of Anaerobic Sludge and Goethite-Rich Limonite/Polyethersulfone Composite Fibers.

Limonite-polyethersulfone (PES) composite fibers were prepared by the wet spinning method to suppress hydrogen sulfide (H2S) generation from anaerobic microbial sludge. The H2S adsorption of the prepared limonite composite fibers followed the Langmuir type, with a maximum adsorption capacity of 3.7-4.4 g H2S/g, indicating mesopore adsorption. The in vitro H2S fermentation environment with anaerobic microbial sludge with the coexistence of limonite composite fibers exhibited suppression of H2S generation. The coexistence of limonite composite fibers also suppressed the amount of CO generation produced by microbial fermentation, so the fibers also affected the metabolism of anaerobic microorganisms. During the anaerobic digestion process, particularly at 672-840 h (28-35 days), the mesopores of limonite in the composite fibers disappeared and changed to macropore adsorption, and the reaction of limonite with hydrogen sulfide produced pyrite (FeS2) and iron sulfate (Fe2(SO4)3) as products, which remained in the fiber with conversion efficiencies of 6.8 and 32.4%, respectively. The in vitro hydrogen disulfide action of limonite composite fibers was found to be able to suppress the generated environment of about 300 ppm to about 0.4 ppm.

Anaerobic co-digestion of elephant dung and biological pretreated Napier grass: Synergistic effect and kinetics of methane production

Elephant dung (ED) and Napier grass (NP) are used as waste-to-energy resources that help in waste management and reduce greenhouse gas emissions, in accordance with Sustainable Development Goal 13 (SDG 13). In this study, anaerobic co-digestion of ED and NP (with and without biological pretreatment) was conducted under various mixing ratios. The biological pretreatment of NP was performed using the microbial activator super LDD1 (mixed culture microbial sludge) enzyme before further biogas production. The results confirmed that LDD1 enzymatic microbes increased methane production capacity by 1.95 times compared to untreated NP. Moreover, the data revealed that a NP/ED ratio of 1:1 with a 14-day pretreatment duration yielded the highest cumulative methane production of 234.8 ± 5.9 mL-CH4/g-VS, a 99.2% increase. The synergistic index results showed that substrate pretreatment had the most significant effect on methane yield, with the maximum synergistic index equaling 1.53. The Gompertz model outperformed the Logistic model in determining the reaction's kinetic parameters, with R2 > 0.995.

Reason for the increased electroactivity of extracellular polymeric substances with electrical stimulation: Structural change of α-helix peptide of protein

Electroactivity is an important parameter to assess the ability of the extracellular polymeric substance (EPS) of microorganisms to participate in extracellular respiration. Many reports have found that the electroactivity of microbial sludge could be enhanced with electrical stimulation, but the reason remains unclear. The results of this study showed that the current generation of the three microbial electrolysis cells increased by 1.27–1.76 times during 49 days of electrical stimulation, but the typical electroactive microorganisms were not enriched. Meanwhile, the capacitance and conductivity of EPS of sludge after the electrical stimulation increased by 1.32–1.83 times and 1.27–1.32 times, respectively. In-situ FTIR analysis indicated that the electrical stimulation could lead to the polarization of amide groups in the protein, likely affecting the protein structure related to the electroactivity. Accordingly, the dipole moment of the α-helix peptide of protein of sludge increased from 220 D to 280 D after the electrical stimulation, which was conducive to electron transfer in the α-helix peptide. Moreover, the vertical ionization potential and ELUMO-EHOMO energy gap of the C-terminal in the α-helix peptide decreased from 4.43 eV to 4.10 eV and 0.41 eV to 0.24 eV, respectively, which indicated that the α-helix was easier to serve as the electron transfer site of electron hopping. These results meant that the enhancement of the dipole moment of the α-helix peptide unchoked the electron transfer chain of the protein, which was the main reason for the increased electroactivity of EPS protein.

