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The purified terephthalic and isophthalic acids production process was improved through the exergy analysis approach demonstrated in this work. The overall exergy losses and low-exergy-efficient units were first identified and presented using visualised exergetic flowsheets. Recommendations were then proposed to reduce losses based on the main cause(s) of irreversibility. Three out of the five constituent blocks contained the highest exergy losses. The oxidation block was the main player where it was suggested that using several reactors in series with gradually decreasing temperatures could lower losses. The product refining block had the second-largest irreversibilities, where improving coolers' performances were recommended. The crude terephthalic acid crystallisation block was the third-largest loss producer, where isothermal and isobaric mixing in the solvent dehydrator was suggested to reduce losses. The approach used in this work can be adapted to improve the energy footprint of other chemical processes.

Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient CBP yeast. Despite showing a close relatedness to commercial wine yeast and the benchmark Ethanol Red, a unique profile of gene copy number variations (CNVs) was found in L20, mainly encoding membrane transporters and secretion pathway proteins but also the fermentative metabolism. Moreover, the genome annotation disclosed seven open reading frames (ORFs) in L20 that are absent in the reference S288C genome. Genome engineering was successfully implemented for amylase production. However, with equal amylase gene copies, L20 proved its proficiency as a good enzyme secretor by exhibiting a markedly higher amylolytic activity than Ethanol Red, in compliance to the findings of the genomic exploration. The recombinant L20 dT8 exhibited the highest amylolytic activity and produced more than 4 g/L of ethanol from 2% starch in a CBP setting without the addition of supplementary enzymes. Based on the performance of this strain, an amylase/glucoamylase ratio of 1:2.5 was suggested as baseline for further improvement of the CBP ability. Overall, L20 showed important traits for the future construction of a proficient CBP yeast. As such, this work shows that natural S. cerevisiae strains can be used for the expression of foreign secreted enzymes, paving the way to strain improvement for the starch-to-bioethanol route.

For eight backslopping steps, eight series of water kefir fermentation processes differing in backslopping time and rinsing of the grains during each backslopping step and eight series of fermentation processes differing in incubation temperature and backslopping time were followed. Short backslopping times resulted in high relative abundances of Liquorilactobacillus nagelii and Saccharomyces cerevisiae, intermediate backslopping times in high relative abundances of Leuconostoc pseudomesenteroides, and long backslopping times in high relative abundances of Oenococcus sicerae and Dekkera bruxellensis. When the grains were rinsed during each backslopping step, the relative abundances of Lentilactobacillus hilgardii and Leuc. pseudomesenteroides increased and those of D. bruxellensis and Liql. nagelii decreased. Furthermore, rinsing of the grains during each backslopping step resulted in a slightly higher water kefir grain growth and lower metabolite concentrations. The relative abundances of Liquorilactobacillus mali were highest at 17°C, those of Leuc. pseudomesenteroides at 21 and 25°C, and those of Liql. nagelii at 29°C. With a kinetic modeling approach, the impact of the temperature and rinsing of the grains during the backslopping step on the volumetric production rates of the metabolites was determined.

The main aim of the present study was to evaluate the effects of the presence or absence of trace elements and yeast extract on growth and ethanol production by Clostridium ragsdalei grown on C1 gas dominated syngas (60% CO, 10% CO2, 10% CH4, 10% H-2, 10% N-2). Comparison of whey powder and glucose syrup as low-cost substitutes helped develop a more cost-effective medium formulation to produce ethanol from syngas with significantly higher yields, achieving up to a 3.8-fold improvement with co-utilisation of syngas and glucose syrup. In addition, the results provided further insights into medium manipulation and possible modifications to decrease total costs for increased ethanol production by solventogenic acetogens via syngas fermentation by achieving a 15.6% reduction in the cost of fermentation medium., TUBITAK-CAYDAG [118Y305], The authors wish to thank TUBITAK-CAYDAG under project number 118Y305.

In the present work, palm wood was pretreated using hydrothermal technique in conjunction with chemical method for removal of lignin. Pretreated palm wood was subjected to hydrolysis using Trichoderma reesei MTCC 4876. Subsequently bioethanol was produced using palm wood hydrolysate by Kluveromyces marxianus MTCC 1389. RSM was used to identify the non-linear relationship and optimize various process parameters such as parameters such as pH, temperature, agitation rate, substrate concentration and inoculum size for bioethanol production. ANN constructed with 5-2-1 topology was also used to optimize process parameters. The experimental bioethanol yield of 22.90 g/l was obtained at ANN optimum conditions of temperature 45 degrees C, agitation rate 156 rpm, pH 5, substrate concentration 8% (v/v) and inoculum size 3.2% (v/v).

The depletion of fossil fuel resources and the negative impact of their use on the climate have resulted in the need for alternative sources of clean, sustainable energy. One available alternative, bioethanol, is a potential substitute for, or additive to, petroleum-derived gasoline. In the lignocellulose-to-bioethanol process, the cellulose hydrolysis step represents a major hurdle that hinders commercialization. To achieve economical production of bioethanol from lignocellulosic materials, the rate and yield of the enzymatic hydrolysis of cellulose, which is preferred over other chemically catalyzed processes, must be enhanced. To achieve this, product inhibition and enzyme loss, which are two major challenges, must be overcome. The implementation of membranes, which can permeate molecules selectively based on their size, offers a solution to this problem. Membrane bioreactors (MBRs) can enhance enzymatic hydrolysis yields and lower costs by retaining enzymes for repeated usage while permeating the products. This paper presents a critical discussion of the use of MBRs as a promising approach to the enhanced enzymatic hydrolysis of cellulosic materials. Various MBR configurations and factors that affect their performance are presented.

