Moderate High-Pressure Superdormancy in Bacillus Spores: Properties of Superdormant Spores and Proteins Potentially Influencing Moderate High-Pressure Germination.

Resistant bacterial spores are a major concern in industrial decontamination processes. An approach known as pressure-mediated germination-inactivation strategy aims to artificially germinate spores by isostatic pressure to mitigate their resistance to inactivation processes. The successful implementation of such a germination-inactivation strategy relies on the germination of all spores. However, germination is heterogeneous, with some "superdormant" spores germinating extremely slowly or not at all. The present study investigated potential underlying reasons for moderate high-pressure (150 MPa; 37°C) superdormancy of Bacillus subtilis spores. The water and dipicolinic acid content of superdormant spores was compared with that of the initial dormant spore population. The results suggest that water and dipicolinic acid content are not major drivers of moderate high-pressure superdormancy. A proteomic analysis was used to identify proteins that were quantified at significantly different levels in superdormant spores. Subsequent validation of the germination capacity of deletion mutants revealed that the presence of protein YhcN is required for efficient moderate high-pressure germination and that proteins MinC, cse60, and SspK may also play a role, albeit a minor one. IMPORTANCE Spore-forming bacteria are ubiquitous in nature and, as a consequence, inevitably enter the food chain or other processing environments. Their presence can lead to significant spoilage or safety-related issues. Intensive treatment is usually required to inactivate them; however, this treatment harms important product quality attributes. A pressure-mediated germination-inactivation approach can balance the need for effective spore inactivation and retention of sensitive ingredients. However, superdormant spores are the bottleneck preventing the successful and safe implementation of such a strategy. An in-depth understanding of moderate high-pressure germination and the underlying causes of superdormancy is necessary to advance the development of mild high pressure-based spore control technologies. The approach used in this work allowed the identification of proteins that have not yet been associated with reduced germination at moderate high pressure. This research paves the way for further studies on the germination and superdormancy mechanisms in spores, assisting the development of mild spore inactivation strategies.

Impact of the electric field intensity and treatment time on whey protein aggregate formation.

Whey proteins are being integrated as high-value food product ingredients due to their versatile and tunable techno-functionality. To meet high food quality and clean label expectations by consumers, electric field (EF) technologies have been proposed to open new frontiers in this field. Despite a variety of studies, it remains ambiguous which EF parameters are crucial to achieving targeted whey protein modifications. Reconstituted liquid whey protein concentrate (WPCL) and filtered, non-heat-treated liquid whey (WPL_filt) at low protein dry weight concentrations (0.4% wt/wt) were exposed to microsecond pulsed electric field (µsPEF) treatments at EF intensities between 1.25 and 12.5 kV/cm, pulse repetition frequencies between 0.38 and 85 Hz, and pulse lengths set to 10 or 100 µs. Protein aggregations were quantified spectroscopically. We report here that aggregates formed at lower temperatures for µsPEF compared with purely thermal treatments in identical treatment geometries at similar time-temperature profiles. We suggest that the observed increase in absorbance is linked to protein migration, the isoelectric point, local deprotonation phenomena of thiol groups, and cation precipitation. The µsPEF treatment time, which is dependent on the pulse repetition frequency, pulse length, and time of process, is the main driver of the increase in absorbance. High EF intensities balanced with shorter pulse repetition frequencies to ensure similar energy inputs resulted in no aggregate formation. For WPL_filt, 12.5 kV/cm, 10 µs, 0.38 Hz (620 ± 96 kJ/kg; ± standard deviation) did not result in an increase in absorbance, whereas 1.25 kV/cm, 10 µs, 50 Hz (634 ± 57 kJ/kg) with similar time-temperature profiles increased the absorbance at a wavelength of 380 nm by a factor of 8.2 ± 1.7 compared with untreated WPL_filt. In conclusion, the treatment time seems to dominate over high EF intensities at similar energy inputs for aggregate formation and increase in absorbance.

