Influence of Inhibitory Compounds on Biofuel Production from Oxalate

Article Inﬂuence of Inhibitory Compounds on Biofuel Production from Oxalate-Rich Rhubarb Leaf Hydrolysates Using Thermoanaerobacter thermohydrosulfuricus Strain AK91

Johann Orlygsson 1,* and Sean Michael Scully 1,2

1 Faculty of Natural Resource Science, School of Business and Science, University of Akureyri, Borgir v. Nordurslod, 600 Akureyri, Iceland; [email protected] 2 Faculty of Education, School of Humanities and Social Sciences, University of Akureyri, Solborg v. Nordurslod, 600 Akureyri, Iceland * Correspondence: [email protected]

Abstract: The present investigation is on bioethanol and biohydrogen production from oxalate-rich rhubarb leaves which are an underutilized residue of rhubarb cultivation. Rhubarb leaves can be the feedstock for bioethanol and biohydrogen production using thermophilic, anaerobic bacteria. The fermentation of second-generation biomass to biofuels by Thermoanaerobacter has already been reported as well as their high ethanol and hydrogen yields although rhubarb biomass has not been examined for this purpose. Thermoanaerobacter thermohydrosulfuricus strain AK91 was characterized (temperature and pH optima, substrate utilization spectrum) which demonstrates that the strain can utilize most carbohydrates found in lignocellulosic biomass. Additionally, the influence of specific culture conditions, namely the partial pressure of hydrogen and initial glucose concentration, were Citation: Orlygsson, J.; Scully, S.M. investigated in batch culture and reveals that the strain is inhibited. Additionally, batch experiments Influence of Inhibitory Compounds containing common inhibitory compounds, namely carboxylic acids and aldehydes, some of which on Biofuel Production from are present in high concentrations in rhubarb. Strain AK91 is not affected by alkanoic carboxylic acids Oxalate-Rich Rhubarb Leaf and oxalate up to at least 100 mM although the strain was inhibited by 40 mM of malate. Interestingly, Hydrolysates Using Thermoanaerobacter strain AK91 demonstrated the ability to reduce alkanoic carboxylic acids to their primary alcohols; thermohydrosulfuricus Strain AK91. more detailed studies with propionate as a model compound demonstrated that AK91’s growth Fuels 2021, 2, 71–86. https://doi.org/ is not severally impacted by high propionate loadings although 1-propanol titers did not exceed 10.3390/fuels2010005 8.5 mM. Additionally, ethanol and hydrogen production from grass and rhubarb leaf hydrolysates was investigated in batch culture for which AK91 produced 7.0 and 6.3 mM g−1, respectively. Academic Editor: Martin Olazar Keywords: second generation ethanol production; lignocellulosic biomass; rhubarb; Thermoanaerobacter Received: 28 December 2020 Accepted: 20 February 2021 Published: 8 March 2021 1. Introduction Publisher’s Note: MDPI stays neutral There has been an increased emphasis on developing alternatives to grain-based first with regard to jurisdictional claims in generation biofuels such as lignocellulosic and macro algal biomass, neither of which published maps and institutional affil- directly compete with food and feed production, using fermentative microbes such as iations. yeasts [1–3] as well as ethanol-producing bacteria [4–6]. The selection of crops as a raw material for bioprocessing is highly dependent upon local growing conditions as well as crop traits and composition. Recent work has demonstrated that the use of perennial crops grown on marginal lands that are not otherwise suitable for cultivation can bypass the Copyright: © 2021 by the authors. conflict between the environmental impacts associated with land usage while minimizing Licensee MDPI, Basel, Switzerland. carbon footprint [7]. While first generation biomass includes biomass that contains a large This article is an open access article fraction of easily fermentable sugars, second-generation biomass uses complex lignocellu- distributed under the terms and losic biomass such as organic agricultural waste (e.g., stems, straw, leaves, husks), industry conditions of the Creative Commons waste (e.g., woodchips, skins, pulp), and non-food crops (e.g., grass) as a raw material [6]. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ A potential second generation biomass widely cultivated in Iceland is the oxalate- and 4.0/). malate-rich leaves of perennial plant Rhubarb (Rheum rhabarbarum) which are discarded

Fuels 2021, 2, 71–86. https://doi.org/10.3390/fuels2010005 https://www.mdpi.com/journal/fuels Fuels 2021, 2 72

