Biofuels and Biochemicals Production Thaddeus Ezeji www.mdpi.com/journal/fermentation Edited by Printed Edition of the Special Issue Published in Fermentation Biofuels and Biochemicals Production Special Issue Editor Thaddeus Ezeji MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade Special Issue Editor Thaddeus Ezeji The Ohio State University USA Editorial Office MDPI AG St. Alban - Anlage 66 Basel, Switzerland This edition is a reprint of the Special Issue published online in the open access journal Fermentation (ISSN 2311- 5637 ) from 2016 – 2017 (available at: http://www.mdpi.com/journal/fermentation/special_issues/biofuels_production ). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: Author 1; Author 2. Article title. Journal Name Year , Article number , page range. 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The book taken as a whole is © 2017 MDPI, Basel, Switzerland, distributed under the terms and conditions of the Creative Commons license CC BY - NC -ND ( http://creativecommons.org/licenses/by - n c- nd/4.0/ ). iii Table of Contents About the Special Issue Editor ..................................................................................................................... v Thaddeus Chukwuemeka Ezeji Production of Bio - Derived Fuels and Chemicals Reprinted from: Fermentation 2017 , 3 (3), 42; doi: 10.3390/fermentation3030042 .................................. 1 Bin Lai, Manuel R. Plan, Mark P. Hodson and Jens O. Krömer Simultaneous Determination of Sugars, Carboxylates, Alcohols and Aldehydes from Fermentations by High Performance Liquid Chromatography Reprinted from: Fermentation 2016 , 2 (1), 6; doi: 10.3390/fermentation2010006 .................................... 5 Patrick T. Sekoai Modelling and Optimization of Operational Setpoint Parameters for Maximum Fermentative Biohydrogen Production Using Box - Behnken Design Reprinted from: Fermentation 2016 , 2 (3), 15; doi: 10.3390/fermentation2030015 .................................. 13 Parushi Nargotra, Surbhi Vaid and Bijender Kumar Bajaj Cellulase Production from Bacillus subtilis SV1 and Its Application Potential for Saccharification of Ionic Liquid Pretreated Pine Needle Biomass under One Pot Consolidated Bioprocess Reprinted from: Fermentation 2016 , 2 (4), 19; doi: 10.3390/fermentation2040019 .................................. 23 Hong-Sheng Zeng, Chi-Ruei He, Andy Tien-Chu Yen, Tzong-Ming Wu and Si-Yu Li Assessment of Acidified Fibrous Immobilization Materials for Improving Acetone - Butanol - Ethanol (ABE) Fermentation Reprinted from: Fermentation 2017 , 3 (1), 3; doi: 10.3390/fermentation3010003 .................................... 39 Minliang Yang, Weitao Zhang and Kurt A. Rosentrater Anhydrous Ammonia Pretreatment of Corn Stover and Enzymatic Hydrolysis of Glucan from Pretreated Corn Stover Reprinted from: Fermentation 2017 , 3 (1), 9; doi: 10.3390/fermentation3010009 .................................... 47 José Miguel Oliva, María José Negro, Paloma Manzanares, Ignacio Ballesteros, Miguel Ángel Chamorro, Felicia Sáez, Mercedes Ballesteros and Antonio D. Moreno A Sequential Steam Explosion and Reactive Extrusion Pretreatment for Lignocellulosic Biomass Conversion within a Fermentation - Based Biorefinery Perspective Reprinted from: Fermentation 2017 , 3 (2), 15; doi: 10.3390/fermentation3020015 .................................. 59 Úrsula Fillat, David Ibarra, María E. Eugenio, Antonio D. Moreno, Elia Tomás-Pejó and Raquel Martín-Sampedro Laccases as a Potential Tool for the Efficient Conversion of Lignocellulosic Biomass: A Review Reprinted from: Fermentation 2017 , 3 (2), 17; doi: 10.3390/fermentation3020017 .................................. 74 Christopher Chukwudi Okonkwo, Victor C. Ujor, Pankaj K. Mishra and Thaddeus Chukwuemeka Ezeji Process Development for Enhanced 2,3 - Butanediol Production by Paenibacillus polymyxa DSM 365 Reprinted from: Fermentation 2017 , 3 (2), 18; doi: 10.3390/fermentation3020018 .................................. 106 iv Daneal C. S. Rorke and Evariste Bosco Gueguim Kana Kinetics of Bioethanol Production from Waste Sorghum Leaves Using Saccharomyces cerevisiae BY4743 Reprinted from: Fermentation 2017 , 3 (2), 19; doi: 10.3390/fermentation3020019 .................................. 121 Mamatha Devarapalli, Randy S. Lewis and Hasan K. Atiyeh Continuous Ethanol Production from Synthesis Gas by Clostridium ragsdalei in a Trickle- Bed Reactor Reprinted from: Fermentation 2017 , 3 (2), 23; doi: 10.3390/fermentation3020023 .................................. 131 Raffaela Cutzu and Laura Bardi Production of Bioethanol from Agricultural Wastes Using Residual Thermal Energy of a Cogeneration Plant in the Distillation Phase Reprinted from: Fermentation 2017 , 3 (2), 24; doi: 10.3390/fermentation3020024 .................................. 