Bacillus spore germination at moderate high pressure: A review on underlying mechanisms, influencing factors, and its comparison with nutrient germination.

Spore-forming bacteria are resistant to stress conditions owing to their ability to form highly resistant dormant spores. These spores can survive adverse environmental conditions in nature, as well as decontamination processes in the food and related industries. Bacterial spores may return to their vegetative state through a process called germination. As spore germination is critical for the loss of resistance, outgrowth, and development of pathogenicity and spoilage potential, the germination pathway has piqued the interest of the scientific community. The inhibition and induction of germination have critical applications in the food industry. Targeted germination can aid in decreasing the resistance of spores and allow the application of milder inactivation procedures. This germination-inactivation strategy allows better maintenance of important food quality attributes. Different stimuli are reported to trigger germination. Among those, isostatic high pressure (HP) has gained increasing attention due to its potential applications in industrial processes. However, pressure-mediated spore germination is extremely heterogeneous as some spores germinate rapidly, while others exhibit slow germination or do not undergo germination at all. The successful and safe implementation of the germination-inactivation strategy, however, depends on the germination of all spores. Therefore, there is a need to elucidate the mechanisms of HP-mediated germination. This work aimed to critically review the current state of knowledge on Bacillus spore germination at a moderate HP of 50-300 MPa. In this review, the germination mechanism, heterogeneity, and influencing factors have been outlined along with knowledge gaps.

Characterization of Chlorella vulgaris (Trebouxiophyceae) associated microbial communities1.

Microalgae exhibit extensive potential for counteracting imminent challenges in the nutraceutical, pharmaceutical, and biomaterial sectors, but lack economic viability. Biotechnological systems for contamination control could advance the economic viability of microalgal feedstock, but the selection of suitable strains that sustainably promote microalgal productivity remains challenging. In this study, total diversity in phototrophic Chlorella vulgaris cultures was assessed by amplicon sequencing comparing cultures subjected to five different cultivation conditions. Overall, 12 eukaryotic and 53 prokaryotic taxa were identified; Alphaproteobacteria (36.7%) dominated the prokaryotic and C. vulgaris (97.2%) the eukaryotic community. Despite altering cultivation conditions, 2 eukaryotic and 40 prokaryotic taxa remained stably associated with C. vulgaris; diversity between systems did not significantly differ (P > 0.05). Among those, 20 cultivable taxa were isolated and identified by 16S rDNA sequencing. Subsequently, controlled co-cultures were investigated showing stable associations of C. vulgaris with Sphingopyxis sp. and Pseudomonas sp.. Out-competition of C. vulgaris due to ammonium or phosphate limitation was not observed, despite significantly elevated growth of Sphingopyxis sp. and Tistrella sp.. (P < 0.05). Nevertheless, C. vulgaris growth was impaired by Tistrella sp.. Hence, the study provides a selection of stable indigenous prokaryotes and eukaryotes for artificially tailoring microbial biocenoses. Following a bottom-up approach, it provides a base for controlled co-cultures and thus the establishment of even more complex biocenoses using interkingdom assemblages. Such assemblages can benefit from functional richness for improved nutrient utilization, as well as bacterial load control, which can enhance microalgal feedstock production through improved culture stability and productivity.

Role of DNA repair in Bacillus subtilis spore resistance to high energy and low energy electron beam treatments.

Bacillus subtilis spore inactivation mechanisms under low energy electron beam (LEEB) and high energy electron beam (HEEB) treatment were investigated using seven mutants lacking specific DNA repair mechanisms. The results showed that most of the DNA repair-deficient mutants, including ΔrecA, ΔKu ΔligD, Δexo Δnfo, ΔuvrAB and ΔsbcDC, had reduced resistances towards electron beam (EB) treatments at all investigated energy levels (80 keV, 200 keV and 10 MeV) compared to their wild type. This result suggested DNA damage was induced during EB treatments. The mutant lacking recA showed the lowest resistance, followed by the mutant lacking Ku and ligD. These findings indicated that recA, Ku and ligD and their associated DNA repair mechanisms, namely, homologous recombination and non-homologous end joining, play important roles in spore survival under EB treatment. Furthermore, exoA, nfo, uvrAB, splB, polY1 and polY2, which are involved in nucleotide damage repair/removal, showed different levels of effects on spore resistance under EB treatment. Finally, the results suggested that HEEB and LEEB inactivate B. subtilis spores through similar mechanisms. This research will provide a better understanding of how EB technologies inactivate B. subtilis spores and will contribute to the application of these technologies as a non-thermal, gentle spore control approach.