after the petiole is utilized for its nutritional value although the presence of potentially inhibitory compounds such as oxalate and malate poses a challenge for bioprocessing. The main components of lignocellulose are lignin, hemicellulose, and cellulose, all of which are tightly bound together necessitating pre-treatment prior to enzymatic decon- struction and fermentation. Pre-treatment regiments routinely consist of a combination of physical, chemical, physio-chemical, or enzymatic methods to liberate fermentable carbohydrates but employ elevated temperatures and acidic conditions [8]. The pre-treatment process is often accompanied by the generation of inhibitory compounds that later often negatively affect the fermentation process by hindering the growth of microorganisms; alternately, inhibitory compounds generated during biomass pre-treatment can be removed with an extra detoxification step. Thus, the goals of pre-treatment are to minimize the formation of unwanted compounds and maximize sugar extraction. Inhibitory furans, such as 2-furfuraldehyde (2-FF) and 5-hydroxymethyl-2-furfuraldehyde (5-HMF), which are formed from five and six carbon monosaccharides, respectively, under conditions commonly found in acidic pre-treatment [8]. 2-Furfuraldhyde has been shown to strongly inhibit alcohol dehydrogenases in yeast while 5-HMF has been found to be inhibitory for some important metabolic enzymes [9]. Additionally, the hemicellulose fraction is often partially acylated and these acyl groups can undergo hydrolysis liberating organic acids under common pre-treatment conditions; carboxylic acids that are fully protonated can cross the cell membrane and cause inhibition by lowering the intracellular pH. The lignin fraction is composed of a random heteropolymeric material consisting of aromatic residues, namely hydroxyphenyl, guaiacyl, and syringyl monomers such as p-coumaryl, coniferyl, and sinapyl alcohols, respectively [8]. While lignin is highly resistant to degradation, the partial hydrolysis of the lignin can liberate these toxic aromatic alcohols, aldehydes, and carboxylic acids which can negatively impact microbial growth and alter fermentation performance. Rhubarb is a perennial species which produces long fleshy edible stalks and large leaves that are poisonous due to the presence of many compounds including, oxalic acid, and maleic acid thus making it a potentially challenging raw material for bioprocessing. While rhubarb stalks are rich in sugars, namely sucrose, and are regarded as a food source, the leaves contain several organic acids making them unfit for human and animal consumption. Oxalic acid is a strong dicarboxylic acid that can be corrosive with a pKa values of 1.25 and 4.14 and a high solubility in water (143 g/L at 25 ◦C); it has notable toxicity with an approximate LD50 of 0.6 g/kg (human) [10]. The typical value of oxalic acid in rhubarb is about 0.5% w/w but may be minimized by cooking the leaves [10,11]. Due to their relative abundance, ease of cultivation, and low cost, rhubarb leaves are a potential renewable feedstock for biofuel production that does not compete with food production. In Iceland, rhubarb has been harvested in Eyjafjörður (N-Iceland) and in Árnessýsla (S-Iceland) for its sugar-rich stem used in the food industry although harvested quantities are not available. Additionally, Iceland’s annual import of rhubarb is 50–60 tons which is used to supplement locally grown crops in order to meet market demand although rhubarb producers in Eyjafjörður are aiming to increase production and ultimately export rhubarb. While a wide range of bioprocessing organisms have been considered for the fermentation of lignocellulosic biomass to bioethanol, namely yeasts [1,3] and highly ethanologic bacteria such as Zymomonas, a commonly encountered drawback of these microorganisms is their limited ability to ferment components of lignocellulosic biomass. In this regard, thermophilic bioprocessing organisms within the genus of Thermoanaerobacter have demonstrated a diverse applicability to the conversion of cellulosic biomass into biofuels [4,6,12]. Thermoanaerobacter strains degrade a wide variety of substrates; hexoses, pentoses, methylpentoses, disaccharides, and tolerate various extremes of temperature, pH, and can grow in the presence of inhibitory compounds [13]. Conversely, like many thermophilic anaerobes, many strain studies so far demonstrate low tolerances for initial substrate concentration [14–16] but this may be overcome using other fermentation modes. Fuels 2021, 2 73

The purpose of this work was to investigate the production of bioethanol and biohydrogen from unutilized rhubarb leaves using Thermoanaerobacter thermohydrosulfuricus strain AK91 isolated from Icelandic geothermal spring. The effects of various environmental factors were investigated to maximize both ethanol and hydrogen production. Additionally, tolerance of the strain towards various inhibitory compounds, namely aldehydes generated from hexoses and pentoses, and carboxylic acids were investigated. Finally, the ability of strain AK91 to produce bioethanol from lignocellulosic biomass hydrolysates, including oxalate-rich rhubarb, was evaluated.

2. Materials and Methods 2.1. Culture Media and Organisms 13 All materials were pursued from Sigma Aldrich, except for C1-labeled propionic acid which is from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Pure nitrogen gas (<5 ppm O2) was used in all cases. Thermoanaerobacter strain AK91, was isolated from a hot spring 67 ◦C, pH 7.4) in Iceland with methods as previously described [15]. The medium used (BM) was according to [15]. Briefly, the medium was boiled for 15 min, chilled on an ice bath under nitrogen flushing, and dispersed to the experimental bottles to give the appropriate volume during active nitrogen flushing. Finally, the bottles were closed with butyl rubber stopper and aluminum caps, and autoclaved at 121 ◦C for 15 min. Glucose, vitamins, and trace elements were added from a syringe-filtered nitrogen-flushed stock bottle after autoclaving. Hungate tubes or serum bottles were used for cultivations without agitation. Tem- perature and pH were 70 ◦C and 7.0, respectively. In all cases, a liquid–gas phase ration (L-G ratio) of 1.00 (1:1 ratio of liquid and gas) was used for a period of 5 days unless stated otherwise. All experiments were conducted in triplicate unless stated otherwise. The inoculation volume was 2% (v/v) in all experiments.

2.2. Collection of Rhubarb Biomass and Preparation of Hydrolysates The Rhubarb (Rheum rhabarbarum) and Timothy grass (Phleum pratense) biomasses were collected from Eyjafjörður during summer 2015. The leaves were separated from the rhubarb stalks and used. The harvested biomass was dried in an incubator at 45–50 ◦C for 24 h, ground in a Waring blender and subsequently milled to <2 µm particles. The milled biomass was stored in airtight containers at ambient temperature. Then, 25 g of dry, milled biomass were weighed into screw cap bottles and approximately 600 mL of 0.5% (v/v)H2SO4 was added. The bottles were autoclaved for 60 min and titrated to pH 4.5 with 6 M NaOH. The pre-treatment of Whatman paper, Rhubarb leaf, and Timothy grass was as described earlier [17]. Briefly, the biomass received Cellulase (1 mL, 700 U/mL, Sigma) via aseptic addition and was incubated at 47 ◦C for 96 h. The hydrolysates were centrifuged (20 min, 4700 rpm) the resultant supernatant collected, its pH adjusted to 7 with 6 M NaOH, and final volume adjusted to 1 L. The hydrolysates were sequentially vacuum filtered through a 53 µm nylon filter, Whatman #1 filter paper (11 µm), 5 µm nylon filter, and a 0.45 µm filter. Finally, the hydrolysates were sterilized by filtering through a syringe filter (Whatman, PES 0.22 µm) into sterile, nitrogen flushed bottles.

2.3. Characterization and Substrate Spectra To determine temperature optimum for strain AK91, 117.5 mL serum bottles containing BM medium (pH 7.0) and glucose (20 mM) were used according to [15]. The pH optima of the strain was determined at the Topt by cultivating at initial pH values from 3 to 9 in 0.5 pH unit increments.