144 John R. Phillips, Raymond L. Huhnke and Hasan K. Atiyeh Syngas Fermentation: A Microbial Conversion Process of Gaseous Substrates to Various Products Reprinted from: Fermentation 2017 , 3 (2), 28; doi: 10.3390/fermentation3020028 .................................. 152 Rosalie Allard-Massicotte, Hassan Chadjaa and Mariya Marinova Phenols Removal from Hemicelluloses Pre - Hydrolysate by Laccase to Improve Butanol Production Reprinted from: Fermentation 2017 , 3 (3), 31; doi: 10.3390/fermentation3030031 .................................. 178 v About the Special Issue Editor Thaddeus Ezeji is an Associate Professor in the Department of Animal Sciences at Ohio State University (OSU) and Ohio State Agricultural and Development Center (OARDC). Dr. Ezeji received his PhD (Magna cum laude) in Microbiology in 2001 from University of Rostock, Germa ny. During his 6 years as a post- doctoral and Research Assistant Professor at the University of Illinois Urbana - Champaign, he focused on fermentation technologies and metabolic engineering of Clostridium species. In 2007, Dr. Ezeji joined the OSU and OARDC. His current research focuses on the development of non - food substrates for the production of fuels and chemicals, and the design of advanced fermentation systems. Additionally, Dr. Ezeji’s laboratory conducts research activities on genetic and metabolic engineering of bacteria of biotechnological significance. Dr. Ezeji is involved in outreach programs that address emerging issues from Ohio’s expanding bioenergy sector. He has published more than 60 scientific papers, 16 book chapters, and co -edited 2 books. fermentation Editorial Production of Bio-Derived Fuels and Chemicals Thaddeus Chukwuemeka Ezeji ID Department of Animal Sciences and Ohio Agricultural Research and Development Center (OARDC), The Ohio State University, 305 Gerlaugh Hall, 1680 Madison Avenue, Wooster, OH 44691, USA; ezeji.1@osu.edu Received: 28 August 2017; Accepted: 28 August 2017; Published: 30 August 2017 Keywords: lignocellulose; biomass; pretreatment; butanol; ethanol; butanediol; acetone; LDMIC; hydrogen The great demand for, and impending depletion of petroleum reserves, the associated impact of fossil fuel consumption on the environment, and volatility in the energy market have elicited extensive research on alternative sources of traditional petroleum-derived products such as biofuels and bio-chemicals. Fossil oil is largely associated with gasoline, however, approximately 6000 petroleum-derived products currently exist in the market, with diverse applications. Ironically, while biofuels are more popular with the public, the other petroleum-derived products have not attracted similar attention despite the vast economic values for these products. Thus, given the finite nature of petroleum, it is timely to deploy substantial resources and research efforts to the development of renewable chemicals (similar to the efforts devoted to biofuels). Theoretically, bio-production of gasoline-like fuels and the 6000 petroleum-derived products is within the realm of possibility, because aquatic and terrestrial ecosystems harbor an abundance of diverse microorganisms, capable of catalyzing unlimited numbers of chemical reactions. Moreover, the fields of synthetic biology and metabolic engineering have evolved to the point that a wide range of microorganisms can be induced or manipulated to catalyze foreign or vastly improve indigenous biosynthetic reactions. Hence the need for this Special Issue to provide a platform for highlighting recent progress on fuel and chemical production from renewable resources such as lignocellulosic biomass. This Special Issue, titled Biofuels and Biochemicals Production, consists of 13 articles in which eleven and two are research and review articles, respectively. The Special Issue covers themes on the development of different methodologies for efficient conversion of lignocellulosic biomass, agricultural wastes, carbon dioxide, and carbon monoxide to fuels (ethanol, butanol, hydrogen), chemicals (2,3-butanediol, acetone, acetic acid), and enzymes (cellulase). Some of the articles in this Special Issue provide recent advancements on pretreatment and hydrolysis of lignocellulosic biomass (LB) to lignocellulosic biomass hydrolysates (LBH), challenges associated with LBH utilization, and recommended mitigation strategies. Consistent with the Biofuels and Biochemicals Production theme, the research groups of Moreno [ 1 ] and Rosentrater [ 2 ] evaluated different pre-treatment technologies for efficient disruption and separation of lignin from the hemicellulose component of the LB to facilitate enzymatic hydrolysis of the carbohydrate fraction to fermentable sugars. By combining acid-catalyzed steam explosion and alkali-based extrusion process, the protective lignin structure of barley straw was disrupted, which resulted in hydrolysates with significant amounts of glucan and hemicellulose sugars, minimal