Novel Experimental Methods for the Investigation of Hermetia illucens (Diptera: Stratiomyidae) Larvae.

Large-scale insect rearing for food and feed production can be improved by understanding diet digestion and host-microbe interactions. To examine these processes in black soldier fly (Hermetia illucens L.; Diptera: Stratiomyidae) larvae, two protocols were developed. Protocol 1 describes a method to produce viable, sterile black soldier fly larvae and a gentle method for diet sterilization. Sterile black soldier fly larvae can be used to study the diverse role of microbes in larval development. Nutrient requirements of sterile black soldier fly larvae are met only through diet. Viable sterile black soldier fly larvae were consistently generated using a four-step treatment with alternating immersions of eggs for 2 min each in ethanol (70%) and sodium hypochlorite (0.6%), over two cycles. A nonthermal method of diet sterilization, namely high-energy electron beam (HEEB) treatment, was introduced. Subsequently, growth of sterile black soldier fly larvae was observed on the HEEB-treated diets (40, 60, and 40% of replicates with poultry feed, liver pie, and an artificial diet, respectively) but not on autoclaved diets. In Protocol 2, we propose a novel method to collect frass from individual larvae. We then measured the metabolites in frass, using high-pressure liquid chromatography. Results on metabolites confirmed the influence of digestion. For instance, succinate increased from 1 to 2 and 7 µmol/g sample from diet to gut homogenate and frass, respectively. The collection method is a promising tool to estimate the diet and nutrient requirements of black soldier fly larvae, thus increasing the performance and reliability of black soldier fly larvae rearing. We discuss in detail the possible applications and limitations of our methods in black soldier fly larvae research.

Continuous Versus Discontinuous Ultra-High-Pressure Systems for Food Sterilization with Focus on Ultra-High-Pressure Homogenization and High-Pressure Thermal Sterilization: A Review.

High-pressure thermal sterilization (HPTS) and ultra-high-pressure homogenization (UHPH) are two emerging sterilization techniques that have not been implemented in the food industry yet. The two technologies apply different acting principles as HPTS uses isostatic pressure in combination with heat whereas UHPH uses dynamic pressure in combination with shear stress, cavitation, impingement, and heat. Both technologies offer significant benefits in terms of spore inactivation in food production with reduced thermal intensity and minimized effects on sensory and nutritional profiles. These benefits have resulted in relevant research efforts on both technologies over the past few decades. This state of the art of the discontinuous HPTS-based and the continuous UHPH-based sterilization concepts are assessed within this review. Further, various basic principles and promising future preservation applications of HPTS and UHPH for food processing, that are also applicable in the pharmaceutical, biochemical, and biotechnological sectors, are summarized. In addition, the applications and limitations of these technologies in terms of optimizations needed to overcome the identified challenges are emphasized.

In situ investigation of Geobacillus stearothermophilus spore germination and inactivation mechanisms under moderate high pressure.

