Resilience towards organic load and activated sludge variations in co-fermentation for carboxylic acid production.

Two perturbations were investigated in acidogenic co-fermentation of waste activated sludge (WAS) and food waste in continuous mesophilic fermenters: increasing the organic loading rate (OLR) and changing the WAS. A control reactor maintained an OLR of 11 gVS/(L·d), while a test reactor had a prolonged OLR change to 18 gVS/(L·d). For each OLR, two WAS were studied. The change in OLR led to differentiated fermentation product profile without compromising the fermentation yields (∼300 mgCOD/gVS). At 11 gVS/(L·d), the product profile was dominated by acetic, butyric, and propionic acids while at 18 gVS/(L·d) it shifted to acetic acid, ethanol, and caproic acid. Reverting the OLR also reverted the fermentation profile. The biomass immigration with the WAS changed the fermentation microbial structure and introduced acetic acid-consuming methanogens, which growth was only delayed by the OLR increase. Microbial monitoring and post-fermentation tests can be used for early detection of acetic acid-consuming events.

Predatory Bdellovibrio-and-like organism mixtures on efficient MBR in-situ membrane fouling diminishment and mechanisms.

Membrane fouling is a major hindrance that restricts the application of membrane bioreactors (MBRs). Bdellovibrio-and-like organisms (BALOs), as obligatory parasitic bacteria, prey upon various bacteria. In this study, the BALO mixtures were screened and found more effective in membrane fouling mitigation compared to the single BALO species and extended the membrane filtration period by as long as 33.3%. The higher BALO diversity reduced the potential foulants generation in the activated sludge by decreasing the sludge viscosity as high as 13.8 ± 0.6% than the pure culture of BALO. Meanwhile, the mixed BALOs demonstrated superior biofilm predation capabilities, with the content of soluble microbial products and extracellular polymeric substances on the biofilm decreasing by 26.1 ± 0.5% and 38.3 ± 0.2% as the most compared to the single BALO species involved system. Additionally, the BALO mixtures expanded the single strains' host lysis spectrum of both the activated sludge and biofilm. The abundance of membrane-fouling-related bacteria such as Flavobacterium, Rhodobacter, and Labilithrix and pioneer bacteria such as Sphingorhabdus and Pseudomonas was significantly reduced. In summary, this study disclosed the significantly better membrane fouling mitigation effects of the BALOs with higher diversity, suggesting that the expansion of the host range is crucial for the further application of BALOs to enhance the anti-fouling performance of the MBR system.

Comparing the performance of fluidized and fixed granular activated carbon beds as cathodes for microbial electrosynthesis of carboxylates from CO2.

Microbial electrosynthesis (MES) can use renewable electricity to power microbial conversion of carbon dioxide (CO2) into carboxylates. To ensure high productivities in MES, good mass transfer must be ensured, which could be accomplished with fluidization of granular activated carbon (GAC). In this study, fluidized and fixed GAC bed cathodes were compared. Acetate production rate and current density were 42 % and 47 % lower, respectively, in fluidized than fixed bed reactors. Although similar microbial consortium dominated by Eubacterium and Proteiniphilum was observed, lowest biomass quantity was measured with fixed GAC bed indicating higher specific acetate production rates compared to fluidized GAC bed. Furthermore, charge efficiency was the highest and charge recovery in carboxylates the lowest in fixed GAC beds indicating enhanced hydrogen evolution and need for enhancing CO2 feeding to enable higher production rates of acetate. Overall, fixed GAC beds have higher efficiency for acetate production in MES than fluidized GAC beds.

Exploring the potential of Meyerozyma caribbica and its combined application with bacteria for lindane bioremediation.