concentrations of lignocellulose derived microbial inhibitory compounds (LDMICs), and a solid residue with significant amounts of lignin [ 1 ]. In addition, the Low-Moisture Anhydrous Ammonia (LMAA) pre-treatment method enhanced enzymatic hydrolysis of the cellulose component of the LB to glucose, thus, the potential is great for LMAA for LB pre-treatment [ 2 ]. Consistent with enzymatic hydrolysis of the cellulose component of LB, Bajaj’s group contributes an article that highlights the capacity of Bacillus subtilis SV1 to use agroindustrial residues (LB) as carbon and nitrogen sources for growth and ionic liquid (IL) stable cellulase production followed by the hydrolysis of IL-pretreated LB Fermentation 2017 , 3 , 42 1 www.mdpi.com/journal/fermentation Fermentation 2017 , 3 , 42 to fermentable sugars [ 3 ]. Unfortunately, pre-treatment and hydrolysis of LB can result in the formation of a complex mixture of LDMICs that are toxic to fermenting microbes. Examples of LDMICs are furfural, hydroxymethylfurfural (HMF), benzaldehyde, syringaldehyde, and acetic, ferulic, glucuronic, p-coumaric, syringic, levulinic acids, and so on [ 4 ]. Overcoming the barriers imposed by LDMICs motivated the study conducted by Marinova’s group in which LDMICs of phenol origin in LBH were detoxified using nanofiltration, flocculation, laccase, and combinations thereof [ 5 ]. Detoxification of LBH by a combination of flocculation and laccase enzymes before fermentation drastically reduced the concentration of LDMICs in LBH, and significantly improved the fermentation of LBH to butanol [5]. To go beyond conversion of LB to fermentable sugars and produce usable products with lesser carbon footprints, Rorke and Kana [ 6 ] evaluated the feasibility of using Monod and modified Gompertz models to study the kinetic behaviour of a bioethanol fermentation process using sorghum leaves and Saccharomyces cerevisiae as a substrate and fermentation microorganism, respectively. Interestingly, obtained Monod and modified Gompertz coefficients indicated that waste sorghum leaves can serve as an efficient substrate for bioethanol production. Similarly, Bardi and Cutzu [ 7 ] evaluated production of ethanol from agricultural wastes (apple, kiwifruit, peach wastes, and corn threshing residue) using residual thermal energy from ethanol distillation column. Their article recapitulates different concentrations of ethanol obtained from these wastes during ethanolic fermentation with S. cerevisiae With the exception of peach wastes, all the waste substrates assessed had promise for industrial ethanol fermentation, a finding that bodes well with use of non-food crops for biofuel production. Additionally, Krömer’s group contributes a technical note that describes simultaneous quantitation of sugars, carboxylates, alcohols and aldehydes in fermentation broth by High Performance Liquid Chromatography (HPLC) [ 8 ]. The developed method allows quantitation of 21 compounds in a single process, and could be used in LB pretreatment, hydrolysis, and fermentation of LBH to fuels and chemicals’ research. The two articles from Atiyeh’s and Sekoai’s groups focus on production of ethanol and acetic acid from synthesis gas by Clostridium ragsdalei [ 9 ] and optimization of fermentative production of hydrogen using Box–Behnken design [ 10 ]. Notably, Atiyeh’s article is the first study on continuous operation of syngas fermentations in a trickle-bed reactor (TBR) for ethanol and acetic acid production, and the report highlights operational constraints and challenges of continuous syngas fermentation in TBR, and how the bioreactor operation can be restarted after major accidents such as flooding and power shutdown [ 9 ]. Sekoai’s study indicates that there can be an improved biohydrogen production yield of 603.5 mL H 2 /g total volatile solid (TVS) or more which is achievable at optimized operational set point variables of 39.56 g/L, 82.58 h, 5.56, and 37.9 ◦ C for substrate concentration, fermentation time, pH, and temperature, respectively; a finding that could facilitate the use of large-scale biohydrogen production processes [10]. Fermentative production of chiral compounds is currently receiving remarkable attention because of the numerous industrial applications in the biofuel, synthetic rubber, bioplastics, cosmetics, and flavor industries, and high cost of production from chemical synthetic routes. Recognizing the importance of chiral compounds in the biotechnology industry, our group [ 11 ] contributed an article in which process development for enhanced 2,3-butanediol (2,3-BD) production by non-pathogenic bacterium, Paenibacillus polymyxa DSM 365, was emphasized. Indeed, while our group was able to increase the concentration of 2-3-BD from 47 g/L (un-optimized) to 68.5 g/L (optimized) under fed-batch fermentation condition, the results underscore an interaction between medium