Ruminations on sustainable and safe food: Championing for open symbiotic cultures ensuring resource efficiency, eco-sustainability and affordability.

Microbes are powerful upgraders, able to convert simple substrates to nutritional metabolites at rates and yields surpassing those of higher organisms by a factor of 2 to 10. A summary table highlights the superior efficiencies of a whole array of microbes compared to conventionally farmed animals and insects, converting nitrogen and organics to food and feed. Aiming at the most resource-efficient class of microbial proteins, deploying the power of open microbial communities, coined here as 'symbiotic microbiomes' is promising. For instance, a production train of interest is to develop rumen-inspired technologies to upgrade fibre-rich substrates, increasingly available as residues from emerging bioeconomy initiatives. Such advancements offer promising perspectives, as currently only 5%-25% of the available cellulose is recovered by ruminant livestock systems. While safely producing food and feed with open cultures has a long-standing tradition, novel symbiotic fermentation routes are currently facing much higher market entrance barriers compared to axenic fermentation. Our global society is at a pivotal juncture, requiring a shift towards food production systems that not only embrace the environmental and economic sustainability but also uphold ethical standards. In this context, we propose to re-examine the place of spontaneous or natural microbial consortia for safe future food and feed biotech developments, and advocate for intelligent regulatory practices. We stress that reconsidering symbiotic microbiomes is key to achieve sustainable development goals and defend the need for microbial biotechnology literacy education.

Microbial protein production from lactose-rich effluents through food-grade mixed cultures: Effect of carbon to nitrogen ratio and dilution rate.

Overabundant agro-industrial side streams such as lactose-rich effluents from dairy activities offer multiple valorisation opportunities. In the present study, a food-grade mixed culture of bacteria and yeasts was tested under different operational conditions for the treatment and the valorisation of cheese whey permeate (CWP), the residue of whey protein recovery, into microbial protein (MP). Under continuous aerobic fermentation settings, the carbon-to-nitrogen (C/N) ratio showed little to no influence on the system performances and MP quality as compared to dilution rates (D), leading to a final protein content as high as 76%. Under high D values, instead, while biomass productivity increased, N-efficiency and protein content decreased. Unlike the bacterial community, the yeast one proved to be highly stable and less influenced by the increase of D. A preliminary estimate indicated that 2-11% of the future MP-based food production could be satisfied by only valorising lactose-rich dairy residues such as CWP.

Microbial protein production from sulfide-rich biogas through an enrichment of methane- and sulfur-oxidizing bacteria.

This study evaluated the possibility of combining methane oxidizing bacteria (MOB) with sulfur oxidizing bacteria (SOB) to enable the utilization of sulfide-rich biogas for microbial protein production. For this purpose, a MOB-SOB mixed-culture enriched by feeding both methane and sulfide was benchmarked against an enrichment of solely MOB. Different CH4:O2 ratios, starting pH values, sulfide levels and nitrogen sources were tested and evaluated for the two enrichments. The MOB-SOB culture gave promising results in terms of both biomass yield (up to 0.07 ± 0.01 g VSS/g CH4-COD) and protein content (up to 73 ± 5% of VSS) at 1500 ppm of equivalent H2S. The latter enrichment was able to grow also under acidic pH (5.8-7.0), but as inhibited outside the optimal CH4:O2 ratio of 2:3. The obtained results show the capability of MOB-SOB mixed-cultures to directly upcycle sulfide-rich biogas into microbial protein potentially suited for feed, food or biobased product applications.

Biowaste upcycling into second-generation microbial protein through mixed-culture fermentation.

Securing a sustainable protein supply at the global level is among the greatest challenges currently faced by humanity. Alternative protein sources, such as second-generation microbial protein (MP), could give rise to innovative circular bioeconomy practices, synthesizing high-value bioproducts through the recovery and upcycling of resources from overabundant biowastes and residues. Within such a multi-feedstock biorefinery scenario, the wide range of microbial pathways and networks that characterize mixed microbial cultures, offers interesting and not yet fully explored advantages over conventional monoculture-based processes. In this review, we combine a comprehensive analysis of waste recovery platforms for second-generation MP production with a critical evaluation of the research gaps and potentials offered by mixed culture-based MP fermentation processes.