2.4. Inﬂuence of Initial Glucose Concentration and Liquid–Gas Phase Ratio To investigate the effect of different initial substrate concentrations, the strain was cultivated between 5 and 400 mM glucose. To evaluate the inﬂuence of L-G ratio the strain Fuels 2021, 2 74

was cultivated in 117.5 mL serum bottles containing different liquid volumes to yield L-G ratios of 0.017, 0.044, 0.093, 0.34, 1.04, and 3.27 as previously described [16].As an example, a L-G ratio of 1.0 is prepared by adding 59.25 mL of BM medium leaving 59.25 mL of headspace while a L-G ratio of 3.16 can be obtained by using 90.0 mL of medium leaving 28.5 mL of headspace.

2.5. Effect of Inhibitory Compounds on Glucose Fermentation The inﬂuence of inhibitory compounds during fermentation was evaluated for 11 potential inhibitors (acetate, propionate, butyrate, lactate, ethanol, malate, oxalate, levulinic acid, p-coumaric acid, 2-furfuraldehyde, and 5- HMF) at various concentrations (10–100 mM) during glucose (20 mM) fermentation. Experiments were performed in Hun- gate tubes (16 × 150 mm) at a L-G ratio of 1:1 with stock solutions of inhibitory compounds having been adjusted to pH 7. The cultures were incubated at 70 ◦C for ﬁve days under anaerobic conditions without stirring. Finally, end-products were analyzed.

2.6. Kinetic Study of Selected Inhibitory Compounds on Glucose Fermentation by Thermoanaerobacter Strain AK91 The inﬂuence of different concentrations (25, 50, 100 mM) of n-propionate on glucose fermentation kinetics by strain AK91 were performed in Hungate tubes (16 × 150 mm) at pH 7.0 and a L-G ratio of 1.00. Samples were collected after 24, 48, and 168 h for chemical analysis.

2.7. Fermentation of Biomass Hydrolysates The fermentation of biomass HLs were conducted in 117.5 mL bottles with biomass loadings equivalent to 2.5, 5, and 10 g/L In one case, the effect of different L-G ratios was tested for 2.5 g/L of biomass. Three different ratios were used: 0.04, 1.00, and 3.29. Then, 1 mL of fermentation broth was collected after 168 h for analysis.

2.8. Analytical Methods Proximate analysis of dried biomass (ash, protein, and fat) was performed using standard AOAC methods, namely ash residue by heating in an ashing oven, protein by Kjeldahl method, and fat by Soxhlet extraction [18]. The determination of cell biomass was determined by optical density (OD) of cultures using Shimadzu UV-1800 UV-Visible spectrophotometer (600 nm, l = 1 cm). Hydrogen and volatile end-products were analysed using a Clarus 580 (PerkinElmer) gas chromatograph equipped with a thermal conductivity detector (TCD) and ﬂame ion detector (FID), respectively, as reported previously [19]. Glucose concentration was determined colorimetrically using the Anthrone method as described previously [19]. Spectra for 13C NMR were obtained as previously reported [20].

3. Results and Discussion 3.1. Biomass Composition The protein, ash, fat, and carbohydrate content of the Rhubarb leaves and Timothy grass are summarized in Table1. The amounts of carbohydrates and fats in the Rhubarb leaves were similar to the values obtained for Timothy grass while the protein content was lower in Timothy grass.

3.2. Strain Characterization Thermoanarobacter strain AK91 was isolated from Icelandic hot spring (temperature 67 ◦C; pH 7.4) with already described techniques [15]. The strain has more than 99% simi- larity to Thermoanarobacter thermohydrosulfuricus based on the 16S rRNA gene (KR007665) as previously reported [21]. Species of the genus Thermoanaerobacter are strictly anaerobic, fermenting numerous proteins and carbohydrates to various volatile fatty acids, alcohols, carbon dioxide, and hydrogen [22–25]. As of 2020, there are 15 species within the genus [26]. ◦ ◦ ◦ The strain grows from 55 C to 75 C with Topt of 70 C and between pH 4.5 to 8.0 with Fuels 2021, 2, FOR PEER REVIEW 5

Table 1. Biochemical composition of raw biomass; values are presented as the average of three replicates ± standard deviation.

Proximate Analysis (% on a Dry Weight Basis) Biomass Fat Protein Ash Carbohydrates 1 Fuels 2021, 2 Rhubarb leaf 3.29 ± 0.90 10.07 ± 2.32 10.78 ± 0.19 75.87 75 Timothy grass 3.73 ± 0.28 15.72 ± 0.16 5.96 ± 0.04 74.59 Whatman paper 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 100.00 1 Calculated by difference of other analytes subtracted◦ from 100%. pHopt of pH 7.0 (Figure1A,B). Growth at 70 C and pH 7.0 resulted in a generation time of 3.2.1.36 Strain h. Characterization TableThermoanarobacter 1. Biochemical composition strain AK91 of rawwas biomass;isolated valuesfrom Icelandic are presented hot asspring the average (temperature of three 67replicates °C; pH ±7.4)standard with already deviation. described techniques [15]. The strain has more than 99% sim- ilarity to Thermoanarobacter thermohydrosulfuricus based on the 16S rRNA gene (KR007665) as previously reported [21]. SpeciesProximate of the genus Analysis Thermoanaerobacter (% on a Dry Weight are Basis) strictly anaerobic, fermentingBiomass numerous proteins Fat and carbohydrate Proteins to various volatile Ash fattyCarbohydrates acids, alcohols,1 carbonRhubarb dioxide, leaf and hydrogen 3.29 ± 0.90 [22–25]. As 10.07 of ±2020,2.32 there are 10.78 15± species0.19 within 75.87the genus [26].Timothy The strain grass grows 3.73from± 550.28 °C to 75 15.72°C with± 0.16 Topt of 70 5.96°C and± 0.04 between pH 74.59 4.5 to 8.0 withWhatman pHopt of paper pH 7.0 (Figure 0.00 ± 0.001A,B). Growth 0.00 at± 0.0070 °C and pH 0.00 7.0± 0.00resulted in a 100.00generation time1 Calculated of 1.36 by h. difference of other analytes subtracted from 100%.