components and fermentation conditions, which tends to influence 2,3-BD and undesirable exopolysaccharides (EPS) production [ 11 ]. Although butanol is an achiral compound, it is an important chemical with many applications in the production of solvents, butyl acetates, butylamines, plasticizers, amino resins, etc. [ 12 ]. These facts were echoed by Li’s group whose article focused on the feasibility of using acidified fibrous immobilization materials (cotton balls, modal fiber and charcoal fiber) to improve production [ 13 ]. By pre-treating modal fiber materials with 3.5% HCl for 12 h, the structure of modal 2 Fermentation 2017 , 3 , 42 fibers was etched to decrease mass transfer resistance, increased adsorption of C. acetobutylicum to the material, and ultimately, enhanced the kinetics of acetone butanol ethanol (ABE) fermentation [13]. The review articles in this Special Issue provide insights into syngas fermentation [ 14 ] and the significance of laccases in the development of LB as an important substrate for the production of renewable fuels and chemicals [ 15 ]. The review article contributed by Phillips et al. [ 14 ] indicates that integration of thermochemical gasification of LB and wastes to syngas (CO, CO 2 and H 2 ) and syngas fermentation by autotrophic bacteria is a robust and potentially economical process for the production of fuels and chemicals. Important concepts such as Wood–Ljungdahl biochemical pathway reactions and applications, gas solubility, mass transfer, thermodynamics of enzyme-catalyzed reactions, electrochemistry and cellular electron carriers and fermentation kinetics, were highlighted [ 14 ]. The review article contributed by Fillat et al. [ 15 ] provides important studies and perspectives on the use of laccases as a delignification and detoxification tool for efficient conversion of LB into value-added products, with emphasis on lignocellulosic ethanol production; highlighting major challenges and opportunities, and plausible ways to integrate the enzymes in the future lignocellulose-allied industries. In conclusion, it is my hope that this Special Issue will serve as a useful resource for students, teachers, professors, engineers, government personnel, and anyone actively or passively involved in renewable fuels and chemical production and research. In summary, I wish to thank our article contributors, Editorial Board members, Ad Hoc reviewers, and Assistant Editors of this journal, whose contributions made the publication of this Special Issue possible. Conflicts of Interest: The author declares no conflict of interest. References 1. Oliva, J.M.; Negro, M.J.; Manzanares, P.; Ballesteros, I.; Chamorro, M.A.; S á ez, F.; Ballesteros, M.; Moreno, A.D. A Sequential Steam Explosion and Reactive Extrusion Pretreatment for Lignocellulosic Biomass Conversion within a Fermentation-Based Biorefinery Perspective. Fermentation 2017 , 3 , 15. [CrossRef] 2. Yang, M.; Zhang, W.; Rosentrater, K.A. Anhydrous Ammonia Pretreatment of Corn Stover and Enzymatic Hydrolysis of Glucan from Pretreated Corn Stover. Fermentation 2017 , 3 , 9. [CrossRef] 3. Nargotra, P.; Vaid, S.; Bajaj, B.K. Cellulase Production from Bacillus subtilis SV1 and Its Application Potential for Saccharification of Ionic Liquid Pretreated Pine Needle Biomass under One Pot Consolidated Bioprocess. Fermentation 2016 , 2 , 19. [CrossRef] 4. Ezeji, T.C.; Qureshi, N.; Blaschek, H.P. Butanol Production from Agricultural Residues: Impact of Degradation Products on Clostridium beijerinckii Growth and Butanol Fermentation. Biotechnol. Bioeng. 2007 , 97 , 1460–1469. [CrossRef] [PubMed] 5. Allard-Massicotte, R.; Chadjaa, H.; Marinova, M. Phenols Removal from Hemicelluloses Pre-Hydrolysate by Laccase to Improve Butanol Production. Fermentation 2017 , 3 , 31. [CrossRef] 6. Rorke, D.C.S.; Kana, E.B.G. Kinetics of Bioethanol Production from Waste Sorghum Leaves Using Saccharomyces cerevisiae BY4743. Fermentation 2017 , 3 , 19. [CrossRef] 7. Cutzu, R.; Bardi, L. Production of Bioethanol from Agricultural Wastes Using Residual Thermal Energy of a Cogeneration Plant in the Distillation Phase. Fermentation 2017 , 3 , 24. [CrossRef] 8. Lai, B.; Plan, M.R.; Hodson, M.P.; Krömer, J.O. Simultaneous Determination of Sugars, Carboxylates, Alcohols and Aldehydes from Fermentations by High Performance Liquid Chromatography. Fermentation 2016 , 2 , 6. [CrossRef] 9. Devarapalli, M.; Lewis, R.S.; Atiyeh, H.K. Continuous Ethanol Production from Synthesis Gas by Clostridium ragsdalei in a Trickle-Bed Reactor. Fermentation 2017 , 3 , 23. [CrossRef] 10. Sekoai, P. Modelling and Optimization of Operational Setpoint Parameters for Maximum Fermentative Biohydrogen Production Using Box-Behnken Design. Fermentation 2016 , 2 , 15. [CrossRef] 11. Okonkwo, C.C.; Ujor, V.C.; Mishra, P.K.; Ezeji, T.C. Process Development for Enhanced 2,3-Butanediol Production by Paenibacillus polymyxa DSM 365. Fermentation 2017 , 3 , 18. [CrossRef] 3 Fermentation 2017 , 3 , 42 12. Ezeji, T.C.; Qureshi, N.; Blaschek, H.P. Microbial Production of a Biofuel (Acetone-Butanol-Ethanol) in a Continuous Bioreactor: Impact of Bleed and Simultaneous Product Removal. Bioprocess Biosyst. Eng. 2013 , 36 , 109–116. [CrossRef] [PubMed] 13. Zeng, H.