Meat substitutes: Resource demands and environmental footprints.

The modern food system is characterized with high environmental impact, which is in many cases associated with increased rates of animal production and overconsumption. The adoption of alternatives to meat proteins (insects, plants, mycoprotein, microalgae, cultured meat, etc.) might potentially influence the environmental impact and human health in a positive or negative way but could also trigger indirect impacts with higher consumption rates. Current review provides a condensed analysis on potential environmental impacts, resource consumption rates and unintended trade-offs associated with integration of alternative proteins in complex global food system in the form of meat substitutes. We focus on emissions of greenhouse gases, land use, non-renewable energy use and water footprint highlighted for both ingredients used for meat substitutes and ready products. The benefits and limitations of meat substitution are highlighted in relation to a weight and protein content. The analysis of the recent research literature allowed us to define issues, that require the attention of future studies.

Kinetic and Stoichiometric Modeling-Based Analysis of Docosahexaenoic Acid (DHA) Production Potential by Crypthecodinium cohnii from Glycerol, Glucose and Ethanol.

Docosahexaenoic acid (DHA) is one of the most important long-chain polyunsaturated fatty acids (LC-PUFAs), with numerous health benefits. Crypthecodinium cohnii, a marine heterotrophic dinoflagellate, is successfully used for the industrial production of DHA because it can accumulate DHA at high concentrations within the cells. Glycerol is an interesting renewable substrate for DHA production since it is a by-product of biodiesel production and other industries, and is globally generated in large quantities. The DHA production potential from glycerol, ethanol and glucose is compared by combining fermentation experiments with the pathway-scale kinetic modeling and constraint-based stoichiometric modeling of C. cohnii metabolism. Glycerol has the slowest biomass growth rate among the tested substrates. This is partially compensated by the highest PUFAs fraction, where DHA is dominant. Mathematical modeling reveals that glycerol has the best experimentally observed carbon transformation rate into biomass, reaching the closest values to the theoretical upper limit. In addition to our observations, the published experimental evidence indicates that crude glycerol is readily consumed by C. cohnii, making glycerol an attractive substrate for DHA production.

From lignin to nylon: Cascaded chemical and biochemical conversion using metabolically engineered Pseudomonas putida.

Cis,cis-muconic acid (MA) is a chemical that is recognized for its industrial value and is synthetically accessible from aromatic compounds. This feature provides the attractive possibility of producing MA from mixtures of aromatics found in depolymerized lignin, the most underutilized lignocellulosic biopolymer. Based on the metabolic pathway, the catechol (1,2-dihydroxybenzene) node is the central element of this type of production process: (i) all upper catabolic pathways of aromatics converge at catechol as the central intermediate, (ii) catechol itself is frequently generated during lignin pre-processing, and (iii) catechol is directly converted to the target product MA by catechol 1,2-dioxygenase. However, catechol is highly toxic, which poses a challenge for the bio-production of MA. In this study, the soil bacterium Pseudomonas putida KT2440 was upgraded to a fully genome-based host for the production of MA from catechol and upstream aromatics. At the core of the cell factories created was a designed synthetic pathway module, comprising both native catechol 1,2-dioxygenases, catA and catA2, under the control of the Pcat promoter. The pathway module increased catechol tolerance, catechol 1,2-dioxygenase levels, and catechol conversion rates. MA, the formed product, acted as an inducer of the module, triggering continuous expression. Cellular energy level and ATP yield were identified as critical parameters during catechol-based production. The engineered MA-6 strain achieved an MA titer of 64.2 g L-1 from catechol in a fed-batch process, which repeatedly regenerated the energy levels via specific feed pauses. The developed process was successfully transferred to the pilot scale to produce kilograms of MA at 97.9% purity. The MA-9 strain, equipped with a phenol hydroxylase, used phenol to produce MA and additionally converted o-cresol, m-cresol, and p-cresol to specific methylated variants of MA. This strain was used to demonstrate the entire value chain. Following hydrothermal depolymerization of softwood lignin to catechol, phenol and cresols, MA-9 accumulated 13 g L-1 MA and small amounts of 3-methyl MA, which were hydrogenated to adipic acid and its methylated derivative to polymerize nylon from lignin for the first time.

Utilization of protein-rich residues in biotechnological processes.