How can we possibly resolve the planet's nitrogen dilemma?

Nitrogen is the most crucial element in the production of nutritious feeds and foods. The production of reactive nitrogen by means of fossil fuel has thus far been able to guarantee the protein supply for the world population. Yet, the production and massive use of fertilizer nitrogen constitute a major threat in terms of environmental health and sustainability. It is crucial to promote consumer acceptance and awareness towards proteins produced by highly effective microorganisms, and their potential to replace proteins obtained with poor nitrogen efficiencies from plants and animals. The fact that reactive fertilizer nitrogen, produced by the Haber Bosch process, consumes a significant amount of fossil fuel worldwide is of concern. Moreover, recently, the prices of fossil fuels have increased the cost of reactive nitrogen by a factor of 3 to 5 times, while international policies are fostering the transition towards a more sustainable agro-ecology by reducing mineral fertilizers inputs and increasing organic farming. The combination of these pressures and challenges opens opportunities to use the reactive nitrogen nutrient more carefully. Time has come to effectively recover used nitrogen from secondary resources and to upgrade it to a legal status of fertilizer. Organic nitrogen is a slow-release fertilizer, it has a factor of 2.5 or higher economic value per unit nitrogen as fertilizer and thus adequate technologies to produce it, for instance by implementing photobiological processes, are promising. Finally, it appears wise to start the integration in our overall feed and food supply chains of the exceptional potential of biological nitrogen fixation. Nitrogen produced by the nitrogenase enzyme, either in the soil or in novel biotechnology reactor systems, deserves to have a 'renaissance' in the context of planetary governance in general and the increasing number of people who desire to be fed in a sustainable way in particular.

Valorisation of industrial hemp (Cannabis sativa L.) biomass residues through acidogenic fermentation and co-fermentation for volatile fatty acids production.

In line with the emerging circular bioeconomy paradigm, the present study investigated the valorisation of abundant hemp biomass residues (HBRs) such as hurds (HH) and a mix of leaves and inflorescences (Mix), and other organic wastes (i.e., cheese whey and grape pomace) through the volatile fatty acid (VFA) production in mono- and co-acidogenic fermentation. The highest VFA yields, measured as acetic acid (HAc) per unit of volatile solids (VS), were obtained with the untreated Mix in mono-fermentation (185 ± 57 mg HAc/g VS) and with the combination of Mix and CW in co-fermentation (651 ± 65 mg HAc/g VS), while the highest HAc percentage reached up to 94% of total VFAs. Finally, a preliminary techno-economic evaluation revealed that the mono-fermentation of alkali pretreated HH could lead to the highest revenues among HBRs, reaching up to 710-1810, 618-1577 and 766-3722 €/ha∙year for the production of HAc, single cell protein and polyhydroxybutyrates, respectively.

Direct nitrogen stripping and upcycling from anaerobic digestate during conversion of cheese whey into single cell protein.

The environmental impact of the dairy industry is heavily influenced by the overproduction of nitrogen- and carbon-rich effluents. The present study proposes an innovative process to recover waste contaminated nitrogen from anaerobic digestate while treating excess cheese whey (CW) and producing high-quality, clean single cell protein (SCP). By relying on direct aeration stripping techniques, employing an airflow subsequently used in the aerobic cheese whey fermentation step, the investigated process was able to strip 41-80% of the total ammonium nitrogen (N-NH4+) from liquid digestate. The stripped ammonia gas (NH3) was completely recovered as N-NH4+ in the acidic CW, and further upcycled into SCP having a total protein content of 74.7% and a balanced amino acids profile. A preliminary techno-economic analysis revealed the potential to directly recover and upcycle nitrogen into SCP at costs (4.3-6.3 €·kgN-1) and energetic inputs (90-132 MJ·kgN-1) matching those of conventional feed and nitrogen management processes.

Photovoltaic-driven microbial protein production can use land and sunlight more efficiently than conventional crops.