This study explored the degradation potential of a yeast strain, Meyerozyma caribbica, alone and in combination with Bacillus velezensis and Priestia megaterium, found novel for lindane biodegradation. Isolated from hexachlorocyclohexane (HCH)-contaminated sites, M. caribbica, B. velezensis, and P. megaterium demonstrated lindane reduction efficiencies of 86.5%, 78.6%, and 77.5%, respectively, at 750 mg L⁻1 within 10-day incubation period. Kinetic analysis revealed that M. caribbica followed the first-order degradation (r2 = 0.991; T1/2 = 4.3 days). Notably, M. caribbica exhibited the highest dechlorinase activity (9.27 U mL⁻1) in the cell supernatant. Co-cultivation as the mixed culture of M. caribbica and P. megaterium achieved maximum lindane reduction (90%) and dechlorinase activity (9.93 U mL⁻1). Whereas the mixed culture of M. caribbica and B. velezensis resulted in 80.9% reduction at 500 mg L⁻1 lindane with dechlorinase activity of 6.77 U mL⁻1. Growth kinetics, modelled using the Monod equation, showed a maximum specific growth rate of 0.416 h⁻1 for the mixed culture of M. caribbica and P. megaterium at 750 mg L⁻1 lindane. GC-MS analysis confirmed the presence of intermediate metabolites, viz., γ-pentachlorocyclohexane, 1,2,4-trichlorobenzene, 1,4-dichlorobenzene and maleyl acetate, validated successive dechlorination and oxidative-reduction processes during lindane biodegradation. The findings of the study highlighted the potential of these novel microbial strains and their mixed cultures for effective bioremediation of lindane-contamination.

Differentiation and quantification of extracellular polymeric substances from microalgae and bacteria in the mixed culture.

Extracellular polymeric substances (EPS) play significant roles in the formation, function, and interactions of microalgal-bacteria consortia. Understanding the key roles of EPS depends on reliable extraction and quantification methods, but differentiating of EPS from microalgae versus bacteria is challenging. In this work, cation exchange resin (CER) and thermal treatments were applied for total EPS extraction from microalgal-bacteria mixed culture (MBMC), flow cytometry combined with SYTOX Green staining was applied to evaluate cell disruption during EPS extraction, and auto-fluorescence-based cell sorting (AFCS) was used to separate microalgae and bacteria in the MBMC. Thermal extraction achieved much higher EPS yield than CER, but higher temperature and longer time reduced cell activity and disrupted the cells. The highest EPS yield with minimal loss of cell activity and cell disruption was achieved using thermal extraction at 55℃ for 30 min, and this protocol gave good results for MBMC with different microalgae:bacteria (M:B) mass ratios. AFCS combined with thermal treatment achieved the most-efficient biomass differentiation and low EPS loss (<4.5 %) for the entire range of M:B ratios. EPS concentrations in bacteria were larger than in microalgae: 42.8 ± 0.4 mg COD/g TSS versus 9.19 ± 0.38 mg COD/g TSS. These findings document sensitive and accurate methods to extract and quantify EPS from microalgal-bacteria aggregates.

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.

Enrichment performance and salt tolerance of polyhydroxyalkanoates (PHAs) producing mixed cultures under different saline environments.

The key to the resource recycling of saline wastes in form of polyhydroxyalkanoates (PHA) is to enrich mixed cultures with salt tolerance and PHA synthesis ability. However, the comparison of saline sludge from different sources and the salt tolerance mechanisms of salt-tolerant PHA producers need to be clarified. In this study, three kinds of activated sludge from different salinity environments were selected as the inoculum to enrich salt-tolerant PHA producers under aerobic dynamic feeding (ADF) mode with butyric acid dominated mixed volatile fatty acid as the substrate. The maximum PHA content (PHAm) reached 0.62 ± 0.01, 0.62 ± 0.02, and 0.55 ± 0.03 g PHA/g VSS at salinity of 0.5%, 0.8%, and 1.8%, respectively. Microbial community analysis indicated that Thauera, Paracoccus, and Prosthecobacter were dominant salt-tolerant PHA producers at low salinity, Thauera, NS9_marine, and SM1A02 were dominant salt-tolerant PHA producers at high salinity. High salinity and ADF mode had synergistic effects on selection and enrichment of salt-tolerant PHA producers. Combined correlation network with redundancy analysis indicated that trehalose synthesis genes and betaine related genes had positive correlation with PHAm, while extracellular polymeric substances (EPS) content had negative correlation with PHAm. The compatible solutes accumulation and EPS secretion were the main salt tolerance mechanisms of the PHA producers. Therefore, adding compatible solutes is an effective strategy to improve PHA synthesis in saline environment.

Exploring the untapped practices in bacterial-fungal mixed-based cultures for acidic treatment of metal-enriched printed circuit board waste.