Background Presence of inhibitory chemicals in lignocellulose hydrolysates is a major hurdle for production of second-generation bioethanol. Especially cheaper pre-treatment methods that ensure an economical viable production process generate high levels of these inhibitory chemicals. The effect of several of these inhibitors has been extensively studied with non-xylose-fermenting laboratory strains, in synthetic media, and usually as single inhibitors, or with inhibitor concentrations much higher than those found in lignocellulose hydrolysates. However, the relevance of individual inhibitors in inhibitor-rich lignocellulose hydrolysates has remained unclear. Results The relative importance for inhibition of ethanol fermentation by two industrial second-generation yeast strains in five lignocellulose hydrolysates, from bagasse, corn cobs and spruce, has now been investigated by spiking higher concentrations of each compound in a concentration range relevant for industrial hydrolysates. The strongest inhibition was observed with industrially relevant concentrations of furfural causing partial inhibition of both D-glucose and D-xylose consumption. Addition of 3 or 6 g/L furfural strongly reduced the ethanol titer obtained with strain MD4 in all hydrolysates evaluated, in a range of 34 to 51% and of 77 to 86%, respectively. This was followed by 5-hydroxymethylfurfural, acetic acid and formic acid, for which in general, industrially relevant concentrations caused partial inhibition of D-xylose fermentation. On the other hand, spiking with levulinic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid or vanillin caused little inhibition compared to unspiked hydrolysate. The further evolved MD4 strain generally showed superior performance compared to the previously developed strain GSE16-T18. Conclusion The results highlight the importance of individual inhibitor evaluation in a medium containing a genuine mix of inhibitors as well as the ethanol that is produced by the fermentation. They also highlight the potential of increasing yeast inhibitor tolerance for improving industrial process economics.

Ethanol production from non-food substrate is strongly recommended to avoid competition with food production. Whey, which is rich in nutrients, is one of the non-food substrate for ethanol production by Kluyveromyces spp. The purpose of this study was to optimize ethanol from different crude (non-deproteinized, non-pH adjusted, and non-diluted) whey using K. marxianus ETP87 which was isolated from traditional yoghurt. The sterilized and non-sterilized whey were employed for K. marxianus ETP87 substrate to evaluate the yeast competition potential with lactic acid and other microflora in whey. The effect of pH and temperature on ethanol productivity from whey was also investigated. Peptone, yeast extract, ammonium sulfate ((NH4)2SO4), and urea were supplemented to whey in order to investigate the requirement of additional nutrient for ethanol optimization. The ethanol obtained from non-sterilized whey was slightly and statistically lower than sterilized whey. The whey storage at 4 °C didn’t guarantee the constant lactose presence at longer preservation time. Significantly high amount of ethanol was attained from whey without pH adjustment (3.9) even if it was lower than pH controlled (5.0) whey. The thermophilic yeast, K. marxianus ETP87, yielded high ethanol between 30 and 35 °C, and the yeast was able to produce high ethanol until 45 °C, and significantly lower ethanol was recorded at 50 °C. The ammonium sulfate and peptone enhanced ethanol productivity, whereas yeast extract and urea depressed the yeast ethanol fermentation capability. The K. marxianus ETP87, the yeast isolated from traditional yoghurt, is capable of producing ethanol from non-sterilized and non-deproteinized substrates.

Most of the crop plants contain about 30% of hemicelluloses comprising D-xylose and D-arabinose. One of the major limitation for the use of pentose sugars is that high purity grade D-xylose and D-arabinose are yet to be produced as commodity chemicals. Research and developmental activities are going on in this direction for their use as platform intermediates through economically viable strategies. During chemical pretreatment of biomass, the pentose sugars were generated in the liquid stream along with other compounds. This contains glucose, proteins, phenolic compounds, minerals and acids other than pentose sugars. Arabinose is present in small amounts, which can be used for the economic production of value added compound, xylitol. The present review discusses the recent trends and developments as well as challenges and opportunities in the utilization of pentose sugars generated from lignocellulosic biomass for the production of value added compounds.

Microscopy and microanalysis 24(5), 517-525 (2018). doi:10.1017/S1431927618015143, Published by Cambridge University Press, New York, NY

Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria, which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-inducible rhaBAD promoter of Escherichia coli into the model freshwater cyanobacterium Synechocystis sp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer.

Funding Information: The authors would like to thank Pitk?nen Leena, Hatakka Rita and Linnekoski Juha for their skilful assistance in HP-SEC, HPAEC-PAD and GC-FID measurements. This work made use of the Aalto University NMR premises. This work was a part of the Academy of Finland's Flagship Programme under Projects No. 318890 and 318891 (Competence Center for Materials Bioeconomy, FinnCERES). Publisher Copyright: © 2021 The Author(s) Aiming to understand the importance of debarking on the controlled utilization of phenolic-rich willow biomass, biobutanol was produced from it by using Clostridium acetobutylicum. Acid-catalysed steam explosion and enzymatic hydrolysis (EH) were investigated before the acetone-butanol-ethanol (ABE) fermentation. The hydrolysable sugar yield and ABE fermentation efficiency were found to decline progressively from willow wood (WW) to HWE WB (hot water extracted willow biomass), WB (willow biomass) and the WW + HWE (willow wood plus the artificial willow bark water extracts), indicating that the pre-removal of water extracts or the bark can significantly improve ABE yield. Notably, the ABE productivity of WW achieved 12.7 g/L at the solvent yield of 31%, and the butanol concentration (i.e. 8.5 g/L) generated by WW is relatively high among the reported lignocellulosic-derived biomass. Additionally, it is hypothesized that under acidic conditions and high temperatures the fructose present in willow water extractsform hydroxymethylfurfural during steam explosion, which then spontaneously condenses with phenolic substances of willow bark to form a solid furanic precipitate. The formed furanic precipitates play inhibitory role in the enzymatic hydrolysis and are thereby deleterious to the ABE fermentation.

