Toward optimal use of biomass as carbon source for chemical bioproduction.

Biomass is widely identified as a promising, renewable replacement for fossil feedstocks in the production of energy, fuels, and chemicals. However, the sustainable supply of biomass is limited. Economic and ecological criteria support prioritization of biomass as a carbon source for organic chemicals; however, utilization for energy currently dominates. Therefore, to optimize the use of available biomass feedstock, biorefining development must focus on high carbon efficiencies and enabling the conversion of all biomass fractions, including lignin and fermentation-derived CO2. Additionally, novel technological platforms should allow the incorporation of nontraditional, currently underutilized carbon feedstocks (e.g. manure) into biorefining processes. To this end, funneling of waste feedstocks to a single product (e.g. methane) and subsequent conversion to chemicals is a promising approach.

Towards Net Zero Greenhouse Gas Emissions in the Energy and Chemical Sectors in Switzerland and Beyond - A Review.

In today's societies, climate-damaging and finite fossil resources such as oil and natural gas serve a dual purpose as energy source and as carbon source for chemicals and plastics. To respond to the finite availability and to meet international climate goals, a change to a renewable energy and raw material basis is inevitable and represents a highly complex task. In this review, we assess possible technology paths for Switzerland to reach these goals. First, we provide an overview of Switzerland's current energy demand and discuss possible renewable technologies as well as proposed scenarios to defossilize the current energy system. In here, electric vehicles and heat pumps are key technologies, whereas mainly photovoltaics replace nuclear power to deliver clean electricity. The production of chemicals also consumes fossil resources and for Switzerland, the oil demand for imported domestically used chemicals and plastics corresponds to around 20% of the current energetic oil demand. Thus, we additionally summarize technologies and visions for a sustainable chemical sector based on the renewable carbon sources biomass, CO2 and recycled plastic. As biomass is the most versatile renewable energy and carbon source, although with a limited availability, aspects and proposed strategies for an optimal use are discussed.

A heterogeneous microbial consortium producing short-chain fatty acids from lignocellulose.

Microbial consortia are a promising alternative to monocultures of genetically modified microorganisms for complex biotransformations. We developed a versatile consortium-based strategy for the direct conversion of lignocellulose to short-chain fatty acids, which included the funneling of the lignocellulosic carbohydrates to lactate as a central intermediate in engineered food chains. A spatial niche enabled in situ cellulolytic enzyme production by an aerobic fungus next to facultative anaerobic lactic acid bacteria and the product-forming anaerobes. Clostridium tyrobutyricum, Veillonella criceti, or Megasphaera elsdenii were integrated into the lactate platform to produce 196 kilograms of butyric acid per metric ton of beechwood. The lactate platform demonstrates the benefits of mixed cultures, such as their modularity and their ability to convert complex substrates into valuable biochemicals.

Impacts of biofilms on the conversion of cellulose.

Lignocellulose is a widely available renewable carbon source and a promising feedstock for the production of various chemicals in biorefineries. However, its recalcitrant nature is a major hurdle that must be overcome to enable economic conversion processes. Deconstruction of lignocellulose is part of the global carbon cycle, and efficient microbial degradation systems have evolved that might serve as models to improve commercial conversion processes. Biofilms-matrix encased, spatially organized clusters of microbial cells and the predominating lifestyle in nature-have been recognized for their essential role in the degradation of cellulose in nature, e.g., in soils or in the digestive tracts of ruminant animals. Cellulolytic biofilms allow for a high concentration of enzymes at the boundary layer between the solid substrate and the liquid phase and the more complete capture of hydrolysis products directly at the hydrolysis site, which is energetically favorable. Furthermore, enhanced expression of genes for carbohydrate active enzymes as a response to the attachment on solid substrate has been demonstrated for cellulolytic aerobic fungi and anerobic bacteria. In natural multispecies biofilms, the vicinity of different microbial species allows the creation of efficient food webs and synergistic interactions thereby, e.g., avoiding the accumulation of inhibiting metabolites. In this review, these topics are discussed and attempts to realize the benefits of biofilms in targeted applications such as the consolidated bioprocessing of lignocellulose are highlighted. KEY POINTS: Multispecies biofilms enable efficient lignocellulose destruction in the biosphere. Cellulose degradation by anaerobic bacteria often occurs by monolayered biofilms. Fungal biofilms immobilize enzymes and substrates in an external digestion system. Surface attached cultures typically show higher expression of cellulolytic enzymes.

Engineering of ecological niches to create stable artificial consortia for complex biotransformations.