A drawback of biotechnological processes, where microorganisms convert biomass constituents, such as starch, cellulose, hemicelluloses, lipids, and proteins, into wanted products, is the economic feasibility. Particularly the cost of nitrogen sources in biotechnological processes can make up a large fraction of total process expenses. To further develop the bioeconomy, it is of considerable interest to substitute cost-intensive by inexpensive nitrogen sources. The aim of this mini-review was to provide a comprehensive insight of utilization methods of protein-rich residues, such as fish waste, green biomass, hairs, and food waste. The methods described include (i) production of enzymes, (ii) recovery of bioactive compounds, and/or (iii) usage as nitrogen source for microorganisms in biotechnological processes. In this aspect, the utilization of protein-rich residues, which are conventionally considered as waste, allows the development of value-adding processes for the production of bioactive compounds, biomolecules, chemicals, and materials.

Valorization of industrial waste and by-product streams via fermentation for the production of chemicals and biopolymers.

The transition from a fossil fuel-based economy to a bio-based economy necessitates the exploitation of synergies, scientific innovations and breakthroughs, and step changes in the infrastructure of chemical industry. Sustainable production of chemicals and biopolymers should be dependent entirely on renewable carbon. White biotechnology could provide the necessary tools for the evolution of microbial bioconversion into a key unit operation in future biorefineries. Waste and by-product streams from existing industrial sectors (e.g., food industry, pulp and paper industry, biodiesel and bioethanol production) could be used as renewable resources for both biorefinery development and production of nutrient-complete fermentation feedstocks. This review focuses on the potential of utilizing waste and by-product streams from current industrial activities for the production of chemicals and biopolymers via microbial bioconversion. The first part of this review presents the current status and prospects on fermentative production of important platform chemicals (i.e., selected C2-C6 metabolic products and single cell oil) and biopolymers (i.e., polyhydroxyalkanoates and bacterial cellulose). In the second part, the qualitative and quantitative characteristics of waste and by-product streams from existing industrial sectors are presented. In the third part, the techno-economic aspects of bioconversion processes are critically reviewed. Four case studies showing the potential of case-specific waste and by-product streams for the production of succinic acid and polyhydroxyalkanoates are presented. It is evident that fermentative production of chemicals and biopolymers via refining of waste and by-product streams is a highly important research area with significant prospects for industrial applications.

Heterotrophic growth of Galdieria sulphuraria on residues from aquaculture and fish processing industries.

The study aimed at zero-waste utilization of fish processing streams for cultivation of microalgae Galdieria sulphuraria. Wastewater from a fish processing facility, slam (mix of used fish feed and faeces), and dried pellet (sediments after enzymatic hydrolysis of rainbow trout) were investigated as potential sources of carbon, nitrogen, and phosphate for cultivation of G. sulphuraria. The pellet extract was found to support the growth of G. sulphuraria when appropriate diluted, at concentrations below 40 % (v/v). It was revealed that wastewater does not impact the growth negatively, however free amino nitrogen and carbon sources need to be supplied from another source. Therefore, only proteolyzed pellet extract (20 %, v/v) was selected for upscaling and a biomass concentration of 80 g L-1 (growth rate was 0.72 day-1) was achieved in a non-sterile fed-batch culture. Even though biomass was produced under non-sterile conditions no pathogens such as Salmonella sp. could be detected.

Transformation of Seafood Side-Streams and Residuals into Valuable Products.

Seafood processing creates enormous amounts of side-streams. This review deals with the use of seafood side-streams for transformation into valuable products and identifies suitable approaches for making use of it for different purposes. Starting at the stage of catching fish to its selling point, many of the fish parts, such as head, skin, tail, fillet cut-offs, and the viscera, are wasted. These parts are rich in proteins, enzymes, healthy fatty acids such as monounsaturated and polyunsaturated ones, gelatin, and collagen. The valuable biochemical composition makes it worth discussing paths through which seafood side-streams can be turned into valuable products. Drawbacks, as well as challenges of different aquacultures, demonstrate the importance of using the various side-streams to produce valuable compounds to improve economic performance efficiency and sustainability of aquaculture. In this review, conventional and novel utilization approaches, as well as a combination of both, have been identified, which will lead to the development of sustainable production chains and the emergence of new bio-based products in the future.

Effects of phosphorous, nitrogen, and carbon limitation on biomass composition in batch and continuous flow cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii.