Bacterial spores are a major concern for food safety due to their high resistance to conventional preservation hurdles. Innovative hurdles can trigger bacterial spore germination or inactivate them. In this work, Geobacillus stearothermophilus spore high pressure (HP) germination and inactivation mechanisms were investigated by in situ infrared spectroscopy (FT-IR) and fluorometry. G. stearothermophilus spores' inner membrane (IM) was stained with Laurdan fluorescent dye. Time-dependent FT-IR and fluorescence spectra were recorded in situ under pressure at different temperatures. The Laurdan spectrum is affected by the lipid packing and level of hydration, and provided information on the IM state through the Laurdan generalized polarization. Changes in the -CH2 and -CH3 asymmetric stretching bands, characteristic of lipids, and in the amide I' band region, characteristic of proteins' secondary structure elements, enabled evaluation of the impact of HP on endospores lipid and protein structures. These studies were complemented by ex situ analyses (plate counts and microscopy). The methods applied showed high potential to identify germination mechanisms, particularly associated to the IM. Germination up to 3 log10 was achieved at 200 MPa and 55 °C. A molecular-level understanding of these mechanisms is important for the development and validation of multi-hurdle approaches to achieve commercial sterility.

Enhancing protein extraction from heterotrophic Auxenochlorella protothecoides microalgae through emerging cell disruption technologies combined with incubation.

This study evaluated pulsed electric fields (PEF) and ultrasonication (US) combined with incubation to enhance cell disruption and protein extraction from Auxenochlorella protothecoides, comparing them to conventional high-pressure homogenization (HPH). A 5 h incubation enhanced protein yield by 79.4 % for PEF- and 27.2 % for US-treated samples. Extending the incubation to 24 h resulted in a total yield increase of 122 % for PEF (0.25 ± 0.03 kgEP kgTP-1) and 51.9 % for US (0.20 ± 0.02 kgEP-1 kgTP-1). Autofermentation in untreated cells after 24 h resulted in protein release with lower yields than all other treated and incubated samples. While HPH had the highest protein yield (0.58 ± 0.04 kgEP kgTP-1), PEF-incubation after 5 h (56.6 ± 5.3 MJ kgEP-1) and 24 h (49.5 ± 3.7 MJ kgEP-1) were 1.5 and 1.7-times more energy-efficient than HPH (82.9 ± 7.8 MJ kgEP-1). PEF combined incubation is an energy-efficient and targeted protein extraction method in heterotrophic A. protothecoides biorefinery.

Characterization of high-pressure-treated Bacillus subtilis spore populations using flow cytometry - Shedding light on spore superdormancy at 550 MPa.

Mild spore inactivation can be challenging in industry because of the remarkable resistance of bacterial spores. High pressure (HP) can trigger spore germination, which reduces the spore's resistance, and thereby allows mild spore inactivation. However, spore germination is heterogenous. Some slowly germinating or non-germinating spores called superdormant spores remain resistant and can survive. Therefore, superdormant spores need to be characterized to understand the causes of their germination deficiency. Bacillus subtilis spores were pressurized for 50 s - 6 min at a very high pressure (vHP) level of 550 MPa and 60 °C in buffer to trigger germination. For a rapid quantification of the remaining ungerminated superdormant spores, flow cytometry (FCM) analysis was validated using single cell sorting and growth analysis. FCM based on propidium iodide (PI) and SYTO16 can be used for 550 MPa-superdormant spores after short vHP treatments of ≤1 min and post-HP incubation at 37 °C or 60 °C. The need for a post-HP incubation is particular for vHP treatments. The incubation was successful to separate FCM signals from superdormant and germinated spores, thus allowing superdormant spore quantification. The SYTO16 and PI fluorescence levels did not necessarily indicate superdormancy or apparent viability. This highlights the general need for FCM validation for different HP treatment conditions. The ∼7 % of ungerminated, i.e., superdormant, spores were isolated after a vHP treatment (550 MPa, 60 °C, 43-52 s). This allowed the characterization of vHP superdormant spores for the first time. The superdormant spores had a similar dipicolinic acid content as spores of the initial dormant population. Descendants of superdormant spores had a normal vHP germination capacity. The causes of vHP superdormancy were thus unlikely linked to the dipicolinic acid content or a permanent genetic change. Isolated superdormant spores germinated better in a second vHP treatment compared to the initial spore population. This has not been observed for other germination stimuli so far. In addition, the germination capacity of the initial spore population was time-dependent. A vHP germination deficiency can therefore be lost over time and seems to be caused by transient factors. Permanent cellular properties played a minor role as causes of superdormancy under chosen HP treatment conditions. The study gained new fundamental insights in vHP superdormancy which are of applied interest. Understanding superdormancy helps to efficiently develop a strategy to avoid superdormant spores and hence to inactivate all spores. The development of a mild HP spore germination-inactivation process aims at better preserving the food quality.