This study explores the extraction of metals from spent mobile phone printed circuit boards (SMPhPCBs) to address environmental and resource depletion concerns. The challenges in metal recovery from SMPhPCBs arise due to their complex composition and high metal content. While previous research has primarily focused on using bio-cyanide, bio-sulfate, and bio-ferric compounds from acidophilic bacteria, the potential of bio-oxalic acid for SMPhPCBs treatment and the alteration of their complex structure has not yet been explored. Additionally, this study suggests evaluating the untapped potential of Aspergillus niger in oxalic acid production through mixed cultures with bacteria, marking a pioneering approach. A unique culture of Bacillus megaterium and A. niger was created, inducing bio-stress by bacterial metabolites, including gluconic acid (2683 mg/l) and live/dead bacterial cells in a medium with glucose deficiency. Results demonstrated reducing sugar consumption and oxalic acid over-production in mixed cultures compared to pure cultures, ranging from 1350 to 4951 mg/l at an initial glucose concentration (IGC) of 10 g/l and 4276 to 7460 mg/l at IGC 20 g/l. This over-production is attributed to proposed fungal signaling mechanisms to bacteria. Metal extraction using organic acids and siderophores at 10 g/l pulp density, 24 h, and 60 °C yielded Mn (100 %), Pt (100 %), Pd (70.7 %), Fe (50.8 %), Co (48.3 %), Al (21.8 %), among others. The final valuable residue containing copper, gold, and silver holds potential for future recycling. The study concludes with XRD and FTIR analyses to assess the bioleaching effect on the bio-leached powder.

Biohydrogen Production from Waste Black Cumin (Nigella Sativa) Extract Liquid.

Hydrogen creates water during combustion. Therefore, it is expected to be the most promising environmentally friendly energy alternative in the coming years. This study used extract liquid obtained from the waste nigella sativa generated by the black cumin oil industry. The performance of biological hydrogen manufacturing via dark fermentation was investigated in the fluidized bed reactor (FBR) and completely stirred tank reactor (CSTR) under the operation conditions of pH 5.0, 4.0, and 6.0 and a hydraulic retention time (HRT) of 36 and 24 h. The performance of hydrogen manufacturing was determined to be good under an organic loading ratio (OLR) of 6.66 g.nigella sativa extract/L and pH 4.0. According to these conditions, the maximum amount of hydrogen in CSTR and FBR was found to be 20.8 and 7.6 mL H2/day, respectively. The operating process of the reactors displayed that a reduction in HRT augmented biohydrogen manufacturing. The work that used mixed culture found that the dominant microbial population at pH 4.0 involved Hydrogenimonas thermophila, Sulfurospirillum carboxydovorans, Sulfurospirillum cavolei, Sulfurospirillum alkalitolerans, and Thiofractor thiocaminus. No research on waste black cumin extract was found in biohydrogen studies, and it was determined that this substrate source is applicable for biological hydrogen manufacturing.

TUNEL-n-DIFL Method for Detection and Estimation of Apoptosis Specifically in Neurons and Glial Cells in Mixed Culture and Animal Models of Central Nervous System Diseases and Injuries.

Detection of merely apoptosis does not reveal the type of central nervous system (CNS) cells that are dying in the CNS diseases and injuries. In situ detection and estimation of amount of apoptosis specifically in neurons or glial cells (astrocytes, oligodendrocytes, and microglia) can unveil valuable information for designing therapeutics for protection of the CNS cells and functional recovery. A method was first developed and reported from our laboratory for in situ detection and estimation of amount of apoptosis precisely in neurons and glial cells using in vitro and in vivo models of CNS diseases and injuries. This is a combination of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and double immunofluorescent labeling (DIFL) or simply TUNEL-n-DIFL method for in situ detection and estimation of amount of apoptosis in a specific CNS cell type. An anti-digoxigenin (DIG) IgG antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) for blue fluorescence, fluorescein isothiocyanate (FITC) for green fluorescence, or Texas Red (TR) for red fluorescence can be used for in situ detection of apoptotic cell DNA, which is earlier labeled with TUNEL using alkali-stable DIG-11-dUTP. A primary anti-NeuN (neurons), anti-GFAP (astrocytes), anti-MBP (oligodendrocytes), or anti-OX-42 (microglia) IgG antibody and a secondary IgG antibody conjugated with one of the above fluorophores (other than that of ani-DIG antibody) are used for in situ detection of apoptosis in a specific CNS cell type in the mixed culture and animal models of the CNS diseases and injuries.