0.5 A 0.45 0.4 ) −1 0.35 0.3 0.25 0.2 0.15 Growth rate (μ, h 0.1 0.05

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Figure 1. Characteristics of cultivation temperature (A) and pH at 70 ◦C(B) for Thermoanarobacter Figure 1. Characteristics of cultivation temperature (A) and pH at 70 °C (B) for Thermoanarobacter strain AK91. strain AK91. Increased interest in the use of thermophiles for biofuel production is mainly because of theirIncreased ability interest to degrade in the ause wide of thermophiles variety of substrates for biofuel [6 production,14,15]. Thus, is mainly the strain because was ofcultivated their ability on to the degrade main substrates a wide variety present of substrates in lignocellulose [6,14,15]. in Thus, addition the tostrain several was other cul- tivatedcompounds on the (Figure main substrates2). The strain present degrades in lignocellulose many substrates in addition like to hexoses, several xyloseother com- and poundsseveral (Figure disaccharides, 2). The thestrain trisaccharide degrades many rafﬁnose, substrates starch, like grass hexoses, and rhubarb xylose hydrolysates, and several disaccharides,serine, and pyruvate the trisaccharide (Figure2). Theraffinose, main fermentationstarch, grass and products rhubarb were hydrolysates, ethanol and acetateserine, and pyruvate (Figure 2). The main fermentation products were ethanol and acetate (together with CO2 and H2) but lactate was also found in minor amounts (results not shown). The ratio of ethanol and acetate produced from sugars was close to 50:50, with exception of starch where ethanol was found to be the dominant end-product. Serine and pyruvate degradation resulted mainly in the production of acetate, most likely because of their higher oxidation states compared with sugars. No growth above controls was observed on arabinose, rhamnose, sucrose, cellulose, carboxymethylcellulose (CMC), and avicel. This is in common agreement with members of the genus generally not being capable of cellulose degradation but possessing the ability to degrade other polysaccharides such as starch, xylan, and pectin.

Ethanol Acetate Hydrogen

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Figure 1. Characteristics of cultivation temperature (A) and pH at 70 °C (B) for Thermoanarobacter strain AK91.

Increased interest in the use of thermophiles for biofuel production is mainly because of their ability to degrade a wide variety of substrates [6,14,15]. Thus, the strain was cultivated on the main substrates present in lignocellulose in addition to several other com- Fuels 2021, 2 pounds (Figure 2). The strain degrades many substrates like hexoses, xylose and several76 disaccharides, the trisaccharide raffinose, starch, grass and rhubarb hydrolysates, serine, and pyruvate (Figure 2). The main fermentation products were ethanol and acetate (together with CO2 and H2) but lactate was also found in minor amounts (results not shown). The(together ratio of with ethanol CO2 andand acetate H2) but produced lactate was from also sugars found was in minor close to amounts 50:50, with (results exception not shown). The ratio of ethanol and acetate produced from sugars was close to 50:50, with of starch where ethanol was found to be the dominant end-product. Serine and pyruvate exception of starch where ethanol was found to be the dominant end-product. Serine and degradation resulted mainly in the production of acetate, most likely because of their pyruvate degradation resulted mainly in the production of acetate, most likely because higherof their oxidation higher oxidation states compared states compared with suga withrs. sugars.No growth No growthabove controls above controls was observed was onobserved arabinose, on arabinose, rhamnose, rhamnose, sucrose, sucrose,cellulose, cellulose, carboxymethylcellulose carboxymethylcellulose (CMC), (CMC), and andavicel. Thisavicel. is in This common is in common agreement agreement with withmembers members of the ofthe genus genus generally generally not not being being capable of celluloseof cellulose degradation degradation but but possessing possessing the the ability ability to to degrade degrade other other polysaccharides polysaccharides suchsuch as starch,as starch, xylan, xylan, and and pectin. pectin.

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Figure 2. Formation of end-products after 5 day fermentation by Thermoanaerobacter strain AK91 on main substrates (70 ◦C, pH 7.0). The concentration for all substrates evaluated was 20 mM or in case of polymeric substrates 0.2% (w/v). Standard deviation are presented as error bars.

3.3. Effect of Culture Conditions on End-product Formation To gain insight into the effect of various environmental factors the strain was cultivated at various initial glucose concentrations and L-G ratios, as shown in Figure3A,B. Relatively low initial substrate concentrations have been shown to inhibit sugar degradation by thermophilic bacteria. This has indeed been pointed out as the main obstacle to utilizing thermophiles for bioethanol production compared with yeasts. The reasons for this sensitivity may be caused by several factors [4,6]. In addition to ethanol production, thermophiles also produce acids (acetate, lactate) that may cause pH lowering in closed systems with limited buffer capacity. This pH drop may inhibit cell activity and thus substrate degradation. Figure3A shows a good correlation of increased end-product (ethanol and acetate) formation between 5.0 and 20.0 mM initial glucose concentrations. At increased loadings (>20 mM initial glucose concentration), a clear inhibition is observed leading to a less portion of the glucose being degraded and levelling off or a decrease in end-product formation. With the highest glucose initial concentration applied (400 mM), less than 5% of the glucose was degraded. This is well known; other thermophilic anaerobic bacteria are very sensitive towards relatively low initial sugar concentrations, often being inhibited at concentrations above 20–30 mM [6,17]. Another reason for insufﬁcient substrate utilization is the accumulation of hydrogen in closed batch culture systems. The increased partial pressure of hydrogen (pH2) may also change the ﬂow of electrons leading to different end-product formation [16]. Thus, at high L-G ratios, at which pH2 is higher relative to lower L-G ratios the formation of end-products is directed to more reduced compounds (ethanol, lactate), but away from oxidized products (acetate, hydrogen) and vice versa at low pH2 [14–16]. To investigate the Fuels 2021, 2, FOR PEER REVIEW 7

Figure 2. Formation of end-products after 5 day fermentation by Thermoanaerobacter strain AK91 on main substrates (70 Fuels°C,2021 pH, 2 7.0). The concentration for all substrates evaluated was 20 mM or in case of polymeric substrates 0.2% (w/v). Stand- 77 ard deviation are presented as error bars.