The design of controllable artificial microbial consortia has attracted considerable interest in recent years to capitalize on the inherent advantages in comparison to monocultures such as the distribution of the metabolic burden by division of labor, the modularity and the ability to convert complex substrates. One promising approach to control the consortia composition, function and stability is the provision of defined ecological niches fitted to the specific needs of the consortium members. In this review, we discuss recent examples for the creation of metabolic niches by biological engineering of resource partitioning and syntrophic interactions. Moreover, we introduce a complementing process engineering approach to provide defined spatial niches with differing abiotic conditions (e.g. O2, T, light) in stirred tank reactors harboring biofilms. This enables the co-cultivation of microorganisms with non-overlapping abiotic requirements and the control of the strain ratio in consortia characterized by substrate competition.

Two-stage steam explosion pretreatment of softwood with 2-naphthol as carbocation scavenger.

BACKGROUND:  Lignocellulosic biomass is considered as a potential source for sustainable biofuels. In the conversion process, a pretreatment step is necessary in order to overcome the biomass recalcitrance and allow for sufficient fermentable sugar yields in enzymatic hydrolysis. Steam explosion is a well known pretreatment method working without additional chemicals and allowing for efficient particle size reduction. However, it is not effective for the pretreatment of softwood and the harsh conditions necessary to achieve a highly digestible cellulose fraction lead to the partial degradation of the hemicellulosic sugars. Previous studies showed that the autohydrolysis pretreatreatment of softwood can benefit from the addition of 2-naphthol. This carbocation scavenger prevents lignin repolymerisation leading to an enhanced glucose yield in the subsequent enzymatic hydrolysis. RESULTS:  In order to prevent the degradation of the hemicellulose, we investigated in this study a two-stage 2-naphthol steam explosion pretreatment. In the first stage, spruce wood is pretreated at a severity which is optimal for the autocatalytic hydrolysis of the hemicellulose. The hydrolyzate containing the solubilized sugars is withdrawn from the reactor and the remaining solids are pretreated with different amounts of 2-naphthol in a second stage at a severity that allows for high glucose yields in enzymatic hydrolysis. The pretreated spruce was subjected to enzymatic hydrolysis and to simultaneous saccharification and fermentation (SSF). In the first stage, the maximal yield of hemicellulosic sugars was 47.5% at a pretreatment severity of log R 0 = 3.75 at 180 °C. In the second stage, a 2-naphthol dosage of 0.205 mol/mol lignin C9-unit increased the ethanol yield in SSF with a cellulose loading of 1% using the whole second stage pretreatment slurry by 17% from 73.6% for the control without 2-naphthol to 90.4%. At a higher solid loading corresponding to 5% w/w cellulose, the yields decreased due to higher concentrations of residual 2-naphthol in the biomass and the pretreatment liquor, but also due to higher concentrations of potential inhibitors like HMF, furfural and acetic acid. Experiments with washed solids, vacuum filtered solids and the whole slurry showed that residual 2-naphthol can inhibit the fermentation as a single inhibitor but also synergistically together with HMF, furfural and acetic acid. CONCLUSIONS:  This work shows that a two-stage pretreatment greatly enhances the recovery of hemicellulosic sugars from spruce wood. The presence of 2-naphthol in the second pretreatment stage can enhance the ethanol yield in SSF of steam explosion pretreated softwood at low cellulose concentrations of 1% w/w. However, with higher solid loadings of 5% w/w cellulose, the ethanol yields were in general lower due to the solid effect and a synergistic inhibition of HMF, furfural, acetic acid with residual 2-naphthol. The concentration of residual 2-naphthol tolerated by the yeast decreased with increasing concentrations of HMF, furfural, and acetic acid.

Consolidated bioprocessing of lignocellulosic biomass to lactic acid by a synthetic fungal-bacterial consortium.

Consolidated bioprocessing (CBP) of lignocellulosic feedstocks to platform chemicals requires complex metabolic processes, which are commonly executed by single genetically engineered microorganisms. Alternatively, synthetic consortia can be employed to compartmentalize the required metabolic functions among different specialized microorganisms as demonstrated in this work for the direct production of lactic acid from lignocellulosic biomass. We composed an artificial cross-kingdom consortium and co-cultivated the aerobic fungus Trichoderma reesei for the secretion of cellulolytic enzymes with facultative anaerobic lactic acid bacteria. We engineered ecological niches to enable the formation of a spatially structured biofilm. Up to 34.7 gL-1 lactic acid could be produced from 5% (w/w) microcrystalline cellulose. Challenges in converting pretreated lignocellulosic biomass include the presence of inhibitors, the formation of acetic acid and carbon catabolite repression. In the CBP consortium hexoses and pentoses were simultaneously consumed and metabolic cross-feeding enabled the in situ degradation of acetic acid. As a result, superior product purities were achieved and 19.8 gL-1 (85.2% of the theoretical maximum) of lactic acid could be produced from non-detoxified steam-pretreated beech wood. These results demonstrate the potential of consortium-based CBP technologies for the production of high value chemicals from pretreated lignocellulosic biomass in a single step.