Temperature-driven carboxylic acid production from waste activated sludge and food waste: Co-fermentation performance and microbial dynamics.

This work aims to improve the continuous co-fermentation of waste activated sludge (WAS) and food waste (FW) by investigating the long-term impact of temperature on fermentation performance and the underpinning microbial community. Acidogenic co-fermentation of WAS and FW (70:30 % VS-basis) to produce volatile fatty acids (VFA) was studied in continuous fermenters at different temperatures (25, 35, 45, 55 °C) at an organic loading rate of 11 gVS/(L·d) and a hydraulic retention time of 3.5 days. Two batches of WAS (A and B) were collected from the same wastewater treatment plant at different periods to understand the impact of the WAS microbioota on the fermenters' microbial communities. Solubilisation yield was higher at 45 °C (575 ± 68 mgCOD/gVS) followed by 55 °C (508 ± 45 mgCOD/gVS). Fermentation yield was higher at 55 °C (425 ± 28 mgCOD/gVS) followed by 35 °C (327 ± 17 mgCOD/gVS). Temperature also had a noticeable impact on the VFA profile. At 55 °C, acetic (40 %) and butyric (40 %) acid dominated, while acetic (37 %), butyric acid (31 %), and propionic acid (17 %) dominated at 35 °C. At 45 °C, an accumulation of caproic acid was detected which did not occur at other temperatures. Each temperature had a distinct microbial community, where the WAS microbiota played an important role. The biomass mass-balance showed the highest growth of microorganisms (51 %) at 35 °C and WAS_B, where a consumption of acetic acid was observed. Therefore, at 35 °C, there is a higher risk of acetic acid consumption probably due to the proliferation of methanogens imported from WAS.

Mixed Culture Cultivation in Microbial Bioprocesses.

Mixed culture cultivation is well renowned for industrial applications due to its technological and economic benefits in bioprocess, food processing, and pharmaceutical industries. A mixed consortium encompasses to achieve growth in unsterile conditions, robustness to environmental stresses, perform difficult functions, show better substrate utilization, and increase productivity. Hence, mixed cultures are being valorized currently and has also augmented our understanding of microbial activities in communities. This chapter covers a wide range of discussion on recent improvements in mixed culture cultivation for microbial bioprocessing and multifarious applications in different areas. The history of microbial culture, microbial metabolism in mixed culture, biosynthetic pathway studies, isolation and identification of strains, along with the types of microbial interactions involved during their production and propagation, are meticulously detailed in the current chapter. Besides, parameters for evaluating mixed culture performance, large-scale production, and challenges associated with it are also discussed vividly. Microbial community, characteristics of single and mixed culture fermentation, and microbe-microbe interactions in mixed cultures have been summarized comprehensively. Lastly, various challenges and opportunities in the area of microbial mixed culture that are obligatory to improve the current knowledge of microbial bioprocesses are projected.

Mixed culture biotechnology and its versatility in dark fermentative hydrogen production.

Over the years, extensive research has gone into fermentative hydrogen production using pure and mixed cultures from waste biomass with promising results. However, for up-scaling of hydrogen production mixed cultures are more appropriate to overcome the operational difficulties such as a metabolic shift in response to environmental stress, and the need for a sterile environment. Mixed culture biotechnology (MCB) is a robust and stable alternative with efficient waste and wastewater treatment capacity along with co-generation of biohydrogen and platform chemicals. Mixed culture being a diverse group of bacteria with complex metabolic functions would offer a better response to the environmental variations encountered during biohydrogen production. The development of defined mixed cultures with desired functions would help to understand the microbial community dynamics and the keystone species for improved hydrogen production. This review aims to offer an overview of the application of MCB for biohydrogen production.

Recent Advances in 2-Keto-l-gulonic Acid Production Using Mixed-Culture Fermentation and Future Prospects.