We have used phosphate, nitrogen, or carbon limited batch and continuous flow cultures to study how growth and biochemical composition of the dinoflagellate Crypthecodinium cohnii CCMP 316 is affected by nutrient limitation. Specific contents of phosphorous, proteins, and starch were differently affected by nutrient limitation. The specific phosphorous content in C. cohnii varied 10-20 times depending on phosphate availability in the medium. When phosphate was available it was taken up in excess and stored to be re-utilized during phosphate limitation. The specific protein content varied twofold. At most conditions, proteins made up 12-15% of the biomass dry weight but when cells were nitrogen limited, the specific protein content was only half this value. Floridean starch was the major cell constituent of C. cohnii accounting for 40-50% of the biomass dry weight. Only during carbon limitation did the specific starch content decrease. In contrast was the specific lipid content almost unaffected by nutrient availability and lipids accounted for 12-15% of the biomass dry weight irrespectively of which nutrient that was limiting. Lipid production does therefore not depend on nutrient limitation in C. cohnii and lipids are produced even by carbon limited cells. Cultures grown under phosphate limitation resulted in formation of cells with maximal specific contents of all the three major cell constituents; starch, lipid, and protein.

An automated, modular system for organic waste utilization using heterotrophic alga Galdieria sulphuraria: Design considerations and sustainability.

Large amounts of food are wasted and valuable contents are not utilized completely. Methods to process such wastes into biomass of defined composition automatically and in decentralized locations are lacking. Thus, this study presents a modular design for residue utilization and continuous production of the heterotrophic alga Galdieria sulphuraria. A life cycle and economic assessment are carried out on the hypothetical design to define whether the proposed system can be ecologically and economically viable. Producing one kg of dried biomass would cost 4.38 € and be associated with 3.8 kg CO2 eq emitted, 69.9 MJ of non-renewable energy use, and 0.09 m2 of land occupation. Sustainability is comparable to conventional protein sources, with further improvement foreseen through avoidance of drying. These results demonstrate how circular bioeconomy potentials of residues could be realized using heterotrophic G. sulphuraria. It highlights key issues of developing an environmentally and economically sustainable concept.

Quantification of amino acids in fermentation media by isocratic HPLC analysis of their α-hydroxy acid derivatives.

In this paper we describe a novel method for quantification of amino acids. First, α-hydroxy acid derivatives of amino acids were formed after reaction with dinitrogen trioxide by the van Slyke reaction. Second, the α-hydroxy acid derivatives were separated on an Aminex HPX-87H column (Bio-Rad) eluted isocratically with 5 mM H(2)SO(4) and quantified by refractive index detection. We were able to measure the reaction products of 13 of the 20 classical amino acids: glycine, l-alanine, l-valine, l-leucine, l-isoleucine, l-methionine, l-serine, l-threonine, l-asparagine, l-glutamine, l-aspartic acid, l-glutamic acid, and l-proline. We obtained linear relationships between the product peak areas and initial amino acid concentration, whereby the concentrations of these amino acids could be quantified on the basis of the quantification of their products. The method can be used to analyze amino acids in parallel with other small molecules, such as sugars or short chain fatty acids, and was used for parallel quantification of glycine, l-alanine, or l-glutamic acid, and glucose uptake in cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii . The method can also be used to quantify other amines, as demonstrated by detection of Tris (2-amino-2-(hydroxymethyl)propane-1,3-diol).

Life cycle assessment of hetero- and phototrophic as well as combined cultivations of Galdieria sulphuraria.

Microalgae cultivation for food purposes could have high environmental impacts. The study performed life cycle assessment (LCA) of hypothetical model combining phototrophic and heterotrophic cultivations, exchanging produced gases (carbon dioxide from heterotrophic and oxygen from autotrophic) as a potential strategy to reduce the environmental impact of microalgae cultivation. The LCA indicated that the production of Galdieria sulphuraria in a combined cultivation system has environmental benefits compared with the separate phototrophic cultivation and an almost twice lower carbon footprint than phototrophic cultivation. The benefits are based on the lower volume of culture broth and consequently reduced energy demand as well as less demanding wastewater treatment of the heterotrophic cultivation. Such combination of cultivation activities could be recommended to the producers dealing with phototrophic cultivation as a sustainable strategy for the environmental impact reduction.

Heterotrophic cultivation of Galdieria sulphuraria under non-sterile conditions in digestate and hydrolyzed straw.