Preservation of agri-food byproducts by acidification and fermentation in black soldier fly larvae bioconversion.

Maintaining a consistent supply of feedstock for efficient bioconversion of black soldier fly larvae (BSFL) presents challenges due to the fluctuating availability of biowastes and agri-food products. To address the challenge of consistent feedstock supply for BSFL, this study investigated the influence of three preservation methods: wild fermentation, inoculated fermentation, and acidification on agri-food by-products applied over three storage durations (1, 7, and 14 days), evaluating their impact on BSFL bioconversion, and feedstock nutrient and microbiota composition. The preserved feedstocks were characterized for gross nutrient, sugar, fermentation metabolite, and bacterial community analyses. All feedstock preservation methods and storage durations had a high bioconversion rate (21-25 % dry mass) and wet larval mass (170-196 mg). Notably, 7-and-14-day acidified feedstock had a significantly higher bioconversion rate compared to fermented feedstock. Acidification preserved feedstock nutrients best with only a 10 % difference compared to initial nutrient values. Fermentation produced typical lactic acid fermentation metabolites with reducing sugar contents; however, adding a lactic acid bacterial inoculum (7 log10 CFU kg feedstock-1) had no benefit, presumably due to the high nutrient content and existing richness in lactic acid bacteria. Preservations had little influence on Enterobacteriaceae (6.2-7.5 log10 CFU g-1) in freshly harvested larvae. Future research should assess the acidification and fermentation of different BSFL feedstocks and investigate the roles of feedstock pH, organic acids, and fermentation metabolites in more detail. Therefore, this study advances toward reliable and efficient insect-based nutrient recovery from agri-food by-products within the food system.

Physical pretreatment of three biowastes to improve black soldier fly larvae bioconversion efficiency.

Black soldier fly larvae (BSFL, Hermetia illucens (L.)) are recognized for efficient biowaste reduction while yielding valuable proteins and fats for animals. However, lignocellulosic fibers in biowastes are difficult to digest by biowaste and larval digestive tract microorganisms as well as the larvae themselves. This study investigated two biowaste physical pretreatments (thermal, mechanical) for improving BSFL processing of fibrous biowastes. Cow manure, spent grain, and grass clippings were thermally pretreated at 90 °C for three durations (0.5, 1 and 4 h). Contrary to expectations, thermal pretreatment resulted in either no improvement or decreased larval performance on all substrates, regardless of treatment duration. In contrast, mechanical pretreatment of spent grain and grass clippings, involving milling with three screen sizes (0.5, 1 and 2 mm) showed promising results. Specifically, bioconversion rates on 0.5 mm-milled spent grain and grass clippings increased by 0-53 % and 25-44 % dry mass, respectively compared to untreated. Additionally, larval protein conversion increased by 41 % and 23 % on spent grain and grass clippings, respectively. However, mechanical pretreatment did not affect fiber degradation by larval conversion, as hemicellulose decreased by 25 % and 75 % for spent grain and grass clippings, respectively, regardless of particle size. Particle size reduction influenced substrate microbial respiration (CO2 mg/min), with 0.5-mm milled grass clippings exhibiting higher respiration compared to untreated, although this effect was not observed for spent grain. This study highlights mechanical pretreatment's potential in enhancing BSFL bioconversion of fibrous biowastes and the importance of understanding substrate physical properties influencing substrate microorganisms and BSFL.

Valorization of cocoa pod side streams improves nutritional and sustainability aspects of chocolate.