Vitamin C, also known as ascorbic acid, is an essential vitamin that cannot be synthesized by the human body and must be acquired through our diet. At present, the precursor of vitamin C, 2-keto-l-gulonic acid (2-KGA), is typically produced via a two-step fermentation process utilizing three bacterial strains. The second step of this traditional two-step fermentation method involves mixed-culture fermentation employing 2-KGA-producing bacteria (Ketogulonicigenium vulgare) along with associated bacteria. Because K. vulgare has defects in various metabolic pathways, associated bacteria are needed to provide key substances to promote K. vulgare growth and 2-KGA production. Unlike previous reviews where the main focus was the interaction between associated bacteria and K. vulgare, this Review presents the latest scientific research from the perspective of the metabolic pathways associated with 2-KGA production by K. vulgare and the mechanism underlying the interaction between K. vulgare and the associated bacteria. In addition, the dehydrogenases that are responsible for 2-KGA production, the 2-KGA synthesis pathway, strategies for simplifying 2-KGA production via a one-step fermentation route, and, finally, future prospects and research goals in vitamin C production are also presented.

Comparison of SYBR® Green qPCR assay and plate count method to describe growth of Weissella viridescens and Leuconostoc mesenteroides in pure and mixed cultivation.

The current study was conducted to statistically compare the SYBR® Green quantitative polymerase chain reaction (qPCR) assay and the conventional plate counting (PC) method to construct growth curves of a cocktail of Weissella viridescens in pure culture under different isothermal storage conditions (4, 8, 14, and 30 °C) and in mixed culture with Leuconostoc mesenteroides at 8 °C. The efficiency and specificity of the qPCR standard curves were confirmed, and both methods were adequate to quantify the growth kinetics of W. viridescens at all isothermal temperatures, demonstrating a good correlation and agreement. The efficiencies of the standard curves varied between 98% and 102%. The SYBR® Green qPCR assay was also able to differentiate the growth curves of W. viridescens and L. mesenteroides in the mixed culture at 8 °C. Additionally, the SYBR® Green qPCR method was considered a faster and more sensitive alternative to construct growth curves under different isothermal conditions and differentiate morphologically similar lactic acid bacteria. Overall, the results suggest that the SYBR® Green qPCR method is a reliable and efficient tool to study microbial growth kinetics in pure and mixed cultures.

Enrichment of mixed methanotrophic cultures producing polyhydroxyalkanoates (PHAs) from various environmental sources.

Methanotrophic bacteria can use atmospheric methane (CH4) as a sole carbon source for the growth and production of polyhydroxyalkanoates (PHA). The development of CH4 bioconversion processes relies heavily on the selection of an efficient methanotrophic culture. This research assessed the effect of selected growth conditions, such as nitrogen sources on the enrichment of methanotrophic cultures from various environments for PHA accumulation. Nitrate-based medium favoured the culture growth and selection for PHA-producing methanotrophic cultures with Methylocystis sp. as a major genus and accumulation of up to 27 % polyhydroxybutyrate (PHB) in the biomass. Three PHB-producing cultures: enriched from waste activated sludge (AS), peat bog soil (PB) and landfill biocover soil (LB) were then tested for their ability to produce PHA copolymer at different CH4:O2 ratios. All enriched cultures were able to utilise valeric acid as a cosubstrate for the accumulation of PHA with a 3-hydroxyvaleric (3HV) fraction of 21-41 mol% depending on the inoculum source and CH4 concentration. The process performance of selected cultures was evaluated and compared to the culture of reference strain Methylocystis hirsuta DSM 18500. All mixed cultures irrespective of their inoculum source had similar levels of 3HV fraction in the PHA (38 ± 2 mol%). The highest poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production was observed for AS culture at 10 % CH4 with an accumulation of 27 ± 3 % of dry cell weight (DCW), 3HV fraction of 39 ± 2 mol% and yield of 0.42 ± 0.02 g-PHA/g-substrate.

Simultaneous medium chain carboxylic acids and 1,3-propanediol production in a bioaugmented lactate-based chain elongation induced with glycerol.