Lignocellulosic energy crops are promising feedstocks for producing renewable fuels, such as methane, that can replace diminishing fossil fuels. However, there is a major handicap in using lignocellulosic sources to produce biofuels, which is their low biodegradability. In this study, the application and the optimization of a lignocellulose pretreatment process, named alkaline hydrogen peroxide, was investigated for the enhancement of methane production from the energy crop switchgrass. Four independent process variables, solid content (3-7%), reaction temperature (50-100 degrees C), H2O2 concentration (1-3%), and reaction time (6-24 h), and three response variables, soluble reducing sugar, soluble chemical oxygen demand, and biochemical methane potential were used in process optimization and modeling. The optimization was performed by two different approaches as maximum methane production and cost minimization. The optimum conditions for the highest methane production were found as 6.65 wt% solid content, 50.6 degrees C reaction temperature, 2.94 wt% H2O2 concentration, and 16.05 h reaction time. The conditions providing the lowest cost were 6.43 wt% solid content, 50 degrees C reaction temperature, 1.83 wt% H2O2 concentration, and 6.78 h reaction time. For maximum methane production and cost minimization, specific methane yields of 338.52 mL CH4/g VS and 291.34 mL CH4/g VS were predicted with 62.4 % and 39.8 % enhancements compared to untreated switchgrass, respectively. Finally, it was found that the predicted methane production for the maximum methane production represents 77 % of the theoretical methane yield and 82.22 % energy recovery., Project Management Unit of Akdeniz University from Turkey [FYL-2015-711], This research has been financed (Grant no: FYL-2015-711) by the Project Management Unit of Akdeniz University from Turkey. The authors would like to express their gratitude to Prof. Dr. Suleyman Soylu from Selcuk University and Prof. Dr. Osman Yaldiz from Akdeniz University for supplying switchgrass samples.

Pretreatment and hydrolysis of lignocellulosic biomass are affected by several uncertainties, which must be systematically considered for a robust process design. In this work, stochastic simulations for expected uncertainties in feedstock composition, kinetic parameter values, and operational parameter values for these two steps were performed. The results indicated that these uncertainties significantly impacted the concentration profiles, which could also affect the optimal batch time. Global sensitivity analysis was then used to identify the critical uncertain parameters. In the feedstock components, cellulose and xylan fractions for acid pretreatment and cellulose fraction for enzymatic hydrolysis were important. Temperature was the most sensitive operating parameter for both acid pretreatment and hydrolysis. The activation energies for different reactions were ranked in terms of their impact on process output. The selected parameters were used to develop stochastic process models using Ito process and mean reverting process for feed composition and kinetic parameter uncertainty. (C) 2017 Elsevier Ltd. All rights reserved.

Hxt2 is a glucose repressed, high affinity glucose transporter of the yeast Saccharomyces cerevisiae and is subjected to high glucose induced degradation. Hxt11 is a sugar transporter that is stably expressed at the membrane irrespective the sugar concentration. To transfer this property to Hxt2, the N-terminal tail of Hxt2 was replaced by the corresponding region of Hxt11 yielding a chimeric Hxt11/2 transporter. This resulted in the stable expression of Hxt2 at the membrane and improved the growth on 8% D-glucose and 4% D-xylose. Mutation of N361 of Hxt11/2 into threonine reversed the specificity for D-xylose over D-glucose with high D-xylose transport rates. This mutant supported efficient sugar fermentation of both D-glucose and D-xylose at industrially relevant sugar concentrations even in the presence of the inhibitor acetic acid which is normally present in lignocellulosic hydrolysates. Biotechnol. (C) 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Lignocellulosic feedstock is one of the potential renewable sources for producing ethanol for transportation. The process steps viz., acid pretreatment and enzymatic hydrolysis in bio-chemical process route are intended to produce fermentable sugars, which can be readily fermented for producing ethanol. However, the dilute acid pre-treatment and enzymatic hydrolysis process steps are found to be economically inefficient. The present work aims at optimizing these process steps for improving the process performance. Such optimization is expected to increase conversion, reduce energy or material requirement, thereby improving the economics. The kinetic models of acid pretreatment and enzymatic hydrolysis for lignocellulosic feedstock processing are adapted from literature. Subsequently, these kinetic models are augmented by associated mass and energy balances, to develop a batch reactor model and fed-batch reactor model for dilute acid pretreatment and enzymatic hydrolysis processes, respectively. Optimal control with Pontryagin's maximum principle has been implemented to determine the optimal time dependent profiles of heating and cooling fluid flow rates and operating temperatures for acid pretreatment and substrate feed rate profile for enzymatic hydrolysis to optimize the respective processes performance. Different objective functions such as maximizing concentration of desired product, minimizing the batch time, and maximizing profit have been considered. The simulation results yielded an increase of 6.7% and 8.8% in final concentration of desired product; 43% and 42.5% reduction in batch processing time for pretreatment and enzymatic hydrolysis processes, respectively. Finally, the simulation results have also provided optimal operating policies which have increased the profit of pretreatment by 124% and enzymatic hydrolysis by 150%, thereby improving the techno-economic feasibility for processing lignocellulosic feedstock. (C) 2017 Elsevier Ltd. All rights reserved.

The socio-economic effects of bio-energy are not unequivocally positive, although it is one of the main arguments for supporting its expansion. An ex-ante quantification of the impacts is necessary for transparently presenting the benefits and burdens of bioenergy before they occur, and for minimising unwanted outcomes. In this article, the status, limitations, and possibilities for improvements in ex-ante quantitative research methods for investigating socio-economic impacts of bioenergy are mapped. For this, a literature review to identify relevant indicators, analyse the latest quantitative ex-ante research methods, and to assess their ability and suitability to measure these indicators was performed. The spatial aggregation of existing analyses was specifically considered because quantitative information on different spatial scales shows the geographic distribution of the effects. From the 236 indicators of socio-economic impacts spread over twelve impact categories that were found in this review, it becomes evident that there are clear differences in the ex-ante quantification of these indicators. The review shows that some impact categories receive more attention in ex-ante quantification studies, such as project-level economic feasibility and national-level macroeconomic impacts, while other relevant indicators have not been ex-ante quantified, such as community impacts and public acceptance. Moreover, a key blind spot regarding food security impacts was identified in the aggregation level at which food security impacts are quantified, which does not match the level at which the impacts occur. The review also shows that much more can be done in terms of ex-ante quantification of these impacts. Specifically, spatial disaggregation of models and model collaboration can extend the scope of socio-economic analyses. This is demonstrated for food security impacts, which shows the potential for future household-level analysis of food security impacts on all four pillars of food security.