3.3. Effect of Culture Conditions on End-product Formation inﬂuenceTo gain of pinsightH2 on hydrogeninto the effect production, of various the environmental strain was grown factors in serum the strain bottles was at various culti- vatedL-G ratios.at various The maximuminitial glucose hydrogen concentrations yield from and 1 mole L-G ofratios, glucose as shown is 4 moles in Figure of hydrogen 3A,B. Relativelywhen acetate low isinitial the onlysubstrate volatile concentrations end-product have produced. been shown Figure to3 Binhibit shows sugar that thedegrada- strain tionproduces by thermophilic maximally bacteria. 2.8 mol HThis2 per has mole indeed glucose been (70.0%pointed of out the as theoretical the main yield)obstacle at theto utilizinglowest L-G thermophiles ratio, but drops for bioethanol to 0.2 mol producti H2 per molon compared glucose (5.0% with of yeasts. theoretical The yields)reasons at for the highest L-G ratio used. Maximum ethanol yield is 2 mol from 1 mol of glucose. The highest this sensitivity may be caused by several factors [4,6]. In addition to ethanol production, ethanol yields were obtained at the highest L-G ratio used, or 72.5% of the theoretical yield. thermophiles also produce acids (acetate, lactate) that may cause pH lowering in closed Thus, as stated before, it is more likely that the effect of culture conditions can have a more systems with limited buffer capacity. This pH drop may inhibit cell activity and thus sub- dramatic effect on determining if a strain is a good ethanol or good hydrogen producer strate degradation. rather than intrinsic features of the strain. A Ethanol Acetate Hydrogen 25

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Figure 3. The effect of initial glucose concentration (A) and L-G ratio (at 20 mM glucose) (B) on Figure 3. The effect of initial glucose concentration (A) and L-G ratio (at 20 mM glucose) (B) on fermentation by Thermoanaerobacter strain AK91. End-products were quantiﬁed after 5 days. Values fermentation by Thermoanaerobacter strain AK91. End-products were quantified after 5 days. Val- represent averages of triplicate fermentations ± standard deviation (n = 3). ues represent averages of triplicate fermentations ± standard deviation (n = 3).

Figure 3A shows a good correlation of increased end-product (ethanol and acetate) formation between 5.0 and 20.0 mM initial glucose concentrations. At increased loadings (>20 mM initial glucose concentration), a clear inhibition is observed leading to a less portion of the glucose being degraded and levelling off or a decrease in end-product formation. With the highest glucose initial concentration applied (400 mM), less than 5% of the glucose was degraded. This is well known; other thermophilic anaerobic bacteria are very sensitive towards relatively low initial sugar concentrations, often being inhibited at concentrations above 20–30 mM [6,17]. Another reason for insufficient substrate utilization is the accumulation of hydrogen in closed batch culture systems. The increased partial pressure of hydrogen (pH2) may also change the flow of electrons leading to different end-product formation [16]. Thus, at high L-G ratios, at which pH2 is higher relative to lower L-G ratios the formation of end-products is directed to more reduced compounds (ethanol, lactate), but away from oxidized products (acetate, hydrogen) and vice versa at low pH2 [14–16]. To investigate the influence of pH2 on hydrogen production, the strain was grown in serum bottles at various L- G ratios. The maximum hydrogen yield from 1 mole of glucose is 4 moles of hydrogen when acetate is the only volatile end-product produced. Figure 3B shows that the strain produces maximally 2.8 mol H2 per mole glucose (70.0% of the theoretical yield) at the lowest L-G ratio, but drops to 0.2 mol H2 per mol glucose (5.0% of theoretical yields) at the highest L-G ratio used. Maximum ethanol yield is 2 mol from 1 mol of glucose. The highest ethanol yields were obtained at the highest L-G ratio used, or 72.5% of the theoretical yield. Thus, as stated before, it is more likely that the effect of culture conditions can have a more dramatic effect on determining if a strain is a good ethanol or good hydrogen producer rather than intrinsic features of the strain.

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3.4. Effect of Inhibitory Compounds on Growth The formation of end-products by Thermoanaerobacter strain AK91 at 20 mM glucose initial concentration was investigated in the presence of selected exogenous inhibitory compounds. The compounds tested were acetate, propionate, butyrate, lactate, ethanol, malate, oxalate, levulinic acid, p-coumaric acid, 2-furfuraldehyde, and 5-HMF. Most of these substrates are well known as inhibitory for microorganisms and some originate directly from biomass pre-treatment [13]. The alkanoic carboxylic acids can be liberated from the pre-treatment of hemicellulose while the dicarboxylic acids, oxalate and malate, are abundant in rhubarb leaves. Ethanol and lactate are common fermentation end-products, and were included as Thermoanaerobacter strains produce both compounds. As a proxy for lignin degradation products, p-coumaric acid was selected while the inclusion of 5-HMF, 2-furfuraldehyde, levulinic acid are commonly associated with the degradation of hexoses and pentoses under acidic conditions [4,6]. Based on end-product formation the strain was insensitive to most of the organic acids tested up to the maximum tested value, but showed high sensitivity towards p-coumaric acid, levulinic acid, 2-furfuraldehyde, and 5-HMF (Table2).

Table 2. Minimum inhibitory concentrations of model inhibitory compounds based on growth (optical density) and end-product formation of Thermoanaerobacter strain AK91.