Enhanced simultaneous saccharification and fermentation of pretreated beech wood by in situ treatment with the white rot fungus Irpex lacteus in a membrane aerated biofilm reactor.

The aim of the present study was to investigate the combination of steam pretreatment and biological treatment with lignin degrading fungal strains in order to enable efficient bioprocessing of beech wood to ethanol. In a sequential process of steam and fungal pretreatment followed by enzymatic hydrolysis, Irpex lacteus almost doubled the glucose yield for mildly pretreated beech wood, but could not improve yields for more severely pretreated substrates. However, when simultaneous saccharification and fermentation is combined with in situ I. lacteus treatment, which is enabled by the application of a membrane aerated biofilm reactor, ethanol yields of optimally steam pretreated beech could be improved from 65 to 80%. Generally, in situ fungal treatment during bioprocessing of lignocellulose is an interesting method to harness the versatile abilities of white rot fungi.

Biochemical Conversion Processes of Lignocellulosic Biomass to Fuels and Chemicals - A Review.

Lignocellulosic biomass - such as wood, agricultural residues or dedicated energy crops - is a promising renewable feedstock for production of fuels and chemicals that is available at large scale at low cost without direct competition for food usage. Its biochemical conversion in a sugar platform biorefinery includes three main unit operations that are illustrated in this review: the physico-chemical pretreatment of the biomass, the enzymatic hydrolysis of the carbohydrates to a fermentable sugar stream by cellulases and finally the fermentation of the sugars by suitable microorganisms to the target molecules. Special emphasis in this review is put on the technology, commercial status and future prospects of the production of second-generation fuel ethanol, as this process has received most research and development efforts so far. Despite significant advances, high enzyme costs are still a hurdle for large scale competitive lignocellulosic ethanol production. This could be overcome by a strategy termed 'consolidated bioprocessing' (CBP), where enzyme production, enzymatic hydrolysis and fermentation is integrated in one step - either by utilizing one genetically engineered superior microorganism or by creating an artificial co-culture. Insight is provided on both CBP strategies for the production of ethanol as well as of advanced fuels and commodity chemicals.

Application of a slurry feeder to 1 and 3 stage continuous simultaneous saccharification and fermentation of dilute acid pretreated corn stover.

Continuous operation is often chosen for conceptual designs of biological processing of cellulosic biomass to ethanol to achieve higher volumetric productivities. Furthermore, continuous stirred tank reactors (CSTR) can handle higher solids concentrations than possible in batch mode due to broth thinning at partial conversion in a continuous fermentor. However, experience and literature data are very limited for continuous simultaneous saccharification and fermentation (cSSF) processes. In this work, a slurry feed system was developed and applied to a 3-stage bench-scale cSSF train to convert pretreated corn stover to ethanol and determine the effects of dilution rate and number of fermentation vessels on overall volumetric productivity. The highest productivity of 0.4gL(-1)h(-1) was achieved in a single cSSF vessel with an 8h residence time. Furthermore, productivity at identical total residence times was 12% higher for operation with 3 cSSF stages than for a single CSTR stage for pretreated corn stover.

Co-hydrolysis of hydrothermal and dilute acid pretreated populus slurries to support development of a high-throughput pretreatment system.