Chocolate production faces nutritional, environmental and socio-economic challenges present in the conventional cocoa value chain. Here we developed an approach that addresses these challenges by repurposing the often-discarded pectin-rich cocoa pod endocarp and converting it into a gel. This is done using cocoa pulp juice concentrate to replace traditional sugar from sugar beets. Although swelling of fibres, proteins and starches can limit gel incorporation, our proposed chocolate formulation contains up to 20 wt% gel. It also has comparable sweet taste as traditional chocolate while offering improved nutritional value with higher fibre and reduced saturated fatty acid content. A cradle-to-factory life cycle assessment shows that large-scale production of this chocolate could reduce land use and global warming potential compared with average European dark chocolate production. The process also provides opportunities for diversification of farmers' income and technology transfer, offering potential socio-economic benefits for cocoa-producing regions.

Post-high-pressure temperature and time - Overlooked parameters in high pressure treatment of bacterial spores.

High pressure (HP) processing has high potential for bacterial spore inactivation with minimal thermal input. To advance HP germination and subsequent inactivation of spores, this study explored the physiological state of HP-treated spores using flow cytometry (FCM). Bacillus subtilis spores were treated at 550 MPa and 60 °C (very HP (vHP)) in buffer, incubated after the HP treatment, and stained for FCM analysis with SYTO16 indicating germination and propidium iodide (PI) indicating membrane damage. FCM subpopulations were analyzed depending on the HP dwell time (≤20 min), post-HP temperature (ice, 37 °C, 60 °C) and time (≤4 h), germination-relevant cortex-lytic enzymes (CLEs) and small-acid-soluble-proteins-(SASP)-degrading enzymes by using deletion strains. The effect of post-HP temperatures (ice, 37 °C) was additionally studied for moderate HP (150 MPa, 38 °C, 10 min). Post-HP incubation conditions strongly influenced the prevalence of five observed FCM subpopulations. Post-HP incubation on ice did not or only slowly shifted SYTO16-positive spores to higher SYTO16 levels. At 37 °C post-HP, this shift accelerated, and a shift to high PI intensities occurred depending on the HP dwell time. At 60 °C post-HP, the main shift was from SYTO16-positive to PI-positive subpopulations. The enzymes CwlJ and SleB, which are CLEs, seemed both necessary for PI or SYTO16 uptake, and to have different sensitivities to 550 MPa and 60 °C. Different extents of SASP degradation might explain the existence of two SYTO16-positive subpopulations. Shifts to higher SYTO16 intensities during post-HP incubation on ice or at 37 °C might rely on the activity and recovery of CLEs, SASP-degrading enzymes or their associated proteins from reversible HP-induced structural changes. These enzymes seemingly become active only during decompression or after vHP treatments (550 MPa, 60 °C). Based on our results, we provide a refined model of HP germination-inactivation of B. subtilis spores and an optimized FCM method for quantification of the safety-relevant subpopulation, i.e., vHP (550 MPa, 60 °C) superdormant spores. This study contributes to the development of mild spore inactivation processes by shedding light on overlooked parameters: post-HP incubation conditions. Post-HP conditions significantly influenced the physiological state of spores, likely due to varying enzymatic activity. This finding may explain inconsistencies in previous research and shows the importance of reporting post-HP conditions in future research. Furthermore, the addition of post-HP conditions as HP process parameter may open up new possibilities to optimize HP-based inactivation of spores for potential industrial applications in the food industry.

Evaluation of ammonia pretreatment of four fibrous biowastes and its effect on black soldier fly larvae rearing performance.