Minimum Inhibitory Concentration (mM) Acetate >80 mM Propionate >80 mM n-Butyrate >80 mM Lactate >50 mM Ethanol >100 mM Malate >40 mM Oxalate >80 mM Levulinic acid >20 mM p-Coumaric acid <10 mM 2-furfuraldehyde <20 mM 5-HMF <30 mM

There is rather little data available on the effects of inhibitory compounds on thermophilic bacteria. Thermoanaerobacterium strain AK17 was shown to be inhibited completely by 2-furfuraldehyde and 5-HMF at concentrations of 20 mM and 32 mM respectively, when grown on glucose [15]. Figure4 shows the effect of increased propionate concentrations on glucose catabolism and formation of end-products by Thermoanaerobacter strain AK91. Interestingly, production of acetate and hydrogen are not inhibited, their production actually increases a little, by increased initial concentrations of propionate, while ethanol production gradually decreases (Figure4). Surprisingly, increased amounts of 1-propanol were observed to be produced with the addition of propionate although the ratio of 1- propanol to propionate added decreased above 20 mM. There is no simple explanation for the increase in hydrogen and acetate up to 20 mM but it is well known that the production of acetate and hydrogen are ATP yielding reactions whereas the production of ethanol does not give energy. A similar phenomenon was found for n-butyrate addition; 1-butanol was formed (results not shown). This seeming conversion of the acid to alcohol was thus tested in an NMR study (see below). Fuels 2021Fuels, 22021, FOR, 2 PEER REVIEW 79 10

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5 20

0 0 Control 0 10 20 40 60 80 Initial Propionate Concentration (mM)

Figure 4. Inﬂuence of different initial propionate concentrations of end-product formation from glucose (20 mM) after Figure5 4. days Influence of cultivation. of different Standard initial deviation propionate are presented concentrations as error bars. of end-product formation from glucose (20 mM) after 5 days of cultivation. Standard deviation are presented as error bars. 3.5. Kinetic Experiment on Glucose and Propionate Interestingly,To examine production the impact of of both acetate inhibitory and hydrogen effects of are propionate not inhibited, during their glucose production fer- actuallymentation, increases three a different little, by concentrations increased initial of the concentrations acids were used of (20, propionate, 50, and 100 while mM) and ethanol productiongrowth followed gradually kinetically decreases over (Figure a period 4). Su ofrprisingly, 7 days. During increased growth amounts on 20 mM of 1-propanol propi- wereonate, observed end-product to be formationproduced waswith similar the addi as withouttion of anypropionate addition ofalthough an acid (datathe ratio with of 1- propanolonly glucose; to propionate Figure2), namelyadded decreased formation of above both ethanol20 mM. (17.0 There mM) is and no acetatesimple (13 explanation mM) for togetherthe increase with in hydrogen hydrogen (Figure and5 acetateA). By increasingup to 20 mM propionate but it is to well 50 and known 100 mM, that less the pro- ductionethanol of (acetate10.8 mM andand hydrogen 6.2 mM, respectively) are ATP yiel wasding produced reactions but whereas slightly higherthe production acetate of ethanolconcentrations does not weregive observedenergy. A (15.2 similar and 18.8 phen mM,omenon respectively). was found This isfor in n-butyrate good agreement addition; with the data presented in Figure4, where ethanol decreased with increasing propionate 1-butanol was formed (results not shown). This seeming conversion of the acid to alcohol concentrations but acetate increased. Most interesting, however, was the conversion of waspropionate thus tested to in propanol. an NMR Increased study (see propionate below). concentrations resulted in increased formation of propanol in all cases. Recently, our research group has shown the capacity of 3.5.Thermoanaerobacter Kinetic Experimentspecies on Glucose to convert and Propionate fatty acids to their corresponding alcohols [20,27,28] underTo examine speciﬁc conditions.the impact Insteadof both of inhibitory dispersing effects reducing of equivalentspropionate toduring pyruvate glucose and fermentation,produce three only ethanoldifferent (or concentrations lactate), these bacteria of the acids used were the electrons used (20, produced 50, and to100 reduce mM) and fatty acids to alcohols. A similar trend was observed on glucose using three increasing growth followed kinetically over a period of 7 days. During growth on 20 mM propionate, concentrations of butyrate; less ethanol was produced with higher butyrate concentrations end-productbut acetate formation production remainedwas similar similar as orwithout was slightly any addition higher (results of an not acid shown). (data Finally, with only glucose;butyrate Figure was converted2), namely to formation butanol as wasof both the case ethanol for propionate (17.0 mM) conversion and acetate to propanol. (13 mM) together with hydrogen (Figure 5A). By increasing propionate to 50 and 100 mM, less ethanol (10.8 mM and 6.2 mM, respectively) was produced but slightly higher acetate concentrations were observed (15.2 and 18.8 mM, respectively). This is in good agreement with the data presented in Figure 4, where ethanol decreased with increasing propionate concentrations but acetate increased. Most interesting, however, was the conversion of propionate to propanol. Increased propionate concentrations resulted in increased formation of propanol in all cases. Recently, our research group has shown the capacity of Thermoan- aerobacter species to convert fatty acids to their corresponding alcohols [20,27,28] under specific conditions. Instead of dispersing reducing equivalents to pyruvate and produce only ethanol (or lactate), these bacteria used the electrons produced to reduce fatty acids

Fuels 2021, 2, FOR PEER REVIEW 11

to alcohols. A similar trend was observed on glucose using three increasing concentrations of butyrate; less ethanol was produced with higher butyrate concentrations but acetate Fuels 2021, 2 production remained similar or was slightly higher (results not shown). Finally, butyrate80 was converted to butanol as was the case for propionate conversion to propanol. A EtOH Acetate 1-PrOH 1-Propionate Glucose OD 0.5

20 0.45

0.4

15 0.35 0.3

0.25 10 0.2 Analyte (mM) 0.15

5 nm) (600 Density Optical 0.1

0.05

0 0 0 20 40 60 80 100 120 140 160 180 Time (h)

B EtOH Acetate 1-PrOH 1-Propionate Glucose OD 50 0.5

45 0.45

40 0.4

35 0.35

30 0.3

25 0.25

20 0.2 Analyte (mM) 15 0.15 Optical Density (600 nm) (600 Density Optical 10 0.1

5 0.05

0 0 0 20 40 60 80 100 120 140 160 180 Time (h)

Figure 5. Cont.