Non-sterile heterotrophic cultivation of Galdieria sulphuraria in presence of digestate as well as straw after hydrolysis was investigated. G. sulphuraria can be grown in pure digestate at rates of 0.9 day-1 with glucose. However, a proteolytic treatment of digestate resulted in increased growth rates (1.2 day-1) and doubled cell concentrations. Furthermore, G. sulphuraria can utilize glucose obtained after straw hydrolysis. Biomass yields in glucose limited cultures were around 0.9 g per g glucose, while only 0.2 g biomass was formed per g glucose in glucose sufficient cultures. Biomass composition (w/w) of G. sulphuraria grown in digestate supplemented with straw hydrolysate consisted of 20% carbohydrates, 37% proteins and 3% lipids. This study revealed the potential to utilize agricultural waste streams to form algal biomass rich in proteins and may pave the way to novel utilization strategies to be implemented in rural areas.

Cultivation of the heterotrophic microalga Galdieria sulphuraria on food waste: A Life Cycle Assessment.

The aim of this study was to perform a Life Cycle Assessment of a production process of 1 kg dry algal biomass powder (Galdieria sulphuraria) with 27 % (w/w) protein content for human consumption for optimizing the production regarding global warming potential and resource efficiency in combination with food waste utilization. It was investigated, underpinned by a comparison of the use of conventional glucose, whether and to what extent the environmental impact/global warming potential can be reduced by changing to food waste hydrolysate and how this can lead to a more sustainable use of resources and a sustainable development. Overall, the results showed that hydrolysis, along with freeze-drying, caused most of the overall impact. The carbon footprint associated with the use of hydrolyzed food waste was 11% higher than using conventional glucose and supplementary nutrients mainly driven by the high demand of energy for hydrolysis.

Bioremediation of chlorinated pesticide-contaminated soil using anaerobic sludges and surfactant addition.

Methanogenic granular sludge and wastewater fermented sludge were used as inocula for batch tests of anaerobic bioremediation of chlorinated pesticide contaminated soil. Results obtained for both types of biomass were similar: 80 to over 90% of gamma -hexachlorocyclohexane (gamma-HCH), 1,1,1-trichloro-2,2-bis-(4-methoxyphenyl)ethane (methoxychlor) and 1,1,1-trichloro-2,2-bis-(4-chlorophenyl)ethane (DDT) removed in 4-6 weeks. Residual fractions of these pesticides persisted till the end of the 16-week experiment. DDT was degraded through 1,1-dichloro-2,2-bis-(4-chlorophenyl)ethane (DDD). Accumulation of this product corresponded stoichiometrically only to 34-53% of removed DDT, supposedly due to its further transformations, finally resulting in formation of detected 4,4'-dichlorobenzophenone (DBP). Addition of 0.5 mM Tween 80 nonionic surfactant resulted in about a twofold decrease of gamma -HCH and methoxychlor residual concentrations, as well as considerably lower DDD accumulation (7-29%) and higher DBP production. However, 1.25 mM dose of this surfactant applied together with granular sludge brought DDD levels back to that observed for treatments with the sludge alone, also impairing DBP formation.

Green Chemistry and Its Contribution to Industrial Biotechnology.

Sustainable chemistry is a broad framework that starts with the function that a chemical product is offering. Not only chemical but also economic and ethical aspects come into focus throughout the complete lifecycle of chemical products. Green chemistry is an important building block for sustainable chemistry and addresses the issue of greener synthesis and, to a certain degree, the more benign properties of chemicals. The principles of green chemistry clearly aim at making chemical reactions and processes more environmentally friendly. Aspects such as atom efficiency, energy efficiency, harmless reactants, renewable resources, and pollution prevention are considered. Despite the progress made toward a "greener" chemistry, biotechnological processes, as processes for the conversion of biomass into value-added products, have not been properly adapted to new developments. Processes used in industrial biotechnology are predominantly linear. This review elaborates on the potential contributions of green chemistry to industrial biotechnology and vice versa. Examples are presented of how green chemistry and biotechnology can be connected to make substrate supply, upstream and downstream processing, and product formation more sustainable. The chapter ends with a case study of adipic acid production from lignin to illustrate the importance of a strong connection between green chemistry and biotechnology.

Estimation of the economy of heterotrophic microalgae- and insect-based food waste utilization processes.