Many of the sustainability concerns of bioenergy are related to direct or indirect land use change (LUC ) resulting from bioenergy feedstock production. The environmental and socio‐economic impacts of LUC highly depend on the site‐specific biophysical and socio‐economic conditions. The objective of this study is to spatiotemporally assess the potential LUC dynamics resulting from an increased biofuel demand, the related greenhouse gas (GHG ) emissions, and the potential effect of LUC mitigation measures. This assessment is demonstrated for LUC dynamics in Brazil towards 2030, considering an increase in the global demand for bioethanol as well as other agricultural commodities. The potential effects of three LUC mitigation measures (increased agricultural productivity, shift to second‐generation ethanol, and strict conservation policies) are evaluated by using a scenario approach. The novel modelling framework developed consists of the global Computable General Equilibrium model MAGNET , the spatiotemporal land use allocation model PLUC , and a GIS ‐based carbon module. The modelling simulations illustrate where LUC as a result of an increased global ethanol demand (+26 × 109 L ethanol production in Brazil) is likely to occur. When no measures are taken, sugar cane production is projected to expand mostly at the expense of agricultural land which subsequently leads to the loss of natural vegetation (natural forest and grass and shrubland) in the Cerrado and Amazon. The related losses of above and below ground biomass and soil organic carbon result in the average emission of 26 g CO 2‐eq/MJ bioethanol. All LUC mitigation measures show potential to reduce the loss of natural vegetation (18%–96%) as well as the LUC ‐related GHG emissions (7%–60%). Although there are several uncertainties regarding the exact location and magnitude of LUC and related GHG emissions, this study shows that the implementation of LUC mitigation measures could have a substantial contribution to the reduction of LUC ‐related emissions of bioethanol. However, an integrated approach targeting all land uses is required to obtain substantial and sustained LUC ‐related GHG emission reductions in general.

Biofuels are suitable alternatives to conventional and fossil fuels that pose serious environmental adverse effects to society. Bioethanol is the most promising biofuel and can be generated from lignocellulosic biomasses. Forest residues are outside the human food chain and inexpensive raw materials, but they need a previous pretreatment, in order to improve the cellulose accessibility for further bioconversion. The study of bioethanol production from rockrose pretreated by steam explosion (SE) was carried out employing separate enzymatic hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) approaches. Saccharification of untreated rockrose attained only 0.9% of sugars yield. However, the steam explosion pretreatment promoted the disruption of interfibrillar surfaces of fibers with partial degradation of lignin thus enhancing the accessibility of polysaccharides toward enzymatic hydrolysis. Alkaline extraction after steam explosion pretreatment of rockrose residue (R-SE-OH) led to the partial removal of lignin, hemicelluloses, and other degradation products from fibre surface allowing an increase of 75% in the glucose yield. Bioethanol production in SSF mode was faster and slightly more efficient process than SHE providing the best results: ethanol concentration of 16.1 g L-1, fermentation efficiency of 69.8% and a yield of 22.1 g ethanol/100 g R-SE-OH. (C) 2015 Elsevier B.V. All rights reserved.

Xylose isomerase from Piromyces sp. E2 (PirXI) can be used to equip Saccharomyces cerevisiae with the capacity to ferment xylose to ethanol. The biochemical properties and structure of the enzyme have not been described even though its metal content, catalytic parameters, and expression level are critical for rapid xylose utilization. We have isolated the enzyme after high-level expression in Escherichia coli, analyzed the metal dependence of its catalytic properties, and determined 12 crystal structures in the presence of different metals, substrates, and substrate analogues. The activity assays revealed that various bivalent metals can activate PirXI for xylose isomerization. Among these metals, Mn2+ is the most favorable for catalytic activity. Furthermore, the enzyme shows the highest affinity for Mn2+, which was established by measuring the activation constants (Kact) for different metals. Metal analysis of the purified enzyme showed that in vivo the enzyme binds a mixture of metals that is determined by metal availability as well as affinity, indicating that the native metal composition can influence activity. The crystal structures show the presence of an active site similar to that of other xylose isomerases, with a d-xylose binding site containing two tryptophans and a catalytic histidine, as well as two metal binding sites that are formed by carboxylate groups of conserved aspartates and glutamates. The binding positions and conformations of the metal-coordinating residues varied slightly for different metals, which is hypothesized to contribute to the observed metal dependence of the isomerase activity.

The present work focuses firstly on the evaluation of the effect of laccase on enzymatic hydrolysis of hazelnut husk which is one of the most abundant lignocellulosic agricultural residues generated in Turkey. In this respect, the co-enzymatic treatment of hazelnut husk by cellulase and laccase, without a conventional pretreatment step is evaluated. Using 2.75 FPU/g substrate (40 g/L substrate) and a ratio of 131 laccase U/FPU achieved the highest reducing sugars concentration. Gas chromatography mass spectrometry confirmed that the hydrolysate was composed of glucose, xylose, mannose, arabinose and galactose. The inclusion of laccase in the enzyme mixture [carboxymethyl cellulase (CMCase) and beta-glucosidase] increased the final glucose content of the reducing sugars from 20 to 50%. Therefore, a very significant increase in glucose content of the final reducing sugars concentration was obtained by laccase addition. Furthermore, the production of cellulases and laccase by Pycnoporus sanguineus DSM 3024 using hazelnut husk as substrate was also investigated. Among the hazelnut husk concentrations tested (1.5, 6, 12, 18 g/L), the highest CMCase concentration was obtained using 12 g/L husk concentration on the 10th day of fermentation. Besides CMCase, P. sanguineus DSM 3024 produced beta-glucosidase and laccase using hazelnut husk as carbon source. In addition to CMCase and b-glucosidase, the highest laccase activity measured was 2240 +/- 98 U/L (8.89 +/- 0.39 U/mg). To the best of our knowledge, this is the first study to report hazelnut husk hydrolysis in the absence of pretreatment procedures.