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C EtOH Acetate 1-PrOH 1-Propionate Glucose OD 100 0.5

90 0.45

80 0.4

70 0.35

60 0.3

50 0.25

40 0.2 Analyte (mM) 30 0.15 Optical Density (600 nm) (600 Density Optical 20 0.1

10 0.05

0 0 0 20 40 60 80 100 120 140 160 180 Time (h)

Figure 5. End-product formation from glucose (20 mM) in the presence of 20 mM n-propionate (A), 50 mM n-propionate (B) and 100 mM n-propionate (C). Figure 5. End-product formation from glucose (20 mM) in the presence of 20 mM n-propionate (A), 50 mM n-propionate (B) and 100 mM n-propionate (C). 3.6. Fermentation of Biomass Hydrolysates 3.6. FermentationTwo types of of lignocellulosicBiomass Hydrolysates biomass were tested in the present investigation together withTwo a control, types Whatmanof lignocellulosic paper, grass biomass (P. pratense were tested) and in rhubarb the present (R. rhabarbarum investigation). The together focus withwas ona control, rhubarb Whatman as a potential paper, raw grass material (P. pratense for biofuel) and rhubarb production (R. rhabarbarum as the rhubarb). The leaves focus P. pratense wasare anon agriculturalrhubarb as a waste potential material raw frommaterial the fo rhubarbr biofuel industry. production The as grass the rhubarb leaves, was also chosen as a reference since there are substantial data available for both ethanol and are an agricultural waste material from the rhubarb industry. The grass P. pratense, was hydrogen yields on this substrate. Based on the results above, experiments using all three also chosen as a reference since there are substantial data available for both ethanol and types of biomass were performed at three different concentrations, 2.5, 5.0, and 10 g L−1, hydrogen yields on this substrate. Based on the results above, experiments using all three of which the lowest concentrations were also tested at three different L-G ratios: 0.04, 1.0, types of biomass were performed at three different concentrations, 2.5, 5.0, and 10 g L−1, and 3.29. of which the lowest concentrations were also tested at three different L-G ratios: 0.04, 1.0, The reason for using different initial biomass loadings was the sensitivity of the and 3.29. strain towards increased glucose concentrations. In an experiment using three different The reason for using different initial biomass loadings was the sensitivity of the strain concentrations of Whatman paper (2.5, 5.0, and 10.0 g L−1), assuming it was completely towards increased glucose concentrations. In an experiment using three different concen- hydrolysed to glucose, means that the concentration of glucose available should be between trations of Whatman paper (2.5, 5.0, and 10.0 g L−1), assuming it was completely hydro- 15.4 to 61.7 mM. As observed earlier, Thermoanaerobacter strain AK91 is severely inhibited lysed to glucose, means that the concentration of glucose available should be between 15.4 between 20–30 mM initial glucose concentration (Figure3A). Thus, it is not surprising toto 61.7 see thatmM. the As highest observed yields earlier, of ethanol Thermoanaerobacter from Whatman strain paper AK91 are is observed severely inhibited at the lowest be- tweenbiomass 20–30 loading mM usedinitial (2.5 glucose g L−1 concentration), or 14.0 mM (45.4%(Figure yields) 3A). Thus, (Figure it is6 A).not Thesurprising main reason to see thatfor thesethe highest low yields yields is of due ethanol to a large from fractionWhatman of paper the sugar are observed ending up at beingthe lowest converted biomass to −1 loadingacetate underused (2.5 these g L culture), or 14.0 conditions mM (45.4% (11.0 yields) mM). (Figure Together, 6A). acetate The main and ethanolreason for amount these lowto 25 yields mM ofis end-products,due to a large or fraction 81.2% ofof theoreticalthe sugar ending carbon up yields. being The converted rest is presumably to acetate underlactate these and carbonculture storedconditions in cells (11.0 (not mM). analyzed). Together, Carbon acetate yields and ethanol of ethanol amount and acetate to 25 mM on ofWhatman end-products, paper droppedor 81.2% to of 55.2 theoretical and 26.9% carbon at 5.0 yields. and 10.0 The g L re−1sthydrolysate is presumably concentrations, lactate and carbonrespectively stored (Figure in cells6 A),(not most analyzed). likely Carb dueon to inefﬁcientyields of ethanol glucose and degradation acetate on atWhatman higher papersubstrate dropped loadings. to 55.2 Ethanol and 26.9% yields at were 5.0 and thus 10.0 45.5, g 27.6, L−1 hydrolysate and 13.4% at concentrations, 2.5, 5.0 and 10.0 respec- g L−1 tivelyhydrolysate (Figure loadings, 6A), most respectively. likely due to Yields inefficient for acetate glucose at degradation these concentrations at higher weresubstrate 35.7, loadings.27.6 and 13.4%,Ethanol respectively yields were (Figurethus 45.5,6A). 27.6, Yields and of 13.4% hydrogen at 2.5, were5.0 and 23.6, 10.0 14.2, g L− and1 hydroly- 7.5%, saterespectively. loadings, However, respectively. by using Yields a high for acetate L-G ratio at forthese the concentrations lowest hydrolysate were concentrations,35.7, 27.6 and 13.4%, respectively (Figure 6A). Yields of hydrogen were 23.6, 14.2, and 7.5%, respectively. However, by using a high L-G ratio for the lowest hydrolysate concentrations, ethanol