-S.; He, C.-R.; Yen, A.T.-C.; Wu, T.-M.; Li, S.-Y. Assessment of Acidified Fibrous Immobilization Materials for Improving Acetone-Butanol-Ethanol (ABE) Fermentation. Fermentation 2017 , 3 , 3. [CrossRef] 14. Phillips, J.R.; Huhnke, R.L.; Atiyeh, H.K. Syngas Fermentation: A Microbial Conversion Process of Gaseous Substrates to Various Products. Fermentation 2017 , 3 , 28. [CrossRef] 15. Fillat, U.; Ibarra, D.; Eugenio, M.E.; Moreno, A.D.; Tom á s-Pej ó , E.; Mart í n-Sampedro, R. Laccases as a Potential Tool for the Efficient Conversion of Lignocellulosic Biomass: A Review. Fermentation 2017 , 3 , 17. [CrossRef] © 2017 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 4 fermentation Technicalnote Simultaneous Determination of Sugars, Carboxylates, Alcohols and Aldehydes from Fermentations by High Performance Liquid Chromatography Bin Lai 1,2,† , Manuel R. Plan 3,4,† , Mark P. Hodson 3,4 and Jens O. Krömer 1,2, * 1 Centre for Microbial Electrochemical Systems (CEMES), The University of Queensland, Brisbane QLD 4072, Australia; b.lai@uq.edu.au 2 Advanced Water Management Centre (AWMC), The University of Queensland, Brisbane QLD 4072, Australia 3 Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane QLD 4072, Australia; m.plan@uq.edu.au (M.R.P); m.hodson1@uq.edu.au (M.P.H) 4 Metabolomics Australia (Queensland Node), The University of Queensland, Brisbane QLD 4072, Australia * Correspondence: j.kromer@uq.edu.au; Tel.: +61-733-463-222; Fax: +61-733-654-726 † These authors contributed equally to this work. Academic Editor: Thaddeus Ezeji Received: 29 January 2016; Accepted: 24 February 2016; Published: 7 March 2016 Abstract: Despite the rise of ‘omics techniques for the study of biological systems, the quantitative description of phenotypes still rests to a large extent on quantitative data produced on chromatography platforms. Here, we describe an improved liquid chromatography method for the determination of sugars, carboxylates, alcohols and aldehydes in microbial fermentation samples and cell extracts. Specific emphasis is given to substrates and products currently pursued in industrial microbiology. The present method allows quantification of 21 compounds in a single run with limits of quantification between 10 ́ 7 and 10 ́ 10 mol and limits of detection between 10 ́ 9 and 10 ́ 11 mol. Keywords: high performance liquid chromatography; ion-exchange chromatography; metabolite separation; fermentation product quantification 1. Introduction High performance liquid chromatography (HPLC) has been widely used for quantification of compounds in biological samples [ 1 ]. It is precise, quantitative and highly reproducible, but, depending on the analysis, HPLC can be slow and the analysis of different compound classes are best performed with dedicated columns and methods [ 2 ]. To date, numerous HPLC-based methods have been developed for analyzing sugars [ 3 ], organic acids [ 4 , 5 ] and alcohols [ 6 ], respectively. However, running multiple dedicated methods has an impact on sample throughput, unless several instruments are available. In addition, sample throughput can only be increased by reducing chromatographic acquisition time, which may subsequently compromise peak resolution and, thus, data reproducibility. Therefore, a combined method permitting analysis of multiple compound classes is preferable and desirable, permitting the analyst to strike a balance between best possible analysis and throughput. However, the few published combined methods were either operated at high temperatures or achieved lower compound resolutions [7,8]. Despite the progress in column development in other areas of chromatography, such as rapid resolution in reversed phase applications, the method of choice for combined quantification of alcohols, organic acids and sugars is still ion-exchange chromatography [ 9 , 10 ], and due to the use of refractive index detection for sugar and alcohol analysis, this is still mainly based on isocratic elution. Fermentation 2016 , 2 , 6 5 www.mdpi.com/journal/fermentation Fermentation 2016 , 2 , 6 Amongst a wide range of applications for such analyses in the food and chemical industries, one important application is in biotechnology research. In particular, the quantitative analysis of compounds from fermentation samples can serve as an essential tool for the understanding of microbial phenotypes and for the development of improved microbial strains for the production of biofuels, fine chemicals or bulk chemical feedstocks as replacements for petrochemicals. Here, we