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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Enhancing single-cell bioconversion efficiency by harnessing nanosecond pulsed electric field processing.

Nanosecond pulsed electric field (nsPEF) processing is gaining momentum as a physical means for single-cell bioconversion efficiency enhancement. The technology allows biomass yields per substrate (YX/S) to be leveraged and poses a viable option for stimulating intracellular compound production. NsPEF processing thus resonates with myriad domains spanning the pharmaceutical and medical sectors, as well as food and feed production. The exact working mechanisms underlying nsPEF-based enhancement of bioconversion efficiency, however, remain elusive, and a better understanding would be pivotal for leveraging process control to broaden the application of nsPEF and scale-up industrial implementation. To bridge this gap, the study provides the electrotechnological and metabolic fundamentals of nsPEF processing in the bio-based domain to enable a critical evaluation of pathways underlying the enhancement of single-cell bioconversion efficiency. Evidence suggests that treating cells during the rapid proliferating and thus the early to mid-exponential state of cellular growth is critical to promoting bioconversion efficiency. A combined effect of transient intracellular and sublethal stress induction and effects caused on the plasma membrane level result in an enhancement of cellular bioconversion efficiency. Congruency exists regarding the involvement of transient cytosolic Ca2+ hubs in nsPEF treatment responses, as well as that of reactive oxygen species formation culminating in the onset of cellular response pathways. A distinct assignment of single effects and their contributions to enhancing bioconversion efficiency, however, remains challenging. Current applications of nsPEF processing comprise microalgae, bacteria, and yeast biorefineries, but these endeavors are in their infancies with limitations associated with a lack of understanding of the underlying treatment mechanisms, an incomplete reporting, insufficient characterization, and control of processing parameters. The study aids in fostering the upsurge of nsPEF applications in the bio-based domain by providing a basis to gain a better understanding of cellular mechanisms underlying an nsPEF-based enhancement of cellular bioconversion efficiency and suggests best practice guidelines for nsPEF documentation for improved knowledge transfer. Better understanding and reporting of processes parameters and consequently improved process control could foster industrial-scale nsPEF realization and ultimately aid in perpetuating nsPEF applicability within the bio-based domain.

Transfer of Lauric and Myristic Acid from Black Soldier Fly Larval Lipids to Egg Yolk Lipids of Hens Is Low.

Implementing insects, such as the black soldier fly larvae (BSFL), as animal feed commonly includes the previous removal of substantial amounts of fat. This fat may represent an as yet underutilized energy source for livestock. However, transfer of lauric and myristic acid, prevalent in BSFL fat and undesired in human nutrition, into animal-source foods like eggs may limit its implementation. To quantify this, a laying hen experiment was performed comprising five different diets (10 hens/diet). These were a control diet with soybean oil and meal and a second diet with soybean oil but with partially defatted BSFL meal as protein source. The other three diets were based on different combinations of partially defatted BSFL meal and fat obtained by two different production methods. Lauric acid made up half of the BSFL fat from both origins. Both BSFL fats also contained substantial amounts of myristic and palmitic acid. However, in the insect-based diets, the net transfer from diet to egg yolk was less than 1% for lauric acid, whereas the net transfer for myristic and palmitic acid was about 30% and 100%, respectively. The net transfer did not vary between BSFL originating from production on different larval feeding substrates. The results illustrate that hens are able to metabolize or elongate very large proportions of ingested lauric acid and myristic acid, which are predominant in the BSFL lipids (together accounting for as much as 37 mol%), such that they collectively account for less than 3.5 mol% of egg yolk fatty acids.

Automated Online Flow Cytometry Advances Microalgal Ecosystem Management as in situ, High-Temporal Resolution Monitoring Tool.

Microalgae are emerging as a next-generation biotechnological production system in the pharmaceutical, biofuel, and food domain. The economization of microalgal biorefineries remains a main target, where culture contamination and prokaryotic upsurge are main bottlenecks to impair culture stability, reproducibility, and consequently productivity. Automated online flow cytometry (FCM) is gaining momentum as bioprocess optimization tool, as it allows for spatial and temporal landscaping, real-time investigations of rapid microbial processes, and the assessment of intrinsic cell features. So far, automated online FCM has not been applied to microalgal ecosystems but poses a powerful technology for improving the feasibility of microalgal feedstock production through in situ, real-time, high-temporal resolution monitoring. The study lays the foundations for an application of automated online FCM implying far-reaching applications to impel and facilitate the implementation of innovations targeting at microalgal bioprocesses optimization. It shows that emissions collected on the FL1/FL3 fluorescent channels, harnessing nucleic acid staining and chlorophyll autofluorescence, enable a simultaneous assessment (quantitative and diversity-related) of prokaryotes and industrially relevant phototrophic Chlorella vulgaris in mixed ecosystems of different complexity over a broad concentration range (2.2-1,002.4 cells ⋅µL-1). Automated online FCM combined with data analysis relying on phenotypic fingerprinting poses a powerful tool for quantitative and diversity-related population dynamics monitoring. Quantitative data assessment showed that prokaryotic growth phases in engineered and natural ecosystems were characterized by different growth speeds and distinct peaks. Diversity-related population monitoring based on phenotypic fingerprinting indicated that prokaryotic upsurge in mixed cultures was governed by the dominance of single prokaryotic species. Automated online FCM is a powerful tool for microalgal bioprocess optimization owing to its adaptability to myriad phenotypic assays and its compatibility with various cultivation systems. This allows advancing bioprocesses associated with both microalgal biomass and compound production. Hence, automated online FCM poses a viable tool with applications across multiple domains within the biobased sector relying on single cell-based value chains.

