Green electricity and biowastes via biogas to bulk‐chemicals and fuels: The next move toward a sustainable bioeconomy

To deal with the global challenges of limited fossil resources, climate change, and environmental pollution bioeconomy has been identified globally as a strategic development goal. In this regard, many industrial countries and regions have set very ambiguous targets: e.g. within the EU 25–30% of all chemicals and other industrial products as well as 5–10% of transportation fuels should be bio‐based by 2030. These targets are hardly achievable and not sustainable with presently known bio‐production systems, mainly due to constrains in substrate availability, limited product yield, and high processing costs. Thus, new concepts are desperately needed. Against this background, an innovative and sustainable concept is presented and discussed here. The central idea of the concept is the conversion of organic wastes into a widely usable product—biogas (CO₂ +CH₄)—which is then used as a clean and uniform substrate for the synthesis of bulk‐chemicals and/or fuels, especially by using green electricity from wind and solar. Such a concept (shortened as E&G²C) has the potential to overcome major limitations of known bioproduction systems. Biogas as a substrate of biosynthesis has many unique advantages, including sustainability, efficiency, and flexibility. The use of electricity for biosynthesis with biogas represents an ideal system for efficient bioelectrochemical conversion. Here, the rationale behind the concept is illustrated, its sustainability is underpinned with concrete data, and the realization of the concept is discussed by looking at the possible conversion routes and key issues to be solved. The markets and perspectives provided by the concept E&G²C are also briefly addressed. In conclusion, the concept E&G²C provides a unique and innovative path for the next move toward a real sustainable bioeconomy.     

Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures

Grapevine stilbenes, particularly trans‐resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering‐based strategy to produce resveratrol derivatives using resveratrol‐converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O‐methyltransferase (VvROMT) or human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and VvROMT or HsCYP1B1. Grapevine cell cultures transformed with VvROMT produced pterostilbene, which was detected in both intra‐ and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with HsCYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild‐type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.     

Production of C2–C4 diols from renewable bioresources: new metabolic pathways and metabolic engineering strategies

C2–C4 diols classically derived from fossil resource are very important bulk chemicals which have been used in a wide range of areas, including solvents, fuels, polymers, cosmetics, and pharmaceuticals. Production of C2–C4 diols from renewable resources has received significant interest in consideration of the reducing fossil resource and the increasing environmental issues. While bioproduction of certain diols like 1,3-propanediol has been commercialized in recent years, biosynthesis of many other important C2–C4 diol isomers is highly challenging due to the lack of natural synthesis pathways. Recent advances in synthetic biology have enabled the de novo design of completely new pathways to non-natural molecules from renewable feedstocks. In this study, we review recent advances in bioproduction of C2–C4 diols, focusing on new metabolic pathways and metabolic engineering strategies being developed. We also discuss the challenges and future trends toward the development of economically competitive processes for bio-based diol production.     

Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products

Recent progress in synthetic and systems metabolic engineering technologies has explored the potential of microbial cell factories for the production of industrially relevant bulk and fine chemicals from renewable biomass resources in an eco-friendly manner. Corynebacterium glutamicum, a workhorse for industrial amino acid production, has currently evolved into a promising microbial platform for bioproduction of various natural and non-natural chemicals from renewable feedstocks. Notably, it has been recently demonstrated that metabolically engineered C. glutamicum can overproduce several commercially valuable aromatic and related chemicals such as shikimate, 4-hydroxybenzoate, and 4-aminobenzoate from sugars at remarkably high titer suitable to commercial application. On the other hand, overexpression and/or extension of its endogenous metabolic pathways by integrating heterologous metabolic pathways enabled production of structurally intricate and valuable natural chemicals like plant polyphenols, carotenoids, and fatty acids. In this review, we summarize recent advances in metabolic engineering of C. glutamicum for production of those value-added aromatics and other natural products, which highlights high potential and the versatility of this microbe for bioproduction of diverse chemicals.

