Raman spectroscopy-based measurements of single-cell phenotypic diversity in microbial populations

Microbial cells experience physiological changes due to environmental change, such as pH and temperature, the release of bactericidal agents, or nutrient limitation. This has been shown to affect community assembly and physiological processes (e.g., stress tolerance, virulence, or cellular metabolic activity). Metabolic stress is typically quantified by measuring community phenotypic properties such as biomass growth, reactive oxygen species, or cell permeability. However, bulk community measurements do not take into account single-cell phenotypic diversity, which is important for a better understanding and the subsequent management of microbial populations. Raman spectroscopy is a nondestructive alternative that provides detailed information on the biochemical makeup of each individual cell. Here, we introduce a method for describing single-cell phenotypic diversity using the Hill diversity framework of Raman spectra. Using the biomolecular profile of individual cells, we obtained a metric to compare cellular states and used it to study stress-induced changes. First, in two Escherichia coli populations either treated with ethanol or nontreated and then in two Saccharomyces cerevisiae subpopulations with either high or low expression of a stress reporter. In both cases, we were able to quantify single-cell phenotypic diversity and to discriminate metabolically stressed cells using a clustering algorithm. We also described how the lipid, protein, and nucleic acid compositions changed after the exposure to the stressor using information from the Raman spectra. Our results show that Raman spectroscopy delivers the necessary resolution to quantify phenotypic diversity within individual cells and that this information can be used to study stress-driven metabolic diversity in microbial populations. IMPORTANCE Microbial cells that live in the same community can exist in different physiological and morphological states that change as a function of spatiotemporal variations in environmental conditions. This phenomenon is commonly known as phenotypic heterogeneity and/or diversity. Measuring this plethora of cellular expressions is needed to better understand and manage microbial processes. However, most tools to study phenotypic diversity only average the behavior of the sampled community. In this work, we present a way to quantify the phenotypic diversity of microbial samples by inferring the (bio)molecular profile of its constituent cells using Raman spectroscopy. We demonstrate how this tool can be used to quantify the phenotypic diversity that arises after the exposure of microbes to stress. Raman spectroscopy holds potential for the detection of stressed cells in bioproduction

Proof of concept of high-rate decentralized pre-composting of kitchen waste : optimizing design and operation of a novel drum reactor

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Storage, fertilization and cost properties highlight the potential of dried microbial biomass as organic fertilizer

The transition to sustainable agriculture and horticulture is a societal challenge of global importance. Fertilization with a minimum impact on the environment can facilitate this. Organic fertilizers can play an important role, given their typical release pattern and production through resource recovery. Microbial fertilizers (MFs) constitute an emerging class of organic fertilizers and consist of dried microbial biomass, for instance produced on effluents from the food and beverage industry. In this study, three groups of organisms were tested as MFs: a high-rate consortium aerobic bacteria (CAB), the microalgaArthrospira platensis('Spirulina') and a purple non-sulfur bacterium (PNSB)Rhodobactersp. During storage as dry products, the MFs showed light hygroscopic activity, but the mineral and organic fractions remained stable over a storage period of 91 days. For biological tests, a reference organic fertilizer (ROF) was used as positive control, and a commercial organic growing medium (GM) as substrate. The mineralization patterns without and with plants were similar for all MFs and ROF, with more than 70% of the organic nitrogen mineralized in 77 days. In a first fertilization trial with parsley, all MFs showed equal performance compared to ROF, and the plant fresh weight was even higher with CAB fertilization. CAB was subsequently used in a follow-up trial with petunia and resulted in elevated plant height, comparable chlorophyll content and a higher amount of flowers compared to ROF. Finally, a cost estimation for packed GM with supplemented fertilizer indicated that CAB and a blend of CAB/PNSB (85%/15%) were most cost competitive, with an increase of 6% and 7% in cost compared to ROF. In conclusion, as bio-based fertilizers, MFs have the potential to contribute to sustainable plant nutrition, performing as good as a commercially available organic fertilizer, and to a circular economy

Chlorella vulgaris as a green biofuel factory : comparison between biodiesel, biogas and combustible biomass production

Biofuels are viewed as the answer to safeguard the currently challenged energy security. To this end, the present study provides a comparison between approaches regarding microalgal biomass conversion to bioenergy, with a view on sustainable implementation. The energetic valorization of Chlorella vulgaris biomass cultivated under heterotrophic, sulfur-limited conditions was investigated through the biofuels biodiesel, biogas (biomethane) and combustible dry biomass. The lipid productivity can reach the value of 442.9 6.5 mg L-1 d(-1) containing suitable fatty acids for biodiesel production. Next, biochemical methane potential (BMP) assays yielded 360.9 20.2 mL CH4 g VSadlded under mesophilic conditions, while the calorific value of dry C. vulgaris biomass was measured as 24,538 182 kJ kgpw(-1) (5,865 43 kcal kgpw(-1)). Considering the downstream processing required in each approach, the most promising energy valorization method is anaerobic digestion able to reach values up to 20,862 kJ Lreactor 1 day(-1) in continuous systems

Inoculum origin and waste solid content influence the biochemical methane potential of olive mill wastewater under mesophilic and thermophilic conditions

The biological valorization of olive mill wastewater (OMW) is often problematic due to the characteristically high organic load and phenolic compound content of this waste stream. The present study aimed at determining the optimal conditions (i.e. temperature, solid content) for the anaerobic digestion of OMW, striving for the maximum methane yield. Therefore, inocula originating from a wastewater treatment plant and two lab-scale bioreactors treating OMW (mesophilic Up-flow Anaerobic Sludge Blanket (UASB) reactor and thermophilic Up-flow Packed Bed Reactor (UPBR)), were tested in terms of OMW degradation potential and methane yield under mesophilic and thermophilic conditions, through Biochemical Methane Potential (BMP) assays. The methane yields were in all cases higher for the raw OMW (325-472 mL CH4/g VSadded) compared to the centrifuged OMW (219-391 mL CH4/g VSadded). Moreover, the greatest biodegradability extent (94.0-98.5%) was achieved by the mesophilic sludge originating from the UASB reactor. The maximum specific methane production rate (121 mL CH4/g VSSadded.d) was reached by the inoculum obtained from the mesophilic UASB reactor using centrifuged OMW. These results indicate that the mesophilic temperatures are optimal for the anaerobic digestion (AD) of OMW in terms of methane productivity and biodegradation potential, while no solid removal is necessary