Diversity of Activated Sludge Microbial Community Structure in Different Wastewater Treatment Plants

Diversity of Activated Sludge Microbial Community Structure in Different Wastewater Treatment Plants

Properties of alginate-like exopolymers recovered from flocculent and granular microbial sludges of different biological treatment systems treating real municipal wastewater

Treatment of excess microbial sludge and its safe disposal is a major challenge in wastewater treatment plants (WWTPs) due to pathogens, economic and regulatory issues. There is a considerable interest in developing newer technologies for the excess sludge treatment and recovering value-added products. This study investigated recovery of biopolymers from the microbial sludge generated in three different biological treatment systems such as conventional activated sludge process (ASP), activated sludge sequencing batch reactor (AS-SBR) and aerobic granular sludge SBR (AGS-SBR). Alginate-like exopolymer (ALE) was the dominant biopolymer in AS and AGS irrespective of the employed treatment system. ALE content was determined to be 32%, 39% and 44% in the microbial sludge of ASP, AS-SBR and AGS-SBR, respectively. ALE extraction markedly decreased the biosolids content by up to 54%. ALE recovered from AS harvested from ASP had poor gelation properties and did not form beads with calcium. Whereas, the ALE recovered from AS and AGS from the SBRs exhibited gelation properties and formed nice beads with calcium ions. The biopolymer recovered from the microbial sludge developed in the SBRs contained high guluronic (G) acid in the form of poly-guluronic acid (GG) blocks, responsible for excellent gelation property. Extraction of biopolymers mitigates sludge management challenges, pathogens-spread and improves resource recovery in WWTPs.

Removals of endocrine disrupting compounds during landfill leachate treatment in two-stage aerobic sequential batch reactor: Effect of Alcaligenes faecalis no.4 bio-augmentation

The effect of Alcaligenes faecalis no.4 (A. faecalis no.4) in two-stage sequencing batch reactor (SBR) treatment of endocrine disrupting compounds (EDCs), i.e., bisphenol A (BPA), di-n-butyl phthalate (DBP) and bis-2-ethyl hexyl phthalate (DEHP) containing in landfill leachate was investigated. Two aerobic SBRs were operated without (C-SBR) and with A. faecalis no.4 bio-augmentation (B-SBR) at a hydraulic retention time (HRT) and solid retention time (SRT) of 3 and 30 days over 280 days. The removals of BPA, DBP, and DEHP were significantly improved to 91.1%, 84.5%, and 80.9% from their average influent concentrations of 185, 244, and 532 μg/l under the presence of A. faecalis no.4 through their enhanced biodegradation. The A. faecalis no.4 bio-augmentation helped promote a strong consortium of bacterial genera reported as BPA, DBP, and DEHP degraders in microbial sludge. This study demonstrated the technical feasibility of applying SBR with A. faecalis no.4 in landfill leachate treatment with effective EDC removals.

Evaluating the Optimal Oil Concentrations in the Startup Performance of a Membrane Bioreactor Treating Oily Noodle-soup Wastewater

The discharge of high-strength oily wastewater adversely affects the environment; therefore, the treatment of wastewater containing fats, oils, and grease from the food industry is of importance. In this study, we used a membrane bioreactor (MBR) to treat Ramen noodle-soup wastewater, and we evaluated the optimal oil concentration in the wastewater for the startup of the MBR treatment in winter and summer. The MBR system had a sufficient startup in both seasons when fed with a 20-fold dilution of the original oily wastewater, containing approximately 950 to 1,200 mg/L oil and approximately 3,000 to 4,400 mg/L biological oxygen demand (BOD; BOD-SS load of 0.1 to 0.2 kg/kg/d). The reactor performance in winter were relatively stable during the operation. While, activated sludge microbes in summer were not highly active with a 40-fold dilution of wastewater, because of the decreased mixed liquor suspended solid concentration during the operation period. Population shifts in the sludge microbiome with increasing oil concentrations were analyzed using high-throughput sequencing, and the relative abundance of operational taxonomic units belonging to the phylum Bacteroidetes were highest in both winter and summer when fed with 20-fold dilution of the wastewater. In particular, the family Chitinophagaceae was dominant, with relative abundances of 13.5% in winter and 5.1% in summer, suggesting that this family may play important roles in the startup of a MBR treating the wastewater.