Hxt2 is a glucose repressed, high affinity glucose transporter of the yeast Saccharomyces cerevisiae and is subjected to high glucose induced degradation. Hxt11 is a sugar transporter that is stably expressed at the membrane irrespective the sugar concentration. To transfer this property to Hxt2, the N-terminal tail of Hxt2 was replaced by the corresponding region of Hxt11 yielding a chimeric Hxt11/2 transporter. This resulted in the stable expression of Hxt2 at the membrane and improved the growth on 8% D-glucose and 4% D-xylose. Mutation of N361 of Hxt11/2 into threonine reversed the specificity for D-xylose over D-glucose with high D-xylose transport rates. This mutant supported efficient sugar fermentation of both D-glucose and D-xylose at industrially relevant sugar concentrations even in the presence of the inhibitor acetic acid which is normally present in lignocellulosic hydrolysates. Biotechnol. (C) 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

International audience; Dynamic flux balance analysis of a bioreactor is based on the coupling between a dynamic problem, which models the evolution of biomass, feeding substrates and metabolites, and a linear program, which encodes the metabolic activity inside cells. We cast the problem in the language of optimal control and propose a hybrid formulation to model the full coupling between macroscopic and microscopic level. On a given location of the hybrid system we analyze necessary conditions given by the Pontryagin Maximum Principle and discuss the presence of singular arcs. For the multi-input case, under suitable assumptions, we prove that generically with respect to initial conditions optimal controls are bang-bang. For the single-input case the result is even stronger as we show that optimal controls are bang-bang.

The economic performance of biofuels supply chains depends on the interaction of technical characteristics as technological pathways and logistics, and social structures as actor behavior, their interactions and institutions. Traditional approaches focus on the technical problems only. Little attention has been paid to the institutional analysis of biofuel supply chains. This paper aims to extend the analysis of the effect of institutions on the emergence of biofuel supply chains by developing a conceptual framework that combines elements of complex adaptive systems, (neo) institutional economics and sociotechnical systems theory. These elements were formalized into an agent-based model. The proposed method is illustrated by a case study on a biodiesel supply chain in Germany. It was found that the patterns in production capacity result from investors basing their decisions on optimistic-perceptions of the market development that increase with a favorable institutional framework. Conversely, patterns in biodiesel production cannot be completely explained by this mechanism. The proposed framework assisted the model conceptualization phase and allowed the incorporation of social structures into the agent based model. This approach could be developed further to provide insights on the effect of different future deployment strategies on bioenergy systems emergence and development. (C) 2016 The Authors. Published by Elsevier Ltd.

We investigate the economic and employment consequences of introducing a new sugarcane-based biofuel industry into Australia. We model the new biofuel industry on the production recipe of the existing large-scale gasoalcohol and alcohol sectors in the Brazilian economy. To this end we utilise a hybrid IO-LCA (input-output life cycle assessment) approach, which involves inserting data on new processes and/or sectors into an existing IO table. In particular, we develop and test an analytical and a numerical approach for re-balancing an IO table augmented with rows and columns representing large new biofuel industries. We quantify changes in economic output and employment in the Australian economy. We conclude that a future biofuel industry will be employment-positive for Australia. (C) 2014 Elsevier B.V. All rights reserved.

Commonly used feed processing technologies are not sufficient to affect recalcitrant non-starch polysaccharides (NSP) such as arabinoxylans present in maize distillers dried grain with solubles (DDGS). Instead, hydrothermal treatments combined with acid catalysts might be more effective to modify these NSP. The objective of this experiment was to investigate the effects of hydrothermal maleic acid treatment (acid-extrusion) on the degradability of maize DDGS in growing pigs. It was hypothesized that acid-extrusion modifies DDGS cell wall architecture and, thereby, increases fermentability of NSP. Two diets, containing either 40% (wt/wt) unprocessed or acid-extruded DDGS, were restrictedly fed to groups of gilts (n = 11, with 4 pigs per group; initial mean BW: 20.8 ± 0.2 kg) for 18 d and performance and digestibility were analyzed. Acid-extrusion tended to decrease apparent ileal digestibility (AID) of CP (~3 percentage units, P = 0.063) and starch (~1 percentage unit, P = 0.096). Apparent digestibility of CP and starch measured at the mid colon (2 percentage units, P = 0.030 for CP and 0.3 percentage units, P

The socio-economic effects of bio-energy are not unequivocally positive, although it is one of the main arguments for supporting its expansion. An ex-ante quantification of the impacts is necessary for transparently presenting the benefits and burdens of bioenergy before they occur, and for minimising unwanted outcomes. In this article, the status, limitations, and possibilities for improvements in ex-ante quantitative research methods for investigating socio-economic impacts of bioenergy are mapped. For this, a literature review to identify relevant indicators, analyse the latest quantitative ex-ante research methods, and to assess their ability and suitability to measure these indicators was performed. The spatial aggregation of existing analyses was specifically considered because quantitative information on different spatial scales shows the geographic distribution of the effects. From the 236 indicators of socio-economic impacts spread over twelve impact categories that were found in this review, it becomes evident that there are clear differences in the ex-ante quantification of these indicators. The review shows that some impact categories receive more attention in ex-ante quantification studies, such as project-level economic feasibility and national-level macroeconomic impacts, while other relevant indicators have not been ex-ante quantified, such as community impacts and public acceptance. Moreover, a key blind spot regarding food security impacts was identified in the aggregation level at which food security impacts are quantified, which does not match the level at which the impacts occur. The review also shows that much more can be done in terms of ex-ante quantification of these impacts. Specifically, spatial disaggregation of models and model collaboration can extend the scope of socio-economic analyses. This is demonstrated for food security impacts, which shows the potential for future household-level analysis of food security impacts on all four pillars of food security.

Rubberwood (Hevea brasiliensis), a potential raw material for bioethanol production due to its high cellulose content, was used as a novel feedstock for enzymatic hydrolysis and bioethanol production using biological pretreatment. To improve ethanol production, rubberwood was pretreated with white rot fungusCeriporiopsis subvermisporato increase fermentation efficiency. The effects of particle size of rubberwood (1 mm, 0.5 mm, and 0.25 mm) and pretreatment time on the biological pretreatment were first determined by chemical analysis and X-ray diffraction and their best condition obtained with 1 mm particle size and 90 days pretreatment. Further morphological study on rubberwood with 1 mm particle size pretreated by fungus was performed by FT-IR spectra analysis and SEM observation and the result indicated the ability of this fungus for pretreatment. A study on enzymatic hydrolysis resulted in an increased sugar yield of 27.67% as compared with untreated rubberwood (2.88%). The maximum ethanol concentration and yield were 17.9 g/L and 53% yield, respectively, after 120 hours. The results obtained demonstrate that rubberwood pretreated byC. subvermisporacan be used as an alternative material for the enzymatic hydrolysis and bioethanol production.