BACKGROUND: The BioEnergy Science Center (BESC) developed a high-throughput screening method to rapidly identify low-recalcitrance biomass variants. Because the customary separation and analysis of liquid and solids between pretreatment and enzymatic hydrolysis used in conventional analyses is slow, labor-intensive and very difficult to automate, a streamlined approach we term 'co-hydrolysis' was developed. In this method, the solids and liquid in the pretreated biomass slurry are not separated, but instead hydrolysis is performed by adding enzymes to the whole pretreated slurry. The effects of pretreatment method, severity and solids loading on co-hydrolysis performance were investigated. RESULTS: For hydrothermal pretreatment at solids concentrations of 0.5 to 2%, high enzyme protein loadings of about 100 mg/g of substrate (glucan plus xylan) in the original poplar wood achieved glucose and xylose yields for co-hydrolysis that were comparable with those for washed solids. In addition, although poplar wood sugar yields from co-hydrolysis at 2% solids concentrations fell short of those from hydrolysis of washed solids after dilute sulfuric acid pretreatment even at high enzyme loadings, pretreatment at 0.5% solids concentrations resulted in similar yields for all but the lowest enzyme loading. CONCLUSIONS: Overall, the influence of severity on susceptibility of pretreated substrates to enzymatic hydrolysis was clearly discernable, showing co-hydrolysis to be a viable approach for identifying plant-pretreatment-enzyme combinations with substantial advantages for sugar production.

The effect of bovine serum albumin on batch and continuous enzymatic cellulose hydrolysis mixed by stirring or shaking.

Bovine serum albumin (BSA) was applied as a model non-catalytic protein to enzymatic hydrolysis of Avicel and dilute acid pretreated corn stover at different reaction conditions to improve the understanding of its ability to enhance cellulose hydrolysis. Addition of BSA improved the 72 h hydrolysis yields in shake flasks by up to 26% for both substrates by reducing de-activation of the exoglucanases and by facilitating reductions in particle size and crystallinity during a magnetically stirred pre-incubation step. The enzyme stabilizing effect of BSA addition was most striking for batch hydrolysis in a stirred tank reactor, with glucose yields increasing by 76% after 72 h for Avicel and by 40% after 145 h for corn stover. Application of BSA to continuous hydrolysis for a mean residence time of 24h gave 33% and 40% higher glucose yields for corn stover and Avicel compared to the controls.

Engineering of a high-throughput screening system to identify cellulosic biomass, pretreatments, and enzyme formulations that enhance sugar release.

The recalcitrance of cellulosic biomass, the only abundant, sustainable feedstock for making liquid fuels, is a primary obstacle to low cost biological processing, and development of more easily converted plants and more effective enzymes would be of great benefit. Because no single parameter describes recalcitrance, superior variants can only be identified by measuring sugar release from plants subjected to pretreatment and enzymatic hydrolysis. However, genetic modifications of plants coupled with molecular engineering of deconstruction proteins and definition of pretreatment conditions create a very large sample set, and previous methods for biomass pretreatment at elevated temperatures and pressures prevented use of a fully integrated high-throughput (HTP) screening pipeline. Herein, we report on the engineering of a novel HTP pretreatment system employing a 96 well-plate format that withstands extreme pretreatment conditions for rapid screening of biomass-enzyme-pretreatment combinations. This includes the development of new approaches to steam heating and water quenching the system that result in much faster heat up and cool down than previously possible and show consistent temperature histories across the multiwell plate. Coupled pretreatment and enzymatic hydrolysis performance of the well plate pretreatment system is shown to be consistent among the many wells in the device and also with performance of conventional tubular reactors.

Review: Continuous hydrolysis and fermentation for cellulosic ethanol production.

Ethanol made biologically from a variety of cellulosic biomass sources such as agricultural and forestry residues, grasses, and fast growing wood is widely recognized as a unique sustainable liquid transportation fuel with powerful economic, environmental, and strategic attributes, but production costs must be competitive for these benefits to be realized. Continuous hydrolysis and fermentation processes offer important potential advantages in reducing costs, but little has been done on continuous processing of cellulosic biomass to ethanol. As shown in this review, some continuous fermentations are now employed for commercial ethanol production from cane sugar and corn to take advantage of higher volumetric productivity, reduced labor costs, and reduced vessel down time for cleaning and filling. On the other hand, these systems are more susceptible to microbial contamination and require more sophisticated operations. Despite the latter challenges, continuous processes could be even more important to reducing the costs of overcoming the recalcitrance of cellulosic biomass, the primary obstacle to low cost fuels, through improving the effectiveness of utilizing expensive enzymes. In addition, continuous processing could be very beneficial in adapting fermentative organisms to the wide range of inhibitors generated during biomass pretreatment or its acid catalyzed hydrolysis. If sugar generation rates can be increased, the high cell densities in a continuous system could enable higher productivities and yields than in batch fermentations.

High concentrations of clavulanic acid but not of its degradation products decrease glycerol consumption and oxygen uptake rates in cultures of Streptomyces clavuligerus.