present a thoroughly tested method that has broad application in microbiological research, providing quantitative data for a range of common substrates in microbial fermentation including hexoses, pentoses and disaccharides, while at the same time covering a broad range of fermentation products including mono-, di-, tri-alcohols, aldehydes, mono-, di- and tri-carboxylic acids, as well as sugar acids. While still based on cation-exchange, the method provides optimized operation temperature and mobile phase composition for a recently commercialized column. It has been optimized for simultaneous quantification of at least 21 compounds, including carbohydrates to varied alcohol products via central metabolism and has been applied to three very different samples. 2. Experimental Section 2.1. Chemicals A list of 30 compounds was tested, and all chemicals used in the study were of analytical grade and were purchased from Sigma–Aldrich (Sydney, Australia). Aqueous analyte solutions and mobile phase were prepared using high purity water (18.2 k Ω ) generated by an Elga Lab water purification system (Veolia Water Solutions and Technologies, Saint Maurice Cedex, France). 2.2. HPLC Set up Separation of compounds was performed on an Agilent 1200 HPLC system using an Agilent Hiplex H column (300 ˆ 7.7 mm, PL1170-6830, Santa Clara, CA, USA) with a guard column (SecurityGuard Carbo-H, Phenomenex PN: AJO-4490; Lane Cove West, New South Wales, Australia) for extended column life. Moreover, to extend column life, the column is cleaned with 0.2 mL/min of high purity water (18.2 M Ω ) at 60 ̋ C overnight and then regenerated with the same conditions using 25 mM sulfuric acid for a few hours, which is ideally performed after each batch of analysis. With regular column maintenance and careful sample preparation (e.g., samples pre-filtered using 0.22 μ m PES syringe filter (Millipore: Cork, Ireland) and pre-diluted microbial fermentation samples) we have been able to make more than 200 injections per batch of analysis without change in column performance ( i.e. , without significant RT drift or increase in back pressure). Sugars and alcohols were monitored using a refractive index detector (Agilent RID, G1362A) set on positive polarity and optical unit temperature of 40 ̋ C with mobile phase in the reference cell, while organic acids were monitored using RID and/or ultraviolet detector at 210 nm (Agilent MWD, G1365B). A sample volume of 30 μ L was injected onto the column using an autosampler (Agilent HiP-ALS, G1367B) and the column temperature was maintained at 40 ̋ C using a thermostatically controlled column compartment (Agilent TCC, G1316A). Analytes were eluted isocratically with 14 mM H 2 SO 4 at 0.4 mL/min for 38 or 65 min (elution time was dependent upon whether higher alcohols were present in the sample). Chromatograms were integrated using Agilent ChemStation (Rev B.03.02; Santa Clara, CA, USA). 3. Results and Discussions A series of preliminary experiments were conducted to monitor the interaction of the retention times (RT) of compounds from various classes with column temperature (30, 50 and 65 ̋ C), mobile phase concentration (2, 4, 6, 8, 10, 12 and 14 mM) and flow rate (0.4, 0.5 and 0.6 mL/min) (see supplementary information) and found that a column temperature of 40 ̋ C, aqueous solution of H 2 SO 4 (14 mM) and a flow rate of 0.4 mL/min was the best combination to achieve separation of the highest number of target compounds. 6 Fermentation 2016 , 2 , 6 With the optimized operating parameters, the method developed in this article is suitable for mapping varied metabolic routes from carbohydrates, via carboxylic acids to alcoholic products (Figure 1) and is, thus, highly relevant for fermentation process development. Looking at widely used sugar substrates for fermentation [ 11 ] and sugar products occurring in bioprocesses [ 12 ], our method has the capacity to separate D -trehalose, D -glucose, D -galactose, L -arabinose and D -ribose in the same sample. D -fructose and D -galactose partially overlap, which means they should only be quantified if the other sugar is known to be absent from the sample, the same holds for the disaccharides D -maltose and D -trehalose. Sucrose exhibited partial on/in-column inversion and cannot be analyzed reliably with the presented method, however the same column with water as the mobile phase would be suitable for sucrose quantification (data not shown). In addition to the fermentation substrates, 10 organic acids related to central metabolism were identifiable and quantifiable in a single injection with this method, including two specific sugar acids, gluconic acid and 2-ketogluconic acid, making this method suitable for microbes that favour the Embden–Meyerhof–Parnas, as well as those using the Entner–Doudoroff pathway for sugar utilization. This extends the applicability of the method amongst others