Effects of cultivar choice and nitrogen supply on biomass yield and dry matter partitioning of sweet sorghum [Sorghum bicolor (L.) Moench] were studied under irrigated conditions in the arid temperate climate of north-west China. Two hybrid cultivars, Zaoshu-1 (ZS 1) and Chuntian-2 (CT 2), and two inbred cultivars, Rio and Lvneng-3 (LN 3) were grown in a field trial at two nitrogen fertilizer levels (0 and 150 kg/ha of N) in Urumchi (43°01'N, 88°37'E). Hybrid cultivars produced much higher aboveground dry biomass yields, amounting to on an average 27.2 t/ha than inbred cultivars with on an average 24.3 t/ha. Crop growth rates amounted to 30.8 g/m2/day on an average during the period from elongation to heading as well as from heading to maturity, however; the growth rates of the inbred cultivars slowed down after heading, while those of the hybrids still increased. Inbred cultivar Rio had the highest plant height (328 cm) and stem dry weight (16.5 t/ha). Average grain yield was 11.2 and 5.5 t/ha for hybrids and inbreds, respectively. Partitioning of dry matter differed between the inbred cultivars, but did not for the hybrids. Application of fertilizer nitrogen resulted in a higher plant height, stem dry biomass, dry biomass of leaves and aboveground biomass in inbred and hybrid cultivars. In conclusion, hybrid and inbred cultivars of sweet sorghum performed well in the environment of north-west China when irrigated. Biomass and grain yields of hybrids turned out to be higher than those of inbreds.

International audience; Lignocellulose, the most abundant renewable carbon source on earth, is the logical candidate to replace fossil carbon as the major biofuel raw material. Nevertheless, the technologies needed to convert lignocellulose into soluble products that can then be utilized by the chemical or fuel industries face several challenges. Enzymatic hydrolysis is of major importance, and we review the progress made in fungal enzyme technology over the past few years with major emphasis on (i) the enzymes needed for the conversion of polysaccharides (cellulose and hemicellulose) into soluble products, (ii) the potential uses of lignin degradation products, and (iii) current progress and bottlenecks for the use of the soluble lignocellulose derivatives in emerging biorefineries.

Inhibitors commonly found in lignocellulosic hydrolysates impair yeast metabolism and growth, reducing the productivity of the overall bioethanol production process. FTIR spectroscopy was used to analyze the metabolomic alterations induced by acetic and formic acid, furfural and 5-hydroxymethyl-2-furaldehyde (HMF) on yeast metabolism, using three Saccharomyces cerevisiae strains with different sensitivities. IR spectrum alterations were summarized with synthetic descriptors to rapidly visualize the kinds of molecules displaying the more intense reactions and to evaluate the type of interaction between inhibitors in a mixture, at concentrations close to those found at the industrial scale. The four inhibitors induced different levels of mortality and metabolomic changes. The metabolomic response was proportional to the different strain resistance level, further supporting their original classification. Inhibitor mixtures severely hindered the cell viability with the exception of the lowest concentration tested, which was partially biocidal. Furthermore, for the first time, this study revealed antagonistic interactions exerted by inhibitor mixtures on microbial metabolism, closely strain- and dose-dependent. This confirms that yeast strain resistance to single inhibitors cannot be used to predict behaviour on exposure to mixtures. This finding is worth further studies to explain the underlying antagonistic mechanism and to support the selection of highly tolerant strains.

Quantification of different physiological states of Candida shehatae cells was performed by flow cytometry associated with two fluorescent probes. Propidium iodide and carboxyfluorescein diacetate acetoxymethyl ester fluorescent dyes were chosen based on data from the literature. A staining procedure, developed from the previous works was applied to the yeast. Then, the protocol was improved to fit with fermentation constraints such as no physiological interference between the staining procedure and the cells, shortest preparation time and small amounts of dyes. From this optimisation, propidium iodide was included in the sample at 8 mg/L whereas carboxyfluorescein was first diluted in Pluronic (R) agent and used at 3 mg/L, samples were incubated for 10 min at 40 degrees C. Repeatability and accuracy were evaluated to validate this flow cytometry procedure for viability determination. (C) 2012 Elsevier B.V. All rights reserved.

This review describes different potential sources for amino acids that could be used for the production of bulk chemicals in a biorefinery, such as agricultural byproduct streams. Volumes at which these sources and the amino acids therein are available were determined, and the most interesting amino acids in terms of their potential available quantity were identified. The investigated sources are maize and wheat DDGS, sugarcane vinasse and its leaves, sugar beet vinasse and its leaves, cassava leaves, press cakes of rapeseed, sunflower, soybean, palm oil and Jatropha, animal slaughter waste, microalgae, macroalgae, grass and alfalfa. It can be concluded that there are enough sources available to produce bio-based chemicals such as N-methylpyrrolidone with a market sizes around 100 kt per year from amino acids. Bulk chemicals such as acrylonitrile can partly be replaced in the future by their bio-based equivalent, depending on the amounts of biofuels that will be produced in the future. However, it is still necessary to find cost-effective methods for the isolation of amino acids from the discussed sources.

Saccharomyces cerevisiae was immobilized on gamma-alumina pellets and used for repeated batch fermentations in glucose medium (16.5 g/100 mL) at various temperatures and pressures. An increase in pressure from 3 to 7 atm and a decrease in temperature from 30 to 20 degrees C reduced the ethanol productivity by about 50% and 70%, respectively. Increasing concentrations of volatile by-products were observed at lower fermentation temperatures, while the pressure influence on the concentrations of these by-products was proved to be more complex. Mathematical expressions were established to allow the calculation of the fermentation rate at various pressures and sugar concentrations when the corresponding rate at atmospheric pressure is known. The study showed that the height of bioreactors has to be limited to 19.5 m due to hydrostatic pressure shock at higher fill levels. (C) 2012 Elsevier Ltd. All rights reserved.