Isolation, stability, and characteristics of high-pressure superdormant Bacillus subtilis spores.

Bacterial spores are a major challenge in industrial decontamination processes owing to their extreme resistance. High-pressure (HP) of 150 MPa at 37 °C can trigger the germination of spores, making them lose their extreme resistance. Once their resistance is lost, germinated spores can easily be inactivated by a mild decontamination step. The implementation of this gentle germination-inactivation strategy is hindered by the presence of a subpopulation of so-called high-pressure superdormant (HPSD) spores, which resist germination or germinate only very slowly in response to HP. It is essential to understand the properties of HPSD spores and the underlying causes of superdormancy to tackle superdormant spores and further develop germination-inactivation strategies involving HP. This study investigated factors influencing the prevalence of HPSD spores and successfully isolated them by combining buoyant density centrifugation and fluorescence-activated cell sorting, which allowed further characterisation of HPSD spores for the first time. The prevalence of HPSD spores was shown to be strongly dependent on the HP dwell time, with increasing treatment times reducing their prevalence. Spore mutants lacking major germinant receptors further showed a highly increased prevalence of HPSD spores; 93% of the spores remained dormant even after a prolonged HP dwell time of 40 min. In contrast to nutrient germination, sublethal heat treatment of 75 °C for 30 min prior to pressure treatment did not induce spore activation and increase germination. The isolated HPSD spores did not show visible structural differences compared to the initial dormant spores when investigated with transmission electron microscopy. Re-sporulated HPSD spores showed similar germination capacity compared to the initial dormant spores, indicating that HPSD spores are most likely not genetically different from the rest of the population. Moreover, the majority of HPSD spores germinated when exposed a second time to the same germination treatment; however, the germination capacity was lower than that of the initial population. The fact that the majority of spores lost superdormancy when exposed a second time to the same trigger makes it unlikely that there is one factor that determines whether a spore germinates with a certain HP treatment or not. Instead, it seems possible that there are other reversible or cumulative causes. This study investigated the factors influencing spore HP superdormancy to improve the understanding of HPSD spores with regard to their stability, germination capacity, and potential underlying causes of spore HP superdormancy. This knowledge will contribute to the development of HP-based germination-inactivation strategies for gentle but effective spore control.

Biochemical and Morphological Characterization of Heterotrophic Crypthecodinium cohnii and Chlorella vulgaris Cell Walls.

Microalgae are attractive for the food and cosmetic industries because of their nutrient composition. However, the bioaccessibility and extractability of nutrients in microalgae are limited by the rigid and indigestible cell wall. The goal of this study is to explore the cell wall polysaccharides (CWPSs) composition and morphology in heterotrophic Crypthecodinium cohnii and Chlorella vulgaris biomasses during growth. Our results showed that glucose was the major component of CWPSs and exopolysaccharides in C. cohnii. C. vulgaris CWPSs have a similar sugar profile in exponential and stationary phases, essentially composed of rhamnose and galactose. C. vulgaris cell wall thickness increased from 82 nm in the exponential phase to 114 nm in the stationary phase and consisted of two main layers. C. cohnii's cell wall was 133 nm thick and composed of several membranes surrounding thecal plates. Understanding of the microalgae cell wall helps developing a more efficient and targeted biorefinery approach.

Articulating the effect of food systems innovation on the Sustainable Development Goals.

Food system innovations will be instrumental to achieving multiple Sustainable Development Goals (SDGs). However, major innovation breakthroughs can trigger profound and disruptive changes, leading to simultaneous and interlinked reconfigurations of multiple parts of the global food system. The emergence of new technologies or social solutions, therefore, have very different impact profiles, with favourable consequences for some SDGs and unintended adverse side-effects for others. Stand-alone innovations seldom achieve positive outcomes over multiple sustainability dimensions. Instead, they should be embedded as part of systemic changes that facilitate the implementation of the SDGs. Emerging trade-offs need to be intentionally addressed to achieve true sustainability, particularly those involving social aspects like inequality in its many forms, social justice, and strong institutions, which remain challenging. Trade-offs with undesirable consequences are manageable through the development of well planned transition pathways, careful monitoring of key indicators, and through the implementation of transparent science targets at the local level.