Reduction of greenhouse gas (GHG) emission in the vegetation-activated sludge process (V-ASP) involving decontaminated plants for decentralized wastewater treatment

Wastewater treatment plant (WWTP) is one of the important sources for anthropogenic greenhouse gases (GHGs) emission. The objective of this study was to evaluate vegetation-activated sludge process (V-ASP) that simultaneously achieved the improvement of decentralized wastewater treatment performance and reduction of GHGs emission, in contrast to conventional ASP. With the same organic and nitrogen loading rates and operational modes, V-ASP achieved a decreasing of 19.79% for total GHGs emission in comparison to ASP. From the microbial community analysis on activated sludge microbes and plant roots, V-ASP had a much lower abundance of Chloroflexi, and a comparable abundance of denitrifiers, in contrast to conventional ASP, which likely correlated with the differences of CH4 production and emission. Specifically, it was noted that the relatively higher abundance of mcrA and nirS genes in the V-ASP would take a certain responsible for reducing CH4 and N2O emission, respectively. The V-ASP also demonstrated the increased enzymatic activities, such as ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO), which could help to reduce GHGs emission flux. It was proved that V-ASP process, compared to the traditional ASP process, was an effective decentralized wastewater treatment technique for reducing GHGs emission.

Recovery of High Quality Metagenome-Assembled Genomes From Full-Scale Activated Sludge Microbial Communities in a Tropical Climate Using Longitudinal Metagenome Sampling.

The analysis of metagenome data based on the recovery of draft genomes (so called metagenome-assembled genomes, or MAG) has assumed an increasingly central role in microbiome research in recent years. Microbial communities underpinning the operation of wastewater treatment plants are particularly challenging targets for MAG analysis due to their high ecological complexity, and remain important, albeit understudied, microbial communities that play ssa key role in mediating interactions between human and natural ecosystems. Here we consider strategies for recovery of MAG sequence from time series metagenome surveys of full-scale activated sludge microbial communities. We generate MAG catalogs from this set of data using several different strategies, including the use of multiple individual sample assemblies, two variations on multi-sample co-assembly and a recently published MAG recovery workflow using deep learning. We obtain a total of just under 9,100 draft genomes, which collapse to around 3,100 non-redundant genomic clusters. We examine the strengths and weaknesses of these approaches in relation to MAG yield and quality, showing that co-assembly may offer advantages over single-sample assembly in the case of metagenome data obtained from closely sampled longitudinal study designs. Around 1,000 MAGs were candidates for being considered high quality, based on single-copy marker gene occurrence statistics, however only 58 MAG formally meet the MIMAG criteria for being high quality draft genomes. These findings carry broader broader implications for performing genome-resolved metagenomics on highly complex communities, the design and implementation of genome recoverability strategies, MAG decontamination and the search for better binning methodology.

Effects of perfluorooctanoic acid (PFOA) on activated sludge microbial community under aerobic and anaerobic conditions.

Per- and polyfluoroalkyl substances (PFASs) have received increasing attention due to their widespread presence in diverse environments including wastewater treatment plants (WWTPs) and their potential adverse health effects. Perfluorooctanoic acid (PFOA) is one of the most detected forms of PFASs in WWTPs. However, there is still a paucity of knowledge about the effect of PFASs on microorganisms of the key component of WWTP, activated sludge. In this study, lab-scale microcosm experiments were established to evaluate the influences of PFOA on activated sludge microbes under aerobic and anaerobic conditions. The diversity, structure, and microbe-microbe interaction of microbial community were determined by 16S rRNA gene amplicon sequencing and co-occurrence network analysis. After 90days of exposure to PFOA, activated sludge microbial richness decreased under both aerobic and anaerobic conditions. Specifically, under aerobic condition, Rhodopseudomonas (mean relative abundance 3.6%), Flavobacterium (2.4%), and Ignavibacterium (6.6%) were enriched in PFOA-spiked activated sludge compared with that in the unspiked sludge (2.6%, 0.1%, and 1.9%, respectively). By contrast, after 90days of exposure to PFOA, Eubacterium (2.1%), Hyphomicrobium (1.8%), and Methyloversatilis (1.2%) were enriched under anaerobic condition, and more abundant than that in the control sludge (0.4%, 1.5%, and 0.6%, respectively). These genera were the potential PFOA-resistant members. In addition, Azospirillum and Sporomusa were the most connected taxa in PFOA-aerobic and PFOA-anaerobic networks, respectively. Prediction of the functional gene showed that PFOA inhibited some gene expression of sludge microbes, such as transcription, amino acid transport and metabolism, and energy production and conversion. In summary, continued exposure to PFOA induced substantial shifts of the sludge bacterial diversity and composition under both aerobic and anaerobic conditions.