The objective was to investigate the impact of the bioaugmentation on chain elongation process using glycerol, lactate and lactose as substrates in an open culture fermentation. In the batch trials the highest selectivity for chain elongation product, i.e. caproate, was observed in trials inoculated with co-culture of Megasphaera elsdenii and Eubacterium limosum grown on glycerol (28.6%), and in non-bioaugmented open culture run on lactose + lactate (14.8%). The results showed that E. limosum, out of two bioaugmented strains, was able to survive in the open culture. A continuous open culture fermentation of glycerol led to caproate and 1,3-propanediol (1,3-PDO) formation, while lactate addition led to 1,3-PDO and short chain carboxylates production. Moving the process into batch mode triggered even-carbon chain elongation. Presence of E. limosum promoted odd-carbon chain elongation and valerate production. Imaging flow cytometry combined with machine learning enabled the discrimination of Eubacterium cells from other microbial strains during the process.

Selecting for a high lipid accumulating microalgae culture by dual growth limitation in a continuous bioreactor.

A dual-growth-limited continuous operated bioreactor (chemostat) was used to enhance lipid accumulation in an enrichment culture of microalgae. The light intensity and nitrogen concentration where both limiting factors resulting in high lipid accumulation in the mixed culture. Both conditions of light and nitrogen excess and deficiency were tested. Strategies to selectively enrich for a phototrophic lipid-storing community, based on the use of different nitrogen sources (ammonium vs. nitrate) and vitamin B supplementation in the growth medium, were evaluated. The dual limitation of both nitrogen and light enhanced the accumulation of storage compounds. Ammoniacal nitrogen was the preferred nitrogen source. Vitamin B supplementation led to a doubling of the lipid productivity. The availability of vitamins played a key role in selecting an efficient lipid-storing community, primarily consisting of Trebouxiophyceae (with an 82 % relative abundance among eukaryotic microorganisms). The obtained lipid volumetric productivity (387 mg L-1 d-1) was among the highest reported in literature for microalgae bioreactors. Lipid production by the microalgae enrichment surpassed the efficiencies reported for continuous microalgae pure cultures, highlighting the benefits of mixed-culture photo-biotechnologies for fuels and food ingredients in the circular economy.

Effect of predatory bacterial mixtures on biolysis of waste activated sludge to improve dewatering performance.

The generation of surplus sludge during biological wastewater treatment has become a prevalent issue, necessitating the development of a dewatering approach that is efficient, economically feasible, and ecologically sound. Bdellovibrio-and-like-organisms (BALOs) are obligatory parasitic bacteria that prey on an array of bacteria. In this study, different BALO strains were isolated and purified from waste activited sludge (WAS). Anti-predation host strains were applied to screen the BALO strains with different host-range to minimize the overlap of the biolysis prey spectrum. In addition, the BALO strains with different host preferences were mixed for sludge biolysis treatment efficiency comparison. The results indicated that the capillary suction time and the bound water content in the WAS treated with the mixed BALOs were significantly decreased by 25.9% ± 1.7% and 5.2% ± 1.2%, respectively, compared to those treated with the single BALO strain. The soluble chemical oxygen demand concentration in the mixed BALOs treated group was increased by 31.2% ± 0.7% than that treated with the single strain. The findings indicate that the mixed strains used in the treatment process resulted in a notable enhancement of both sludge dewatering performance and lysis degree. In addition, the abundance of Proteobacteria treated with the BALO mixtures decreased by 69.1% than the single strain treated one which demonstrated that the BALO mixture expanded the sludge host lysis spectrum. This study revealed the different effects of single and mixed strains on sludge community structure, suggesting that the BALO host-range expansion is crucial to further improve sludge dewatering performances.

Increasing student interest with the Bacterial Unknown Identification Project: using mixed cultures to create real-world applications.

Many introductory-level classes teach fundamental concepts, as they are prerequisites for upper-division courses. Therefore, the student body in these classes has diverse interests. To address this breadth of career trajectory, introductory-level laboratory courses often include experiments that demonstrate a wide range of microbiological techniques and processes. One of the experiments that is a standard component of many microbiology classes, the Bacterial Unknown Identification Project (BUIP), is often limited to isolated organisms or a specific environment. Here, we describe an updated method for the BUIP that incorporates the projected student career diversity through the implementation of multiple mixed cultures of microorganisms associated with different environments. This update can be utilized in any microbiology laboratory classroom. We maintained the learning objectives, including applying appropriate microbiological methods to analyze and interpret results, and effectively communicate scientific findings, while modifying the sample composition. Assessment of the modification demonstrated that upon completion of the BUIP, students felt that the project applied to their career and it did not take too much of their free time to complete.