to the group of Pseudomonads , which contains a range of new strains for biotechnology that are currently widely studied for biosynthesis of chemicals [ 13 ]. It has to be noted that citric acid and 2-ketogluconic acid co-elute with this method and should not occur simultaneously. Previously published methods struggled to separate compounds like formic acid and fumaric acid, 2-ketoglutaric acid and citric acid, pyruvic acid and glucose [14]. These can now be successfully resolved and quantified in the same sample. ȱ Figure 1. Substrates, intermediates and products of microbial fermentation captured by the presented method in culture broth. Sugars (blue), organic acids (yellow), alcohols, ketones and aldehydes (green). Looking at target compounds for biotechnology, this method is able to analyze a range of alcohols currently studied as biofuels and chemical feedstock replacements. This includes ethanol, 1-butanol, sec -butanol, iso -butanol, 1-propanol as well as 2-propanol (Table 1). Acetone and its structural isomer propionaldehyde are the metabolic precursors to 2-propanol and 1-propanol, respectively, and can now be analyzed with their respective end product in the same solution. Butyric acid and iso -butyric acid, the main by-products of butanol fermentation, can be quantified simultaneously as well. One problem is the separation of 2-propanol and butyric acid, these will partly overlap with the current chromatographic conditions. In any case, peak identification should be confirmed with alternative means (e.g., mass spectrometry) in complex samples. The calibration curves achieved a good fit and recoveries in the standard matrix were high (Table 1). The achieved peak shape and elution profiles were acceptable for an isocratic HPLC method (Figure 2). 7 Fermentation 2016 , 2 , 6 Table 1. Analytes quantified with the presented method in order of retention time. LOQ: limit of quantification; S/N: Signal-to-Noise ratio; LOD: limit of detection. LOQ and LOD are given both as concentration in the sample, as well as amount injected. LOQ, LOD and S/N were detected and calculated based on the specified detector for quantification for each compound. In other sample matrices LOQ, LOD and S/N might vary. Calibration curves were obtained through linear regression (forced through the origin) of five standard points covering the linear detection range. UV/RT: retention time in UV detector; RI/RT: retention time in RI detector, detectors in series. Peak Compound RT (min) LOQ S/N LOQ LOD S/N LOD Calibration Curve Recovery (%) UV RI mM nmol mM nmol Detector Slope R 2 1 D -Trehalose - 12.53 0.098 2.94 10.9 0.012 0.37 2.8 RI 203,384 0.99999 100.6 D -Maltose - 12.56 0.117 3.52 12.4 0.029 0.88 2.9 RI 218,036 0.99998 100.3 2 2-Ketogluconic acid 13.34 13.70 0.158 4.73 12.3 0.005 0.16 2.9 UV 374 0.99997 102.3 Citric acid 13.42 13.78 0.060 1.80 15.0 0.010 0.30 2.9 UV 827 0.99999 100.0 3 Gluconic acid 14.13 14.49 0.159 4.77 11.4 0.005 0.16 3.2 UV 343 0.99999 100.6 D -Glucose - 14.50 0.391 11.72 9.4 0.049 1.46 1.9 RI 116,091 0.99977 101.1 4 2-Ketoglutaric acid 14.94 15.29 0.025 0.75 10.7 0.003 0.09 2.1 UV 706 0.99996 99.9 D -Galactose - 15.38 0.300 9.00 20.1 0.075 2.24 3.1 RI 119,106 0.99974 96.0 D -Fructose - 15.64 0.365 10.94 14.9 0.024 0.73 3.6 RI 109,365 0.99996 101.8 5 Pyruvic acid 16.33 16.68 0.020 0.59 7.2 0.005 0.15 2.3 UV 1976 0.99992 96.5 L -Arabinose - 16.68 0.365 10.95 13.8 0.091 2.74 1.8 RI a 0.99999 100.0 6 D -Ribose - 17.17 0.266 7.99 11.3 0.018 0.53 1.6 RI 86,258 0.99996 102.7 7 Succinic acid 19.01 19.43 0.196 5.87 9.4 0.024 0.73 1.8 UV 285 0.99996 99.8 8 Lactic acid 19.90 20.27 0.235 7.06 10.9 0.029 0.88 1.7 UV 297 0.99997 99.4 Glycerol - 20.45 0.156 4.68 44.1 0.078 2.34 3.0 RI 199,288 0.99999 98.9 9 1,3-Dihydroxyacetone 20.51 20.88 0.363 10.90 9.2 0.060 1.8 2.6 UV 192 0.9995 105.6 11 Formic acid 21.53 21.89 0.469 14.08 12.3 0.059 1.76 2.0 UV 186 0.99999 99.8 11 Acetic acid 23.46 23.83 0.361 10.84 9.2 0.045 1.36 1.7 UV 147 0.99999 100.2 12 Fumaric acid 25.33 - 0.006 0.19 32.6 0.001 0.02 4.2 UV 44,143 0.99967 95.8 13 1,3-Propanediol - 26.48 0.811 24.33 7.3 0.203 6.08 3.1 RI 37,638 0.99986 98.9 14 Propionic acid 28.22 28.59 0.276 8.27 6.5 0.034 1.03 1.8 UV 177 0.99998 100.2 15 Ethanol - 32.45 9.609 288.26 10.8 0.601 18.02 2.0 RI 12,451 0.99994 99.7 iso -Butyric acid 32.76 33.13 0.394 11.82 17.8 0.049 1.48 2.9 UV 274 0.99987 99.5 16 Propionaldehyde - 34.94 1.634 49.03 9.8 0.204 6.13 1.8 RI 21,298 0.99982 97.5 Acetone - 35.16 1.568 47.05 9.7 0.399 11.97 2.3 RI 16,063 0.99999 101.0 17 2-Propanol - 35.96 1.616 48.48 10.1 0.404 12.12 1.7 RI 23,012 0.99998 101.2 Butyric acid 36.02 36.38 0.450 13.51 12.9 0.074 2.23 2.3 UV 212 0.99998 102.3 18 1-Propanol - 41.38 1.566 46.97 15.5 0.196 5.87 2.0 RI 10,988 0.99966 100.7 19 sec -Butanol - 48.87 1.558 46.73 9.8 0.195 5.84 1.5 RI 33,209 0.99999 99.5 20 iso -Butanol - 52.07 2.286 68.58 13.9 0.377 11.3 2.3 RI 35,461 0.99999 101.1 21 1-Butanol - 59.97 1.565 46.96 11.9 0.391 11.74 2.6 RI 34,911 0.99999 99.3 a The line of best fit for arabinose was quadratic ( y = 497.65*xˆ2 + 64,692.83*x). 