The production of fermentable substrates from barley straw under various process conditions was studied. Pretreatment included chemical pretreatment with dilute-acid followed by enzymatic hydrolysis; the pretreatment conditions were expressed in a combined severity factor, CS, which ranged in the present study from -1.6 to 1.1. Considering the production of fermentable sugars and the release of inhibitory compounds, the optimal pretreatment conditions were 170 °C, 0% sulfuric acid and 60 min, corresponding to CS -0.4. Under these conditions, 21.4 g glucose/L, 8.5 g xylose/L, and 0.5 g arabinose/L were produced, while 0.1 g HMF/L, 0.4 g furfural/L, 0.0 g levulinic acid/L, 0.0 g formic acid/L, and 2.1 g acetic acid/L were released. The ratio of Ssugars/Sinhibitors proved to be a good tool for evaluating the suitability of a hydrolysate for fermentation purposes.

Olive-mill wastes are produced by the industry of olive oil production, which is a very important economic activity, particularly for Spain, Italy and Greece, leading to a large environmental problem of current concern in the Mediterranean basin. There is as yet no accepted treatment method for all the wastes generated during olive oil production, mainly due to technical and economical limitations but also the scattered nature of olive mills across the Mediterranean basin. The production of virgin olive oil is expanding worldwide, which will lead to even larger amounts of olive-mill waste, unless new treatment and valorisation technologies are devised. These are encouraged by the trend of current environmental policies, which favour protocols that include valorisation of the waste. This makes biological treatments of particular interest. Thus, research into different biodegradation options for olive-mill wastes and the development of new bioremediation technologies and/or strategies, as well as the valorisation of microbial biotechnology, are all currently needed. This review, whilst presenting a general overview, focusses critically on the most significant recent advances in the various types of biological treatments, the bioremediation technology most commonly applied and the valorisation options, which together will form the pillar for future developments within this field.

Background The yeast Saccharomyces cerevisiae is unable to ferment pentose sugars like d-xylose. Through the introduction of the respective metabolic pathway, S. cerevisiae is able to ferment xylose but first utilizes d-glucose before the d-xylose can be transported and metabolized. Low affinity d-xylose uptake occurs through the endogenous hexose (Hxt) transporters. For a more robust sugar fermentation, co-consumption of d-glucose and d-xylose is desired as d-xylose fermentation is in particular prone to inhibition by compounds present in pretreated lignocellulosic feedstocks. Results Evolutionary engineering of a d-xylose-fermenting S. cerevisiae strain lacking the major transporter HXT1–7 and GAL2 genes yielded a derivative that shows improved growth on xylose because of the expression of a normally cryptic HXT11 gene. Hxt11 also supported improved growth on d-xylose by the wild-type strain. Further selection for glucose-insensitive growth on d-xylose employing a quadruple hexokinase deletion yielded mutations at N366 of Hxt11 that reversed the transporter specificity for d-glucose into d-xylose while maintaining high d-xylose transport rates. The Hxt11 mutant enabled the efficient co-fermentation of xylose and glucose at industrially relevant sugar concentrations when expressed in a strain lacking the HXT1–7 and GAL2 genes. Conclusions Hxt11 is a cryptic sugar transporter of S. cerevisiae that previously has not been associated with effective d-xylose transport. Mutagenesis of Hxt11 yielded transporters that show a better affinity for d-xylose as compared to d-glucose while maintaining high transport rates. d-glucose and d-xylose co-consumption is due to a redistribution of the sugar transport flux while maintaining the total sugar conversion rate into ethanol. This method provides a single transporter solution for effective fermentation on lignocellulosic feedstocks. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0360-6) contains supplementary material, which is available to authorized users.

A range of energy crops can be grown on marginal land (i.e. land that is not suitable for food crop production or contaminated site) to provide feedstocks for bioenergy, non-food products and biofuels. The food versus fuel debate had a significant negative impact in Europe on first generation biofuels production from food crops (i.e. wheat, rapeseed, etc). A new approach involving the use of marginal land for the production of lignocellulosic species for the production of bioethanol is now pursued in Italy and in many other countries, where the demand for high quality water resources, arable land, food and fossil fuels is rapidly growing. With an emerging “feed versus fuel debate” there is a pressing need to find options for the use of marginal lands and wastewaters or saline ground waters to produce second generation biofuel or bio paper crops. Arundo donax was selected as a potential crop for use in these areas, since it produces more cellulosic biomass and sequesters more contaminants, using less land and pesticides than any other alternative crops reported in the literature. The objective of this paper is to evaluate economically a simplified process for the production of second generation bioethanol from A. donax. Process calculations and economic analyses are performed using the software SuperPro Designer®.

Use of biomass is crucial for a sustainable supply of chemicals and fuels for future generations. Compared to fossil feedstocks, biomass is more functionalized and requires defunctionalisation to make it suitable for use. Deoxygenation is an important method of defunctionalisation. While thermal deoxygenation is possible, high energy input and lower reaction selectivity makes it less suitable for producing the desired chemicals and fuels. Catalytic deoxygenation is more successful by lowering the activation energy of the reaction, and when designed correctly, is more selective. Catalytic deoxygenation can be performed in various ways. Here we focus on decarboxylation and decarbonylation. There are several classes of catalysts: heterogeneous, homogeneous, bio- and organocatalysts and all have limitations. Homogeneous catalysts generally have superior selectivity and specificity but separation from the reaction is cumbersome. Heterogeneous catalysts are more readily isolated and can be utilised at high temperatures, however they have lower selectivity in complex reaction mixtures. While bio-catalysts can operate at ambient temperatures, the volumetric productivity is lower. Therefore it is not always apparent in advance which catalyst is the most suitable in terms of conversion and selectivity under optimal process conditions. Here we compare classes of catalysts for the decarboxylation and decarbonylation of biobased molecules and discuss their limitations and advantages. We mainly focus on the activity of the catalysts and find there is a strong correlation between specific activity (turn over frequency) and temperature for metal based catalysts (homogeneous or heterogeneous). Thus one is not more active than the other at the same temperature. Alternatively, enzymes have a higher turnover frequency but drawbacks (low volumetric productivity) should be overcome.