Population growth and changes in dietary patterns place an ever-growing pressure on the environment. Feeding the world within sustainable boundaries therefore requires revolutionizing the way we harness natural resources. Microbial biomass can be cultivated to yield protein-rich feed and food supplements, collectively termed single-cell protein (SCP). Yet, we still lack a quantitative comparison between traditional agriculture and photovoltaic-driven SCP systems in terms of land use and energetic efficiency. Here, we analyze the energetic efficiency of harnessing solar energy to produce SCP from air and water. Our model includes photovoltaic electricity generation, direct air capture of carbon dioxide, electrosynthesis of an electron donor and/or carbon source for microbial growth (hydrogen, formate, or methanol), microbial cultivation, and the processing of biomass and proteins. We show that, per unit of land, SCP production can reach an over 10-fold higher protein yield and at least twice the caloric yield compared with any staple crop. Altogether, this quantitative analysis offers an assessment of the future potential of photovoltaic-driven microbial foods to supplement conventional agricultural production and support resource-efficient protein supply on a global scale.

Potential of microbial protein from hydrogen for preventing mass starvation in catastrophic scenarios.

Human civilization's food production system is currently unprepared for catastrophes that would reduce global food production by 10% or more, such as nuclear winter, supervolcanic eruptions or asteroid impacts. Alternative foods that do not require much or any sunlight have been proposed as a more cost-effective solution than increasing food stockpiles, given the long duration of many global catastrophic risks (GCRs) that could hamper conventional agriculture for 5 to 10 years. Microbial food from single cell protein (SCP) produced via hydrogen from both gasification and electrolysis is analyzed in this study as alternative food for the most severe food shock scenario: a sun-blocking catastrophe. Capital costs, resource requirements and ramp up rates are quantified to determine its viability. Potential bottlenecks to fast deployment of the technology are reviewed. The ramp up speed of food production for 24/7 construction of the facilities over 6 years is estimated to be lower than other alternatives (3-10% of the global protein requirements could be fulfilled at end of first year), but the nutritional quality of the microbial protein is higher than for most other alternative foods for catastrophes. Results suggest that investment in SCP ramp up should be limited to the production capacity that is needed to fulfill only the minimum recommended protein requirements of humanity during the catastrophe. Further research is needed into more uncertain concerns such as transferability of labor and equipment production. This could help reduce the negative impact of potential food-related GCRs.

Stochasticity in microbiology: managing unpredictability to reach the Sustainable Development Goals.

Pure (single) cultures of microorganisms and mixed microbial communities (microbiomes) have been important for centuries in providing renewable energy, clean water and food products to human society and will continue to play a crucial role to pursue the Sustainable Development Goals. To use microorganisms effectively, microbial engineered processes require adequate control. Microbial communities are shaped by manageable deterministic processes, but also by stochastic processes, which can promote unforeseeable variations and adaptations. Here, we highlight the impact of stochasticity in single culture and microbiome engineering. First, we discuss the concepts and mechanisms of stochasticity in relation to microbial ecology of single cultures and microbiomes. Second, we discuss the consequences of stochasticity in relation to process performance and human health, which are reflected in key disadvantages and important opportunities. Third, we propose a suitable decision tool to deal with stochasticity in which monitoring of stochasticity and setting the boundaries of stochasticity by regulators are central aspects. Stochasticity may give rise to some risks, such as the presence of pathogens in microbiomes. We argue here that by taking the necessary precautions and through clever monitoring and interpretation, these risks can be mitigated.

Decoupling Livestock from Land Use through Industrial Feed Production Pathways.

One of the main challenges for the 21st century is to balance the increasing demand for high-quality proteins while mitigating environmental impacts. In particular, cropland-based production of protein-rich animal feed for livestock rearing results in large-scale agricultural land-expansion, nitrogen pollution, and greenhouse gas emissions. Here we propose and analyze the long-term potential of alternative animal feed supply routes based on industrial production of microbial proteins (MP). Our analysis reveals that by 2050, MP can replace, depending on socio-economic development and MP production pathways, between 10-19% of conventional crop-based animal feed protein demand. As a result, global cropland area, global nitrogen losses from croplands and agricultural greenhouse gas emissions can be decreased by 6% (0-13%), 8% (-3-8%), and 7% (-6-9%), respectively. Interestingly, the technology to industrially produce MP at competitive costs is directly accessible for implementation and has the potential to cause a major structural change in the agro-food system.