     

Isolation,improvement and characterization of an ammonium excreting mutant strain of the heterocytous cyanobacterium,Anabaena variabilis PCC 7937

Besides potential applications in the agriculture field as natural nitrogen fertilizer, N₂-fixing cyanobacteria have recently gained some attentions for new applications linked to the potential production of biologically active molecules or biohydrogen. Ammonium bioproduction is also gaining attention with the potential use of microalgae in biofuels production and the concerns about the increasing needs for nitrogen substrates. This study has investigated some phenotypic traits linked to biomass production and ammonium release in multicellular cyanobacteria, Anabaena variabilis PCC 7937. It confirms that this wild-type strain has no natural ability for ammonium excretion under diazotrophic conditions. A mutant strain, A. variabilis PCC 7937-C9, was obtained after double random mutagenesis treatments with ethyl methane–sulfonate and screening in batch cultures for resistance to the effect of a glutamine synthetase inhibitor, l-methionine-d,l-sulfoximine (MSX). Although significantly characterized by shorter cell filaments, the growth parameters in photobioreactors of the mutant strain cultures were in the same range of values than those of the wild type. In the presence of MSX this strain was shown to produce extracellular ammonium, with specific rates up to 4.9 μmol NH₄⁺ mg Chl a⁻¹ h⁻¹. The efficiency of this strain, estimated by its specific rate of ammonium excretion, was shown to be improved after consecutive batch cultures with increasing concentrations of MSX. Such mutant strains are of potential use for investigating ways to improve extracellular ammonium bioproduction.     

Biosynthesis of butyric acid by Clostridium tyrobutyricum

Butyric acid (C₃H₇COOH) is an important chemical that is widely used in foodstuffs along with in the chemical and pharmaceutical industries. The bioproduction of butyric acid through large-scale fermentation has the potential to be more economical and efficient than petrochemical synthesis. In this paper, the metabolic pathways involved in the production of butyric acid from Clostridium tyrobutyricum using hexose and pentose as substrates are investigated, and approaches to enhance butyric acid production through genetic modification are discussed. Finally, bioreactor modifications (including fibrous bed bioreactor, inner disk-shaped matrix bioreactor, fibrous matrix packed in porous levitated sphere carriers), low-cost feedstocks, and special treatments (including continuous fermentation with cell recycling, extractive fermentation with solvent, using different artificial electron carriers) intended to improve the feasibility of commercial butyric acid bioproduction are summarized.     

Differential separation of thromboxanes from prostaglandins by one and two-dimensional thin layer chromatography

[¹⁴C]-labelled thromboxane B₂ and hydroxy fatty acids were isolated using thin layer and gas chromatographic procedures from human platelets incubated with [1-¹⁴C]-arachidonic acid. A number of TLC solvent systems were evaluated for differential separation of thromboxanes and hydroxy fatty acids from prostaglandins E₂, A₂, D₂ and F_2α. Chromatographic properties in nine different solvent systems are tabulated. Two dimensional TLC procedures suitable for complete resolution of mixtures of these compounds on a single plate were developed. The systems were used to demonstrate conversion of [1-¹⁴C]-arachidonic acid to thromboxane B₂ and prostaglandin E₂ by human lung fibroblasts in tissue culture.     

Bioproduction of prostaglandins in a transgenic liverwort, Marchantia polymorpha

Prostaglandins are biologically active substances used in a wide range of medical treatments. Prostaglandins have been supplied mainly by chemical synthesis; nevertheless, the high cost of prostaglandin production remains a factor. To lower the cost of prostaglandin production, we attempted to produce prostaglandins using a liverwort, Marchantia polymorpha L., which accumulates arachidonic acid, which is known as a substrate of prostaglandins. Here we report the first bioproduction of prostaglandins in plant species by introducing a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla into the liverwort. The transgenic liverworts accumulated prostaglandin F_2α, prostaglandin E₂ and prostaglandin D₂ which were not detected in the wild-type liverwort. Moreover, we succeeded in drastically increasing the bioproduction of prostaglandins using an in vitro reaction system with the extracts of transgenic liverworts.     

Next‐generation bioproduction systems: Cell‐free conversion concepts for industrial biotechnology

Within the last decade, biotechnology gained pace in substituting petro‐based products for the chemical industries. This is visible with the appearance of bio‐based products in the market, from biosurfactants to bio‐based polymers like polylactic acid to bio‐ethylene. These technologies are mainly based on established fermentation technologies fostered by the use of renewable resources, culminating in the establishment of biorefineries that may be connected directly to the existing chemical infrastructure. Besides these large‐scale technologies, the combination of molecular technologies, microfluidic devices, and enzymatic and cell‐free conversions are currently developed to create new bioproduction systems enabling the production of compounds that may not be produced within a cell. This article summarizes some of the current ideas that are currently in development paving the way for a next generation of biotechnology.     