An estimation of the economy of Hermetia illucens and Chlorella pyrenoidosa cultivations as food waste treatment with benefits was carried out. For both organisms, a process scale was assumed to treat 56.3 t of wet food waste per day, which is equivalent to the amount of food waste appearing in a catchment area of 141,000 inhabitants. Using hypothetical insect and heterotrophic microalgae cultivation processes, a daily production of 3.64 t and 7.14 t dried biomass, respectively, can be achieved. For the cultivation of H. illucens, equipment and daily operational costs were estimated at 79,358.15 € and 5,281.56 €, respectively. Equipment and operational costs for the C. pyrenoidosa cultivation was 50 and 6 times higher, respectively. The higher costs reflect the more complex and advanced process compared to H. illucens cultivation. The internal return rate for a plant lifetime of 20 times revealed an economic benefit when C. pyrenoidosa biomass is produced. Nevertheless, both processes were found economically feasible when dried biomass is directly commercialized as food without any further downstream processing. However, extraction and purification of special chemicals, such as unsaturated fatty acids and pigments, can significantly increase the revenue.

Continuous pretreatment, hydrolysis, and fermentation of organic residues for the production of biochemicals.

Agricultural residues pose a valuable resource. Through microbial fermentations, a variety of products can be obtained, ranging from fuels to platform chemicals. Depending on the nature of the organic residue, pretreatment and hydrolysis are needed prior to fermentation in order to release fermentable sugars. Continuous set-ups are common for the production of methane or ethanol from lignocellulosic biomass, however, this does not apply for the fermentative generation of biochemicals, an approach that conserves chemical functionality present in biomass. Certainly, continuous set-ups could beneficially contribute to bioeconomy by providing techniques allowing the production of biochemicals in a sustainable and efficient way. This review summarizes research conducted on the continuous pretreatment, hydrolysis, and fermentation of lignocellulosic biomass, and particularly towards the production of the biobased molecules: Succinic and lactic acid.

Techniques to Control Microbial Contaminants in Nonsterile Microalgae Cultivation.

The aim of this mini-review with own results was an identification of techniques to suppress the growth of microbial contaminants under photo- and mixotrophic conditions. Techniques identified are the modification of environmental conditions, such as pH, oxygen, and nutrient concentrations, as well as the application of pulsed electric field, ultrasonication, and surfactants. In phototrophic cultivations, the mentioned techniques result in a decrease of number of predatory cells, but not in a complete removal. Measures to suppress the growth of contaminations (e.g., bacteria and fungi) in mixotrophic cultivations could not be identified. The co-cultivation of algae and fungi, however, was found to be beneficial for the utilization of unusual carbon compounds (e.g., phenolic compounds).

Non-sterile fermentation of food waste with indigenous consortium and yeast - Effects on microbial community and product spectrum.

This work presents examples of non-sterile mixed culture fermentation of food waste with a cultivated indigenous consortium (IC) gained from food waste, which produces lactic and acetic acids, combined with Saccharomyces cerevisiae, which produces ethanol. All results are flanked by microbial analysis to monitor changes in microbial community. At pH 6 and inoculated with yeast or IC, or both mixed sugars conversion was equal to 71%, 51%, or 67%, respectively. Under pH unregulated conditions metabolic yields were 71%, 67%, or up to 81%. While final titer of acetic acid was not affected by pH (100-200 mM), ethanol and lactic acid titers were. Using mixed culture and pH 6, sugars were almost equally used for formation of ethanol and lactic acid (400-500 mM). However, under pH unregulated conditions 80% of the substrate was converted into ethanol (900-1000 mM).

Assessment of upstream bioprocessing.

Depending on the purpose and use, bioprocesses are carried out in order to reduce, maintain or increase the molar O/C ratio of biomass as the initial substrate. Cascade use considers the holistic and efficient use of biomass. In the current debate of biomass use, however, one may admit that an efficient use of biomass can further be based on the maintenance of initially present molar O/C ratio and functionality. In this regard, what compound should be formed that possesses highest functionality and similar molar O/C ratio as the substrate? How much energy should be spent on bioprocesses for the conversion of biomass under aerobic or anaerobic conditions? This study discusses and contributes to the efficiency assessment of aerobic and anaerobic bioprocesses based on chemical functionality and molar O/C ratio and their scale-depended energy need for creating appropriate environmental conditions for biological agents.