TILAFeed: A bio-based inventory for circular nutrients management and achieving bioeconomy in future aquaponics

Future food systems aim to achieve improved resource use efficiency and minimized environmental footprint through a circular bioeconomy-based approach. Aquaponics is a hallmark of such circular food production. The image of a circular nutrient utilization efficiency in aquaponics is often weakened by the daily use of additional inorganic fertilizers in such systems. As circular bioeconomy greatly emphasizes developing bio-based solutions, the presented novel inventory ‘TilaFeed’ and its associated utility tools is a step towards achieving more circular nutrient utilization and bioeconomy in future aquaponics. Through the formulation of tailored fish feed that is compatible with aquaponic systems’ needs (e.g. plant nutrient requirement, mineralization efficiency of microbial sludge digesters), the objectives of TilaFeed are (i) to solve nutrient constraints in aquaponic systems, both for fish and plants; (ii) to avoid or strongly limit artificial fertilizer use in aquaponics by smartly tailored aquafeeds; and (iii) to equip system managers with decision-making tools for improved nutrient planning of their aquaponic systems. TilaFeed is a bio-based inventory. It integrates material (nutrient) flow information from feed to fish (in-vivo nutrient partitioning, forms of excretion) to environment (in-situ nutrient loading, nutrient forms) and primary producers (mineralization by microbes, available nutrients to plants). Based on TilaFeed-Model, feed for future aquaponics may be more precisely formulated with the principle that nutrients are not only a resource for fish, but excreted nutrients from fish (feed) also fertilize the microbes and plants.

The Fe3O4 nanoparticles-modified mycelium pellet-based anaerobic granular sludge enhanced anaerobic digestion of food waste with high salinity and organic load

The accumulation of volatile fatty acids and high osmotic pressure of food waste usually result in poor operation performance and even failure of anaerobic digestion (AD). A novel Fe3O4 nanoparticles-modified Aspergillus tubingensis (AT) mycelium pellets-based anaerobic granular sludge (NPs@AT-AGS) was therefore developed to enhance energy recovery efficiency of food waste AD treatment. The AT mycelium particles, with three-dimensional spatial structure formed by large amounts of hyphae, served as support to attach activated sludge microbes. The Fe3O4 nanoparticles (NPs) stimulated extracellular polymeric substances (EPS) secretion of AT mycelium pellets to 117.55 mg/gVSS, and further increased the microbe absorption quantity to 1.23 g/gAT. The abundant EPS of the NPs@AT-AGS could protect microbes from high osmotic pressure, which resulted in a methane yield of 583.8 mL/gVS under a high salinity of 20 g/L NaCl, 1.6 times higher than that of the activated flocculent sludge (AFS). Moreover, the methane yield of actual food with high organic load also had a 24% improvement with the NPs@AT-AGS. Therefore, the NPs@AT-AGS is promising in the effective AD treatment of food waste with high salinity and organic load.

Carbon dissipation from surgical cotton production wastewater using macroalgae, microalgae, and activated sludge microbes.

Surgical cotton production has drastically been increased in the past few years due to excessive use by medical health professionals especially in countries like India, which is among the top three exporters of cotton worldwide. The effluent generated from surgical cotton industries differ from textile effluents by the conspicuous absence of dyeing chemicals. This wastewater has a high concentration of suspended particles, COD, dissolved ions, organic carbon, and alkaline pH. Several studies have been published on the treatment of textile effluents and the degradation of dyeing chemicals, while the treatment studies on surgical cotton wastewater have been rarely reported in spite of their potential to cause pollution in receiving land/water bodies. Activated sludge microbes have been extensively studied and well documented in the treatment of several industrial effluent but does not match to the production of valuable biomass from algae. The global energy demand has prompted the scientific community to investigate and explore the possibility of using algae for energy production with simultaneous wastewater treatment. To the best of the authors' knowledge, no research articles have been published which compare the effectiveness of activated sludge microorganisms, microalgae, and macroalgae in removing contaminants from real wastewater. To date, there is a knowledge gap in understanding and selecting the right choice of biological system for effective and economical effluent treatment. In an attempt to minimize this gap, carbon removal by microalgae, macroalgae, and activated sludge microbes were investigated on real effluent from surgical cotton industries. It was observed that the strain of Chlorella vulgaris could dissipate 83% of COD from real wastewater, while consortia of macroalgae (consisting predominantly of Ulvaceae and Chaetomorpha) and activated sludge microbes could remove 81% and 69% of the carbon, respectively. The microalgal growth (in terms of wet weight) increased from 0.15 to 0.3g, whereas the macroalgal wet weight increased from 1.5 to 3g in over 7days of batch experiments conducted in triplicates. This indicated the superlative performance of microalgae over activated sludge microbes in carbon dissipation.