Recent advances in constraint and machine learning-based metabolic modeling by leveraging stoichiometric balances,thermodynamic feasibility and kinetic law formalisms

Understanding the governing principles behind organisms’ metabolism and growth underpins their effective deployment as bioproduction chassis. A central objective of metabolic modeling is predicting how metabolism and growth are affected by both external environmental factors and internal genotypic perturbations. The fundamental concepts of reaction stoichiometry, thermodynamics, and mass action kinetics have emerged as the foundational principles of many modeling frameworks designed to describe how and why organisms allocate resources towards both growth and bioproduction. This review focuses on the latest algorithmic advancements that have integrated these foundational principles into increasingly sophisticated quantitative frameworks.     

Adaptive laboratory evolution and reverse engineering enhances autotrophic growth in Pichia pastoris

Synthetic biology offers several routes for CO₂ conversion into biomass or bio-chemicals, helping to avoid unsustainable use of organic feedstocks, which negatively contribute to climate change. The use of well-known industrial organisms, such as the methylotrophic yeast Pichia pastoris (Komagataella phaffii), for the establishment of novel C1-based bioproduction platforms could wean biotechnology from feedstocks with alternative use in food production. Recently, the central carbon metabolism of P. pastoris was re-wired following a rational engineering approach, allowing the resulting strains to grow autotrophically with a μ_max of 0.008 h⁻¹, which was further improved to 0.018 h⁻¹ by adaptive laboratory evolution. Using reverse genetic engineering of single-nucleotide (SNPs) polymorphisms occurring in the genes encoding for phosphoribulokinase and nicotinic acid mononucleotide adenylyltransferase after evolution, we verified their influence on the improved autotrophic phenotypes. The reverse engineered SNPs lead to lower enzyme activities in putative branching point reactions and in reactions involved in energy balancing. Beyond this, we show how further evolution facilitates peroxisomal import and increases growth under autotrophic conditions. The engineered P. pastoris strains are a basis for the development of a platform technology, which uses CO₂ for production of value-added products, such as cellular biomass, technical enzymes and chemicals and which further avoids consumption of organic feedstocks with alternative use in food production. Further, the identification and verification of three pivotal steps may facilitate the integration of heterologous CBB cycles or similar pathways into heterotrophic organisms.     

Engineered multiple translation initiation sites: a novel tool to enhance protein production in Bacillus licheniformis and other industrially relevant bacteria

Gram-positive bacteria are a nascent platform for synthetic biology and metabolic engineering that can provide new opportunities for the production of biomolecules. However, the lack of standardized methods and genetic parts is a major obstacle towards attaining the acceptance and widespread use of Gram-positive bacterial chassis for industrial bioproduction. In this study, we have engineered a novel mRNA leader sequence containing more than one ribosomal binding site (RBS) which could initiate translation from multiple sites, vastly enhancing the translation efficiency of the Gram-positive industrial strain Bacillus licheniformis. This is the first report elucidating the impact of more than one RBS to initiate translation and enhance protein output in B. licheniformis. We also explored the application of more than one RBS for both intracellular and extracellular protein production in B. licheniformis to demonstrate its efficiency, consistency and potential for biotechnological applications. Moreover, we applied these concepts for use in other industrially relevant Gram-positive bacteria, such as Bacillus subtilis and Corynebacterium glutamicum. In all, a highly efficient and robust broad-host expression element has been designed to strengthen and fine-tune the protein outputs for the use of bioproduction in microbial cell factories.     

Environmental conditions during glycerol bioconversion affect 3-hydroxypropionic acid bioproduction by Limosilactobacillus reuteri DSM 17938

3-Hydroxypropionic acid (3-HP) is a platform molecule whose biological production was carried out by the bacterium Limosilactobacillus reuteri according to a two-step process: first, a growth phase in batch mode on glucose, then a glycerol bioconversion into 3-HP in fed-batch mode. With the objective of improving 3-HP bioproduction, this study aimed at defining the operating conditions during the bioconversion phase that increases the bioproduction performance. A central composite rotatable design allowed testing various pH levels and specific glycerol feeding rates. By establishing response surfaces, optimal conditions have been identified that were different depending on the considered output variable (final 3-HP quantity, 3-HP production yield and production rate). Of them, 3-HP final quantity and 3-HP production yield were maximized at pH 6.0 and at specific glycerol feeding rates of 60 and 55 mg_gly g_CDW⁻¹ h⁻¹, respectively. The specific 3-HP production rate was the highest at the upper limit of the specific substrate feeding rate (80 mg_gly g_CDW⁻¹ h⁻¹) but was not affected by the pH. An additional experiment was carried out at pH 6.0 and a specific glycerol feeding rate of 80 mg_gly g_CDW⁻¹ h⁻¹ to validate the previous observations. In conclusion, the results showed a significant improvement of 3-HP concentration by 13%, of specific production rate by 34% and of 3-HP volumetric productivity by 39%, as compared to the initial values.     