Biowaste treatment with black soldier fly larvae (BSFL, Hermetia illucens L.) can promote a more sustainable food system by reusing nutrients that would otherwise be wasted. However, many agri-food wastes and byproducts are typically high in lignocellulosic fibers (i.e., cellulose, hemicellulose, and lignin), making it resistant to efficient larval and/or microbial degradation. Ammonia pretreatment could be used to partially degrade lignocellulose, making the biowaste more easily degradable by the larvae and/or microorganisms. This study evaluated ammonia pretreatment for lignocellulose degradation and its effect on BSFL performance on four fibrous biowastes: brewers spent grain, cow manure, oat pulp, and grass clippings. First, the optimal ammonia dose (1 % or 5 % dry mass) and pretreatment time (three or seven days) were assessed by measuring fibers after treatment and further examined using Fourier transform infrared spectroscopy (FTIR) spectra and scanning electron microscopy (SEM) images. Second, BSFL rearing performance on ammonia-pretreated substrates was assessed with a 9-day feeding experiment. Three-day pretreatment with 5 % ammonia was chosen as it decreased the total fiber content by 8-23 % for all substrates except cow manure. Contrary to expectations, ammonia pretreatment with all substates decreased BSFL rearing performance metrics by more than half compared to the untreated control. Follow-up experiments suggested that ammonia pretreatment had a dose-dependent toxicity to BSFL. Interestingly, three-day fermentation of cow manure and oat pulp increased bioconversion rate by 25-31 %. This study shows that ammonia pretreatment is not suitable before BSFL rearing. Ammonia toxicity to BSFL and other pretreatments, such as fermentation, should be further studied.

A novel approach for the protein determination in food-relevant microalgae.

Microalgae are gaining interest as food ingredient. Assessments of functional and nutritional properties are necessary to forward their implementation. In this study, protein content and composition of eight commercially available microalgae biomasses were determined and compared to conventional food proteins. A novel procedure for the determination of the true protein content was proposed: Multiplication of proteinic nitrogen with a sample-specific nitrogen-to-protein conversion factor kA. The proteinic nitrogen was derived from the difference of total nitrogen minus non-protein nitrogen. The average kA for microalgae was 5.3 and considerable variation between different microalgae biomasses were detected. In addition, the content of non-protein nitrogen varied between 3.4% and 15.4%. The amino acid profiles of Chlorella samples were nutritionally superior to the tested plant proteins but indicated lower protein interaction tendency, potentially limiting their structuring functionality. In contrast, Auxenochlorella contained lower amounts of indispensable amino acids while showing comparable interaction potential to plant proteins.

A novel strategy to simultaneously enhance bioaccessible lipids and antioxidants in hetero/mixotrophic Chlorella vulgaris as functional ingredient.

Microalgae are a promising source of polyunsaturated fatty acids as well as bioactive antioxidant compounds such as carotenoids, phenolics and tocopherols. However, the accumulation of these biomolecules is often promoted by conflicting growth conditions. In this study, a phased bioprocessing strategy was developed to simultaneously enhance the lipid and antioxidant amounts by tailoring nitrogen content in the cultivation medium and applying light stress. This approach increased the overall contents of total fatty acids, carotenoids, phenolics, and α-tocopherol in Chlorella vulgaris by 2.2-, 2.2-, 1.5-, and 2.1-fold, respectively. Additionally, the bioaccessibility of the lipids and bioactives from the obtained biomasses improved after pulsed electric field (5 µs, 20 kV cm-1, 31.8 kJ kg-1sus) treatment (up to +12%) and high-pressure homogenization (100 MPa, 5-6 passes) (+41-76%). This work represents a step towards the generation of more efficient algae biorefineries, thus expanding the alternative resources available for essential nutrients.

Residues from black soldier fly (Hermetia illucens) larvae rearing influence the plant-associated soil microbiome in the short term.