8 Fermentation 2016 , 2 , 6 Figure 2. HPLC chromatograms of selected standard mixture that is quantifiable in a single injection. Note: Not all compounds listed in Table 1 are included in the mixture and focus should be on peak shape. Abbreviations: Mal (Maltose), 2KGA (2-Ketogluconic acid), GlcA (Gluconic acid), 2KG (2-Ketoglutaric acid), Pyr (Pyruvic acid), Suc (Succinic acid), Lac (Lactic acid), FA (Formic acid), 1,3DHA (1,3-Dihydroxyacetone), 1,3PDO (1,3-Propanediol). Finally, the method was tested on three different samples: (i) fermentation broth of a genetically modified Escherichia coli fermentation during aerobic growth on glucose in minimal medium; (ii) fermentation broth of Saccharomyces cerevisiae growing on the carbon sources glycerol and ethanol in minimal medium and finally; (iii) the culture supernatant of Chinese Hamster Ovary (CHO) cells in complex cell culture medium using glucose and galactose as the main carbon source (Figure 3). In all three samples peak shapes and separation was good and the main substrates and products could successfully be quantified. This demonstrates the robustness and versatility of the described method. Based on the limits of detection, this method should also in principle be applicable to cell extracts. Quantitative data obtained from this method on fermentation samples of Pseudomonas putida was of sufficient quality to close the carbon and redox balances [ 15 ], underlining the value of this method for a range of applications. Figure 3. Cont 9 Fermentation 2016 , 2 , 6 Figure 3. HPLC chromatograms for supernatants of genetically modified E. coli ( A , B ) growing on glucose in minimal medium, genetically modified S. cerevisiae growing on glycerol and ethanol ( C , D ) in minimal medium and Chinese Hamster Ovary (CHO) cells growing on galactose and glucose in defined cell culture medium. Chromatograms serve the purpose of highlighting resolving power and are not a reference chromatogram for the respective organisms. Detection is performed with UV ( A , C , E ) and RI ( B , D , F ). In these samples, no signals were observed beyond 35 min and chromatograms have been shortened for better visibility of the peaks. Injection signals are visible at around 10 min in UV and 10.4 min RI. Complex medium components in the CHO experiment mainly pass through the column ( E , F ). 4. Conclusions In summary, a broad range of metabolites could be separated and quantified in one HPLC injection with LOQ and LOD in ranges that will be suitable for a large range of fermentation samples, including microbial culture broth and cell culture media and could be extended to intracellular samples. The data quality allows drawing of carbon and degree of reduction balances. The method can be extended to other compounds, if presence of co-eluting compounds can be ruled out with alternative methods (e.g., by mass spectrometry on pooled samples). Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/2311-5637/2/1/6/s1. Acknowledgments: Bin Lai acknowledges scholarship support from The University of Queensland. Manuel R. Plan and Mark P. Hodson acknowledge financial support from Metabolomics Australia. Jens O. Krömer was supported by the Australian Research Council (DE120101549). We thank Alex Prima, Nils Averesch, Axayacatl Gonzalez Garcia and Veronica Martinez for the supply of samples. Author Contributions: Bin Lai and Manuel R. Plan designed, performed the experiments and analyzed the data, under the guidance from Mark P. Hodson and Jens O. Krömer. All authors contributed to the drafting and editing of the manuscript. Conflicts of Interest: The authors declare no conflict of interest. 10 Fermentation 2016 , 2 , 6 References 1. Pereira da Costa, M.; Conte-Junior, C.A. Chromatographic methods for the determination of carbohydrates and organic acids in foods of animal origin. Compr. Rev. Food Sci. Saf. 2015 , 14 , 586–600. [CrossRef] 2. Ball, S.; Lloyd, L. Agilent Hi-Plex Columns for Carbohydrates, Alcohols, and Acids. Available online: http://www.agilent.com/cs/library/applications/5990–8264EN.pdf (accessed on 7 March 2016). 3. Agblevor, F.A.; Hames, B.R.; Schell, D.; Chum, H.L. Analysis of biomass sugars using a novel hplc method. Appl. Biochem. Biotechnol. 2007 , 136 , 309–326. [CrossRef] [PubMed] 4. Womersley, C.; Drinkwater, L.; Crowe, J.H. Separation of tricarboxylic acid cycle acids and other related organic acids in insect haemolymph by high-performance liquid chromatography. J Chromatogr. A 1985 , 318 , 112–116. [CrossRef] 5. van Hees, P.A.W.; Dahlén, J.; Lundström, U.S.; Borén, H.; Allard, B. Determination of low molecular weight organic acids in soil solution by hplc. Talanta 1999 , 48 , 173–179. [CrossRe