The Imine‐Based COF TpPa‐1 as an Efficient Cooling Adsorbent That Can Be Regenerated by Heat or Light

Adsorption‐based cooling systems, which can be driven by waste heat and solar energy, are promising alternatives to conventional, compression‐based cooling systems, as they demand less energy and emit less CO₂. The performance of adsorption‐based cooling systems relates directly to the performance of the working pairs (sorbent–water). Accordingly, improvement of these systems relies on the continual discovery of new sorbents that enable greater mass exchange while requiring less energy for regeneration. Here, it is proposed that covalent‐organic frameworks (COFs) can replace traditional sorbents for adsorption‐based cooling. In tests mimicking standard operating conditions for industry, the imine‐based COF TpPa‐1 exhibits a regeneration temperature below 65 °C and a cooling coefficient of performance of 0.77 – values which are comparable to those reported for the best metal–organic framework sorbents described to date. Moreover, TpPa‐1 exhibits a photothermal effect and can be regenerated by visible light, thereby opening the possibility for its use in solar‐driven cooling.     

Faster turnover of new soil carbon inputs under increased atmospheric CO2

Rising levels of atmospheric CO₂ frequently stimulate plant inputs to soil, but the consequences of these changes for soil carbon (C) dynamics are poorly understood. Plant‐derived inputs can accumulate in the soil and become part of the soil C pool (“new soil C”), or accelerate losses of pre‐existing (“old”) soil C. The dynamics of the new and old pools will likely differ and alter the long‐term fate of soil C, but these separate pools, which can be distinguished through isotopic labeling, have not been considered in past syntheses. Using meta‐analysis, we found that while elevated CO₂ (ranging from 550 to 800 parts per million by volume) stimulates the accumulation of new soil C in the short term (<1 year), these effects do not persist in the longer term (1–4 years). Elevated CO₂ does not affect the decomposition or the size of the old soil C pool over either temporal scale. Our results are inconsistent with predictions of conventional soil C models and suggest that elevated CO₂ might increase turnover rates of new soil C. Because increased turnover rates of new soil C limit the potential for additional soil C sequestration, the capacity of land ecosystems to slow the rise in atmospheric CO₂ concentrations may be smaller than previously assumed.     

Bi-ionic potentials of bovine lens capsules and collodion membranes

Concentration dependencies of bi-ionic potentials of well-cleaned bovine lens capsules in vitro, of collodion and of modified collodion membranes were studied. The lens capsules have positively fixed charges, and collodion membranes have negatively fixed charges. As these membranes are partially selectively permeable, both co-ions and counter-ions exist in the membrane. However, many studies on bi-ionic potentials have been limited to systems in which the membrane has extreme ionic selectivity and co-ions are completely excluded from the membrane. Experimental results agreed with theoretical values obtained by assuming the common ion concentration to be constant throughout the membrane for systems such as KCl(C)-membrane (θ>0, or θ<0)-NaCl(C), NaNO₃(C)-membrane (θ>0)-NaCl(C) and CaCl₂(C₁)-membrane (θ>0)-NaCl(C₂) (C₂/C₁ = 2), where C is the bulk concentration. The theoretical reliability of this assumption was checked. When both electrolytes in solution were uni-univalent, the ratio of ionic mobilities of two counter-ions (or two co-ions) in all of these membranes was almost the same as the ratio obtained in bulk solution, while the ratio of ionic mobilities of the counter-ion and the co-ion was almost the same as the ratio obtained in bulk solution for the lens capsule, but different in the case of the collodion and modified collodion membranes.     

Bioconversion of nitriles by Candida guilliermondii CCT 7207 cells immobilized in barium alginate

Nitrile degradation by Candida guilliermondii CCT 7207 using free and immobilized cell systems was compared. Different specific growth rates were observed for immobilized (mumax=0.021 h(-1)) and the free cells (mumax=0.029 h(-1)). The maximum specific rate of acetic acid formation was 0.387 h(-1) and 0.266 h(-1) for free and immobilized cells, respectively. Cell adhesion to the support materials was confirmed by scanning electron microscopy. When immobilized, the yeast was able to use high nitrile and amide concentrations (aliphatic and aromatic) as nitrogen sources. The results suggest that C. guilliermondii CCT 7207 presents a physiological pattern potentially useful for the bioremediation of polluted environments or for the bioproduction of amides and organic acid of high commercial value.