The larvae of the black soldier fly (BSFL, Hermetia illucens) efficiently close resource cycles. Next to the nutrient-rich insect biomass used as animal feed, the residues from the process are promising plant fertilizers. Besides a high nutrient content, the residues contain a diverse microbial community and application to soil can potentially promote soil fertility and agricultural production through the introduction of beneficial microbes. This research assessed the application of the residues on plant-associated bacterial and fungal communities in the rhizosphere of a grass-clover mix in a 42-day greenhouse pot study. Potted soil was amended with BSFL residues (BR+) or conventional compost (CC+) produced by Rwandan waste management companies in parallel to residues and compost sterilized (BR-, CC-) by high-energy electron beam (HEEB) as abiotic controls. The fertilizers were applied at a rate of 150 kg N ha-1. Soil bacterial and fungal communities in both fertilizer and soil were assessed by high-throughput sequencing of ribosomal markers at different times after fertilizer application. Additionally, indicators for soil fertility such as basal respiration, plant yield and soil physicochemical properties were analyzed. Results showed that the application of BSFL residues influenced the soil microbial communities, and especially fungi, stronger than CC fertilizers. These effects on the microbial community structure could partly be attributed to a potential introduction of microbes to the soil by BSFL residues (e.g., members of genus Bacillus) since untreated and sterilized BSFL residues promoted different microbial communities. With respect to the abiotic effects, we emphasize a potential driving role of particular classes of organic matter like fiber and chitin. Indeed, especially taxa associated with decomposition of organic matter (e.g., members of the fungal genus Mortierella) were promoted by the application of BSFL residues. Soil fertility with respect to plant yield (+17% increase compared to unamended control) and basal respiration (+16% increase compared to unamended control) tended to be improved with the addition of BSFL residues. Findings underline the versatile opportunities for soil fertility arising from the application of BSFL residues in plant production and point to further research on quantification of the described effects.

Nanosecond pulsed electric field processing of microalgae based biorefineries governs growth promotion or selective inactivation based on underlying microbial ecosystems.

Nanosecond pulsed electric field treatment (nsPEF) is a technology-driven, resource-efficient approach fostering microalgae biorefineries for transforming them into economically viable scenarios. A processing window of 100 ns, 7 Hz, and 10 kV cm-1 significantly leveraged phototrophic Chlorella vulgaris and bacterial counts up to + 50.1 ± 12.2% and + 77.0 ± 37.4%, respectively (n = 4; p < 0.05) in non-axenic cultures. Applying the same processing window decreased C. vulgaris (-17.1 ± 13.8%) and prokaryotic (-82.7 ± 14.6%) counts owing to alterations in the prokaryotic community diversity. Principle coordinate analysis of prokaryotic phenotypic fingerprints indicated that phenotype or metabolism related diversity changes in the prokaryotic community affected the treatment outcome. The study fosters the upsurge of industrial-scale nsPEF realization and the economic viability of microalgae biorefineries through improved process understanding and thus control. It perpetuates nsPEF applicability for microalgae feedstock production and several other applications within single-cell biorefineries in the bio-based domain.

Nutrient Adequacy of Global Food Production.

A major challenge for countries around the world is to provide a nutritionally adequate diet to their population with limited available resources. A comprehensive analysis that reflects the adequacy of domestic food production for meeting national nutritional needs in different countries is lacking. Here we combined national crop, livestock, aquaculture, and fishery production statistics for 191 countries obtained from UN FAO with food composition databases from USDA and accounted for food loss and waste occurring at various stages to calculate the amounts of calories and 24 essential nutrients destined for human consumption. We then compared the domestic production quantities of all nutrients with their population-level requirements estimated from age- and sex-specific intake recommendations of WHO to assess the nutrient adequacy of the national food production. Our results show inadequate production of seven out of 24 nutrients (choline, calcium, polyunsaturated fatty acids, vitamin A, vitamin E, folate, and iron) in most countries, despite the overall adequacy of the total global production. High-income countries produce adequate amounts of dietary nutrients in general, while the foods produced in low-income countries mainly comprising roots and cereal products often lack in important micronutrients such as choline, calcium, and vitamin B12. South Asian food production barely fulfills half of the required vitamin A. Our study identifies target nutrients for each country whose domestic production should be encouraged for improving nutritional adequacy through interventions such as increasing the production of foods or fortified foods that are rich in these inadequate nutrients while not undermining the local environment. This assessment can serve as an evidence base for nutrition-sensitive policies facilitating the achievement of the Sustainable Development Goals of zero hunger and good health and well-being.