Electrochemical Ammonia Recovery from Source-Separated Urine for Microbial Protein Production.

Conventional plant and meat protein production have low nitrogen usage efficiencies and high energy needs. Microbial protein (MP) is an alternative that offers higher nitrogen conversion efficiencies with low energy needs if nitrogen is recovered from a concentrated waste source such as source-separated urine. An electrochemical cell (EC) was optimized for ammonia recovery as NH3/H2 gas mixtures usable for MP production. Undiluted hydrolyzed urine was fed to the caustic-generating cathode compartment for ammonia stripping with redirection to the anode compartment for additional ammonium extraction. Using synthetic urine at 48 A m-2 the nitrogen removal efficiency reached 91.6 ± 2.1%. Tests with real urine at 20 A m-2, achieved 87.1 ± 6.0% and 68.4 ± 14.6% requiring 5.8 and 13.9 kWh kg N-1 recovered, via absorption in acid or MP medium, respectively. Energy savings through accompanying electrolytic H2 and O2 production were accounted for. Subsequently, MP was grown in fed-batch on MP medium with conventional NH4+ or urine-derived NH3 yielding 3.74 ± 1.79 and 4.44 ± 1.59 g CDW L-1, respectively. Dissolution of gaseous NH3 in MP medium maintained neutral pH in the MP reactor preventing caustic addition and thus salt accumulation. Urine-nitrogen could thus be valorized as MP via electrochemical ammonia recovery.

Microbes and the Next Nitrogen Revolution.

The Haber Bosch process is among the greatest inventions of the 20th century. It provided agriculture with reactive nitrogen and ultimately mankind with nourishment for a population of 7 billion people. However, the present agricultural practice of growing crops for animal production and human food constitutes a major threat to the sustainability of the planet in terms of reactive nitrogen pollution. In view of the shortage of directly feasible and cost-effective measures to avoid these planetary nitrogen burdens and the necessity to remediate this problem, we foresee the absolute need for and expect a revolution in the use of microbes as a source of protein. Bypassing land-based agriculture through direct use of Haber Bosch produced nitrogen for reactor-based production of microbial protein can be an inspiring concept for the production of high quality animal feed and even straightforward supply of proteinaceous products for human food, without significant nitrogen losses to the environment and without the need for genetic engineering to safeguard feed and food supply for the generations to come.

Microbial protein: future sustainable food supply route with low environmental footprint.

Microbial biotechnology has a long history of producing feeds and foods. The key feature of today's market economy is that protein production by conventional agriculture based food supply chains is becoming a major issue in terms of global environmental pollution such as diffuse nutrient and greenhouse gas emissions, land use and water footprint. Time has come to re-assess the current potentials of producing protein-rich feed or food additives in the form of algae, yeasts, fungi and plain bacterial cellular biomass, producible with a lower environmental footprint compared with other plant or animal-based alternatives. A major driver is the need to no longer disintegrate but rather upgrade a variety of low-value organic and inorganic side streams in our current non-cyclic economy. In this context, microbial bioconversions of such valuable matters to nutritive microbial cells and cell components are a powerful asset. The worldwide market of animal protein is of the order of several hundred million tons per year, that of plant protein several billion tons of protein per year; hence, the expansion of the production of microbial protein does not pose disruptive challenges towards the process of the latter. Besides protein as nutritive compounds, also other cellular components such as lipids (single cell oil), polyhydroxybuthyrate, exopolymeric saccharides, carotenoids, ectorines, (pro)vitamins and essential amino acids can be of value for the growing domain of novel nutrition. In order for microbial protein as feed or food to become a major and sustainable alternative, addressing the challenges of creating awareness and achieving public and broader regulatory acceptance are real and need to be addressed with care and expedience.

Autotrophic nitrogen assimilation and carbon capture for microbial protein production by a novel enrichment of hydrogen-oxidizing bacteria.