The correct choice of the specific lignocellulosic biomass pretreatment allows obtaining high biomass conversions for biorefinery implementations and cellulosic bioethanol production from renewable resources. Cynara cardunculus (cardoon) pretreated by steam explosion (SE) was involved in second-generation bioethanol production using separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) processes. Steam explosion pretreatment led to partial solubilisation of hemicelluloses and increased the accessibility of residual polysaccharides towards enzymatic hydrolysis revealing 64% of sugars yield against 11% from untreated plant material. Alkaline extraction after SE pretreatment of cardoon (CSEOH) promoted partial removal of degraded lignin, tannins, extractives and hemicelluloses thus allowing to double glucose concentration upon saccharification step. Bioethanol fermentation in SSF mode was faster than SHF process providing the best results: ethanol concentration 18.7 g L-1, fermentation efficiency of 66.6% and a yield of 26.6 g ethanol/100 g CSEOH or 10.1 g ethanol/100 g untreated cardoon. (C) 2015 Elsevier Ltd. All rights reserved.

Background: Hardwood spent sulfite liquor (HSSL) is a by-product of acid sulfite pulping process that is rich in xylose, a monosaccharide that can be fermented to ethanol by Scheffersomyces stipitis. However, HSSL also contains acetic acid and lignosulfonates that are inhibitory compounds of yeast growth. The main objective of this study was the use of an evolutionary engineering strategy to obtain variants of S. stipitis with increased tolerance to HSSL inhibitors while maintaining the ability to ferment xylose to ethanol. Results: A continuous reactor with gradually increasing HSSL concentrations, from 20% to 60% (v/v), was operated for 382 generations. From the final obtained population (POP), a stable clone (C-4) was isolated and characterized in 60% undetoxified HSSL. C-4 isolate was then compared with both the parental strain (PAR) and POP. Both POP and C-4 were able to grow in 60% undetoxified HSSL, with a higher capability to withstand HSSL inhibitors than PAR. Higher substrate uptake rates, 7% higher ethanol efficiency and improved ethanol yield were obtained using C-4. Conclusion: S. stipitis was successfully adapted to 60% (v/v) undetoxified eucalyptus HSSL. A stable isolate, C-4, with an improved performance in undetoxified HSSL compared to PAR was successfully obtained from POP. Owing to its improved tolerance to inhibitors, C-4 may represent a major advantage for the production of bioethanol using HSSL as substrate.

Two upflow sludge bed reactors (UASB) were operated for 80 days at 55 degreesC with methanol as the substrate with an organic loading rate (OLR) of about 20 g COD l(-1) per day and a hydraulic retention time (HRT) of 10 h. One UASB was operated without sulphate addition (control reactor-R1) whereas the second was fed with sulphate at a COD:SO42- ratio of 10 (sulphate-fed reactor-R2), providing an influent sulphate concentration of 0.6 g l(-1). For both reactors, methanogenesis was the dominant process with no considerable accumulation of acetate. The methanol removal averaged 93% and 83% for R1 and R2, respectively, and total sulphate removal was achieved in the latter. The pathway of methanol conversion for both sludges was investigated by measuring the fate of carbon in the presence and absence of bicarbonate or specific inhibitors for a sludge sample collected at day 72. In both sludges, about 70% of the methanol was syntrophically converted to methane and/or sulphide, via the intermediate H-2/CO2. strong competition between methanogens and sulphidogens took place in the R2 sludge with half of the methanol-COD being used by methane-producing bacteria and the other half by sulphate-reducing bacteria. Acetate was not an important intermediate for both sludges, and played a slightly more important role for the sulphate-adapted sludge (R2), sustained by the higher amount of bicarbonate produced during sulphate-reduction. The pathway study indicates that, although acetate does not represent an important intermediate, the system is susceptible to its accumulation. (C) 2004 Elsevier B.V. All rights reserved.

The thermophilic (55degreesC) anaerobic conversion of methanol was studied in an unbuffered medium (pH 4 +/- 0.2) and in a phosphate buffered medium (pH 6.4 +/- 0.1), in both cases without bicarbonate addition. Our cultivated sludge consortium was unable to degrade methanol under acidic conditions. During the 160 d of continuous operation of an upflow anaerobic sludge blanket (UASB) reactor (R1), at an organic loading rate (OLR) of 6 gCOD/(l (.) d) and pH around 4, only 5% of the applied methanol load was consumed and no methane (CH4) was detected. However, hydrogenotrophic methanogens were found to be resistant to exposure to such conditions. At the end of the trial, the hydrogenotrophic methanogenic activity of the sludge was 1.23 +/- 0.16 gCOD/(gVSS (.) d) at neutral pH. With methanol as the test substrate, the addition of bicarbonate led to acetate accumulation. A second reactor (R2) was operated for 303 d at OLRs ranging from 5.5 to 25.4 gCOD/(l (.) d) in order to assess the conversion of methanol at neutral pH (phosphate buffered) in a bicarbonate deprived medium. The reactor performance was poor with a methanol-COD removal capacity limited to about 9.5 gCOD/(l (.) d). The system appeared to be quite susceptible to any type of disturbance, even at low OLR. The fraction of methanol-COD converted to CH4 and acetate was found to be unaffected by the OLR applied. At the end of the trial, the outcome of the competition was about 50% methanogenesis and 50% homoacetogenesis.

Commonly used feed processing technologies are not sufficient to affect recalcitrant non-starch polysaccharides (NSP) such as arabinoxylans present in maize distillers dried grain with solubles (DDGS). Instead, hydrothermal treatments combined with acid catalysts might be more effective to modify these NSP. The objective of this experiment was to investigate the effects of hydrothermal maleic acid treatment (acid-extrusion) on the degradability of maize DDGS in growing pigs. It was hypothesized that acid-extrusion modifies DDGS cell wall architecture and, thereby, increases fermentability of NSP. Two diets, containing either 40% (wt/wt) unprocessed or acid-extruded DDGS, were restrictedly fed to groups of gilts (n = 11, with 4 pigs per group; initial mean BW: 20.8 ± 0.2 kg) for 18 d and performance and digestibility were analyzed. Acid-extrusion tended to decrease apparent ileal digestibility (AID) of CP (~3 percentage units, P = 0.063) and starch (~1 percentage unit, P = 0.096). Apparent digestibility of CP and starch measured at the mid colon (2 percentage units, P = 0.030 for CP and 0.3 percentage units, P