In scenarios where yeast and bacterial cells coexist, it is of interest to simultaneously quantify the concentrations of both cell types, since traditional methods used to determine these concentrations individually take more time and resources. Here, we compared different methods for quantifying the fuel ethanol Saccharomyces cerevisiae PE-2 yeast strain and cells from the probiotic Lactiplantibacillus plantarum strain in microbial suspensions. Individual suspensions were prepared, mixed in 1:1 or 100:1 yeast-to-bacteria ratios, covering the range typically encountered in sugarcane biorefineries, and analyzed using bright field microscopy, manual and automatic Spread-plate and Drop-plate counting, flow cytometry (at 1:1 and 100:1 ratios), and a Coulter counter (at 1:1 and 100:1 ratios). We observed that for yeast cell counts in the mixture (1:1 and 100:1 ratios), flow cytometry, the Coulter counter, and both Spread-plate options (manual and automatic CFU counting) yielded statistically similar results, while the Drop-plate and microscopy-based methods gave statistically different results. For bacterial cell quantification, the microscopy-based method, Drop-plate, and both Spread-plate plating options and flow cytometry (1:1 ratio) produced no significantly different results (P > 0.05). In contrast, the Coulter counter (1:1 ratio) and flow cytometry (100:1 ratio) presented results statistically different (P < 0.05). Additionally, quantifying bacterial cells in a mixed suspension at a 100:1 ratio wasn\'t possible due to an overlap between yeast cell debris and bacterial cells. We conclude that each method has limitations, advantages, and disadvantages.

Carbon-based products are essential to society, yet producing them from fossil fuels is unsustainable. Microorganisms have the ability to take up electrons from solid electrodes and convert carbon dioxide (CO2) to valuable carbon-based chemicals. However, higher productivities and energy efficiencies are needed to reach a viability that can make the technology transformative. Here, we show how a biofilm-based microbial porous cathode in a directed flow-through electrochemical system can continuously reduce CO2 to even-chain C2-C6 carboxylic acids over 248 days. We demonstrate a threefold higher biofilm concentration, volumetric current density, and productivity compared with the state of the art. Most notably, the volumetric productivity (VP) resembles those achieved in laboratory-scale and industrial syngas (CO-H2-CO2) fermentation and chain elongation fermentation. This work highlights key design parameters for efficient electricity-driven microbial CO2 reduction. There is need and room to improve the rates of electrode colonization and microbe-specific kinetics to scale up the technology.

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.

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.

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.

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.

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.

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.

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.

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.