Domestic used water treatment systems are currently predominantly based on conventional resource inefficient treatment processes. While resource recovery is gaining momentum it lacks high value end-products which can be efficiently marketed. Microbial protein production offers a valid and promising alternative by upgrading low value recovered resources into high quality feed and also food. In the present study, we evaluated the potential of hydrogen-oxidizing bacteria to upgrade ammonium and carbon dioxide under autotrophic growth conditions. The enrichment of a generic microbial community and the implementation of different culture conditions (sequenced batch resp. continuous reactor) revealed surprising features. At low selection pressure (i.e. under sequenced batch culture at high solid retention time), a very diverse microbiome with an important presence of predatory Bdellovibrio spp. was observed. The microbial culture which evolved under high rate selection pressure (i.e. dilution rate D = 0.1 h(-1)) under continuous reactor conditions was dominated by Sulfuricurvum spp. and a highly stable and efficient process in terms of N and C uptake, biomass yield and volumetric productivity was attained. Under continuous culture conditions the maximum yield obtained was 0.29 g cell dry weight per gram chemical oxygen demand equivalent of hydrogen, whereas the maximum volumetric loading rate peaked 0.41 g cell dry weight per litre per hour at a protein content of 71%. Finally, the microbial protein produced was of high nutritive quality in terms of essential amino acids content and can be a suitable substitute for conventional feed sources such as fishmeal or soybean meal.

Reduction of selenite to elemental selenium nanoparticles by activated sludge.

Total selenium removal by the activated sludge process, where selenite is reduced to colloidal elemental selenium nanoparticles (BioSeNPs) that remain entrapped in the activated sludge flocs, was studied. Total selenium removal efficiencies with glucose as electron donor (2.0 g chemical oxygen demand (COD) L(-1)) at neutral pH and 30 °C gave 2.9 and 6.8 times higher removal efficiencies as compared to the electron donors lactate and acetate, respectively. Total selenium removal efficiencies of 79 (±3) and 86 (±1) % were achieved in shake flasks and fed batch reactors, respectively, at dissolved oxygen (DO) concentrations above 4.0 mg L(-1) and 30 °C when fed with 172 mg L(-1) (1 mM) Na2SeO3 and 2.0 g L(-1) COD of glucose. Continuously operated reactors operating at neutral pH, 30 °C and a DO >3 mg L(-1) removed 33.98 and 36.65 mg of total selenium per gram of total suspended solids (TSS) at TSS concentrations of 1.3 and 3.0 g L(-1), respectively. However, selenite toxicity to the activated sludge led to failure of a continuously operating activated sludge reactor at the applied loading rates. This suggests that a higher hydraulic retention time (HRT) or different reactor configurations need to be applied for selenium-removing activated sludge processes. Graphical Abstract Scheme representing the possible mechanisms of selenite reduction at high and low DO levels in the activated sludge process.

Can direct conversion of used nitrogen to new feed and protein help feed the world?

The increase in the world population, vulnerability of conventional crop production to climate change, and population shifts to megacities justify a re-examination of current methods of converting reactive nitrogen to dinitrogen gas in sewage and waste treatment plants. Indeed, by up-grading treatment plants to factories in which the incoming materials are first deconstructed to units such as ammonia, carbon dioxide and clean minerals, one can implement a highly intensive and efficient microbial resynthesis process in which the used nitrogen is harvested as microbial protein (at efficiencies close to 100%). This can be used for animal feed and food purposes. The technology for recovery of reactive nitrogen as microbial protein is available but a change of mindset needs to be achieved to make such recovery acceptable.

Resource recovery from used water: the manufacturing abilities of hydrogen-oxidizing bacteria.

Resources in used water are at present mainly destroyed rather than reused. Recovered nutrients can serve as raw material for the sustainable production of high value bio-products. The concept of using hydrogen and oxygen, produced by green or off-peak energy by electrolysis, as well as the unique capability of autotrophic hydrogen oxidizing bacteria to upgrade nitrogen and minerals into valuable microbial biomass, is proposed. Both axenic and mixed microbial cultures can thus be of value to implement re-synthesis of recovered nutrients in biomolecules. This process can become a major line in the sustainable "water factory" of the future.