Profile of sulfur oxidizing bacteria in full-scale Biotrickling filter to remove H2S in biogas from in cassava starch industry

The success of Next generation sequencing (NGS) technology over the past decades has led to a better understanding of complex microbial communities in such environment. Here, we employed NGS to monitor sulfur oxidizing bacteria (SOB) community from cassava starch industry (BPT), which can oxidize H2S into sulfate or elemental sulfur under aerobic conditions. Microbial sludge was collected from two sampling points comprising starting sludge (BPT1) and recirculating sludge (BPT2) of the biotrickling filter. gDNA was extracted from BPT1 and BPT2 samples and removed other contaminations by using the GenepHarM Gel/PCR Kit (Geneaid). DNA concentrations and DNA purity were analyzed by using gel electrophoresis and NanoDrop™ 1000 Spectrophotometer, respectively. Analysis of SOB species using NGS technique was based on the hypervariable regions V4 on the 16S rRNA gene. The comparative genomic hybridization (CGH) microarray probes for detection and identification of SOB species-strain that high expression of soxAXBYZ and fccAB genes in the cassava starch industry. For the recirculating sludge (BPT2), the bacterial communities were dominated by Sulfurimonas (13.7%) followed by Synechococcus (9.4%), Hydrogenophaga (6.5%), Methanosaeta (2.4%), and Acidithiobacillus (2.1%), the same dominant genera were also found in the starting sludge (BPT1), but with slightly different abundancy. The heatmap revealed that Acidithiobacillus caldus species (33.9%–49.9%) could play a vital role in eliminating H2S in biogas from the cassava starch industry. The CGH microarray indicated that Thiothrix, Paracoccus, Sulfurimonas, Hydrogenophaga, Magnetospirillum, Rhodoplanes, Syntrophomonas, Pseudomonas, Sulfuricurvum, and Arcobacter were dominant genus, that has a high expression of sulfur oxidation genes in biotrickling filter.

Microbial sludge formation in Brazilian marine diesel oil (B0) and soybean methylic biodiesel blends (B10 and B20) during simulated storage

Biofuels are renewable alternatives to fossil fuels mainly due to the lower impact they have in terms of release of air pollutants. Brazilian diesel oil must be composed of 10–15% biodiesel, while the addition of biodiesel to marine diesel oil has not yet been authorized. This paper aimed to evaluate the impact of the addition of biodiesel to marine diesel oil. The biotic and abiotic degradation of pure (B0) marine diesel oil and that with the addition of 10% (B10) and 20% (B20) of soybean methyl biodiesel were evaluated in simulated storage in an aqueous phase of Bushnell Haas mineral medium for 28 days. An uncharacterized inoculum (adapted from the ASTM E1259-16 standard) was prepared from a microbial population found in the marine diesel oil storage tank of a ship. For 28 days, microbial growth was monitored by gravimetry and the composition of the fungal microbial community was determined by next-generation sequencing (NGS). In the aqueous phase of the tests, the nature of the metabolites (pH measurements, headspace-solid phase microextraction [HS-SPME], gas chromatography with mass spectrometry (GC–MS), detection of the lipase enzyme (Rhodamine B Medium) and production of biosurfactants (surface tension measurements) were assessed. The oil phase was evaluated by gas chromatography (GC) and infrared spectroscopy. Higher biomass was observed in the B20 condition and the microbial community analysis revealed the dominance of the genus Pseudallescheria. No significant pH decreases were observed in the aqueous phase; however, the analyses by HS-SPME and GC–MS showed the presence of organic acids, alkanes, and phenols in both conditions, with and without microorganisms. The infrared results with the mixtures (10 and 20%) showed degradation of the biodiesel fraction. GC analysis showed that the linoleic acid methyl ester was the most susceptible to degradation by microorganisms. Thus, the results found showed the impact of adding biodiesel to marine diesel, calling attention to the need for maintenance routines, which will guarantee the final quality of the stored fuel.