Product inhibition of biological production systems is a widely observed phenomenon with prominent implications for the design and ultimately the success of biotechnological processes. In order to investigate whether such effects could limit the maximal concentrations in the production of the beta-lactamase inhibitor clavulanic acid (CA) in Streptomyces clavuligerus cultivations under process-related conditions, we first validated the equivalence of a laboratory scale aerated stirred tank reactor and a medium scale (50 mL) cultivation device, which required optimization of gas transfer in the latter and finally allowed to conduct the required experiments in smaller volumes with correspondingly reduced consumption of compounds. With this, we investigated the effect of CA additions on two global performance parameters: consumption of the carbon source glycerol and oxygen consumption (measured as the oxygen uptake rate, OUR). Increased levels of CA severely interfered with the physiology of the producing strain at least at and above 1.6 g L(-1) CA, as indicated by a dose-dependent decrease in maximal OURs and glycerol consumption rates. As CA is rapidly degraded during cultivation, it was unclear whether CA itself or its decomposition products, a complex mixture containing amongst others several pyrazine derivatives, were responsible for the observed effects. We supplemented S. clavuligerus cultures with mixtures of the decomposition products and found altered OUR and glycerol consumption, but CA production was not influenced. Compared to the drastic effect of CA itself, it is clear that the products of CA degradation exert a much less severe effect on growing S. clavuligerus cultures.

Clavulanic acid decomposition is catalyzed by the compound itself and by its decomposition products.

The decomposition kinetics of the beta-lactamase inhibitor clavulanic acid (CA) was investigated for CA concentrations between 2.5 and 20 g L(-1), which is assumed to represent a characteristic range for an industrial CA production process. For each initial concentration, first order kinetics plots could be obtained, however the kinetic constant increased from 3.8 x 10(-3) to 8.6 x 10(-3) h(-1) with increasing initial CA concentration, indicating that CA accelerates its own decomposition by general acid-base catalysis. Furthermore, the kinetic constant remained approximately constant during the reaction, suggesting that also the decomposition products of CA had to show similar catalytic activity. This was confirmed experimentally by increased CA decomposition rates when CA degradation products were added to the reaction. A kinetic model is proposed, which is able to reliably predict the observed pseudo first order rate constants. The presented results should be considered in any process where highly concentrated CA solutions are employed, for example, during final downstream processing or in industrial fermentations.

Online medium-throughput respirometry-based OTR measurements in magnetically stirred cultures.

Intensified bioprocess development requires parallelized medium- to high-throughput experimentation with high on- and offline data density across all early scales of the development trajectory from microtiter plate via shake flask to lab-scale reactor. We developed a widespread measurement principle for intermediate scales, respirometry, into a parallelized oxygen transfer rate measurement device that could accurately record common process development-relevant effects such as acetate formation, diauxic growth, and nutrient limitations. The device was further equipped with dissolved oxygen measurement capability and sampling ports that allowed repetitive monoseptic sample withdrawal without disturbing the cultivation. Optimization of the operating parameters lead to k(L) a values of up to 160 h(-1) and corresponding oxygen transfer rates of 1 g L(-1) h(-1) for cultivation volumes of 50 mL.

Flow cytometry: interesting tool for studying binding behavior of DNA on inorganic layered double hydroxide (LDH).

BACKGROUND: A new method was established to characterize the binding kinetics of DNA toward layered double hydroxides (LDHs). The setup consisted of a newly developed sampling tube that allows the injection of analyte during the flow cytometric measurement. METHODS: Layered double hydroxides consist of cationic metal hydroxide layers and exchangeable interlayer anions. This negatively charged structure permits biomolecules such as DNA to adsorb, and a so-called DNA-LDH hybrid is formed. The hydroxide layers can be removed in acidic media and the DNA will be released. CERATOFIX (a registered trademark of Sud-Chemie AG NA that belongs to the family of LDHs, produced by Sud-Chemie AG). The chemical structure can be summarized as [Mg(2)Al(OH)(6)](CO(3))(0.5). The binding capacity and kinetic characteristics of different types of CERATOFIX NA for a model DNA was determined by flow cytometry. RESULTS: The static binding capacities of the different LDHs were determined after 1- and 16-h incubation with DNA solution, showing different binding patterns between the LDH materials. The binding kinetics were revealed by flow cytometric measurements in short-term and long-term kinetic experiments, showing that the majority of DNA adsorbs within the first 60 s. CONCLUSIONS: DNA removal from cell culture supernatants is one of the major concerns in downstream processing. Due to the anion exchange capabilities of LDHs it seemed a very interesting approach to use these materials for binding of DNA for elimination purposes.