Fuels 2021, 2, FOR PEER REVIEW 13

yields increased from 45.5 to 75% (Figure 6B). Similarly, hydrogen yields were improved from 23.6 to 55.7% by lowering the L-G ratio (Figure 6B). It is clear from Figure 6A that the amounts of end-products do not increase linearly with increased substrate loadings in the case of Whatman paper. Since both grass and rhubarb contain less glucose but more of other varieties of sugars this “levelling off” phenomenon is not as apparent for this type of biomass. There seems to be less substrate inhibition when using the complex biomass as compared with the glucose present in the homogenous Whatman paper. However, both ethanol and hydrogen yields are lower on grass and rhubarb compared with yields from the Whatman paper hydrolysate. Ethanol concentrations on grass and rhubarb hydrolysates ranged from 8.6 to 12.1 mM from the three different concentrations used, with the highest yields being obtained on the lowest hydrolysate loading of 2.5 g L−1. Similar values for hydrogen were 11.0 to 17.6 mmol L−1 (Figure 6A). Values for rhubarb hydrolysates were similar or a little lower as compared with grass hydrolysates. Ethanol ranged from 5.8 to 10.9 mM, and hydrogen from 8.0 to 15.3 mmol L−1. However, in the experiment using the lowest hydrolysate concentration (2.5 g L−1) and different L-G, these values shifted depending on the compounds and conditions tested (Figure 6B). As for the Whatman paper experiment, ethanol yields on grass hydrolysates were increased to a maximum of 17.4 mM (7.0 mM g dw−1) at the highest L- G phase ratio examined and hydrogen to a maximum of 29.8 mmol/L (1.36 mol mol g dw−1). Similarly, for rhubarb, the highest ethanol concentration obtained was 15.8 mM (6.3 mM g dw−1) and hydrogen of a maximum of 27.2 mmol L−1. The maximum yields of ethanol and hydrogen are comparable with other similar strains. The strain seems to be more sensitive towards high glucose concentrations as compared with Thermoanaerobacter strain J1 [29]. Thermoanaerobacter strain J1 produced 35 mM ethanol from a Whatman paper hydrolysate (4.5 gL-1) but this strain is highly ethanologenic compared to strain AK91. Another strain, Thermoanaerobacterium AK54, that has been investigated for both ethanol and hydrogen production, produced 29.2 mM of ethanol, 18.1 mM of acetate and 37.1 mmol L−1 of hydrogen from grass hydrolysate [30]. Etha- nol yields in the current study on grass and rhubarb were maximized by cultivating the −1 Fuels 2021, 2 strain at low substrate concentration and high pH2 to 7.0 and 6.3 mM g biomass. These 82 yields are 63 and 57% of theoretical yields from complex biomass. Hydrogen yields were also maximized by using low substrate concentrations and L-G ratios, to 11.9 and 10.9 mmol L−1 g−1 biomass. Thus, the strain can be a good choice whether to use it as an ethanol or hydrogenethanol producer yields from increased complex frombiomass. 45.5 Th tois 75% is to (Figure our knowledge6B). Similarly, the first hydrogen time that yields were rhubarb is usedimproved as a potential from 23.6 biofuel to 55.7% feedstock. by lowering the L-G ratio (Figure6B).

A 25 Ethanol Acetate Hydrogen 20 ) −1 15

10 Analyte (mmol L (mmol Analyte 5

0 Fuels 2021, 2, FOR PEER REVIEW 14 2.5 5 10 2.5 5 10 2.5 5 10 Whatman paper Timothy grass Rhubarb leaf

B Ethanol Acetate Hydrogen 40 35

) 30 −1 25 20 15

Analyte (mmol L (mmol Analyte 10 5 0 0.04 1 3.27 0.04 1 3.27 0.04 1 3.27 Whatman paper Timothy grass Rhubarb leaf Liquid-gas phase ratio

Figure 6. End-productFigure formation6. End-product of Thermoanaerobacter formation of Thermoanaerobacterstrain AK91 grown strain on cellulose,AK91 grown grass on and cellulose, rhubarb grass at three and different hydrolysate concentrationsrhubarb at three (2.5, 5.0 different and 10.0 hydrolysate g/L) (A) and concentrations at three different (2.5, L-G5.0 and ratio 10.0 using g/L) 2.5 (A g/L) and hydrolysates at three differ- (B). Values represent averagesent ofL-G triplicates ratio using with 2.5 standard g/L hydrolysates deviation (B represented). Values represent by error averages bars. of triplicates with standard deviation represented by error bars. It is clear from Figure6A that the amounts of end-products do not increase linearly Maximumwith theoretical increased substrateyields of loadingsethanol from in the pure case cellulose of Whatman is 2 mol paper. ethanol Since from both 1 grass and mol of glucose,rhubarb or 11.1 contain mM g less−1. However, glucose butlower more yields of otherare typically varieties observed of sugars from this ligno- “levelling off” cellulosic biomassphenomenon because is notof the as apparentvariety of for sugars this type present of biomass. and because There a seems portion to of be the less substrate sugars are inhibitionlost in the whenpre-treatment using the steps complex of the biomass biomass. as Examples compared of with high the ethanol glucose yields present in the from varioushomogenous lignocellulosic Whatman biomass paper. are However,shown in bothTable ethanol 3. Thermoanaerobacter and hydrogen yields species are lower on have been showngrass and to produce rhubarb high compared ethanol with yiel yieldsds from from complex the Whatman biomass paper in the hydrolysate.literature. Ethanol Thermoanaerobacterconcentrations strain BG1L1 on grass produces and rhubarb between hydrolysates 8.5–9.2 mM ranged g−1 sugar from consumed 8.6 to 12.1 from mM from the wheat strawthree and different corn stover concentrations [31,32] in continuo used, withus culture. the highest Examples yields of being other obtained high yields on the lowest from lignocellulosehydrolysate are loadingthat of Thermoanaerobacter of 2.5 g L−1. Similar mathranii values foron hydrogenwheat straw were [33] 11.0 and to Ther- 17.6 mmol L−1 moanarobacter(Figure strain6A). J1 Valueson various for rhubarb lignocellulosic hydrolysates biomasses were [29]. similar Other or agenera, little lower such asas compared Thermoanaerobacterium,with grass hydrolysates. are also good Ethanol ethanolranged producers from when 5.8 to grown 10.9 mM, on carbohydrate and hydrogen bi- from 8.0 to − omass. As 15.3an example, mmol L Thermoanaeroacterium1. However, in the experiment strain AK17 using produces the lowest 8.6 mM hydrolysate g−1 cellulose concentration hydrolysate and 5.5 mM g−1 grass hydrolysate at very low initial substrate (2.5 g L−1) concentrations [14] and Clostridium thermocellum on paddy straw [34]. Rhubarb has to our knowledge not been used for bioethanol or biohydrogen production before. Some strains within the genus of Thermoanarobacter have also been shown to be good hydrogen producers, such as T. tengcongensis which can reportedly product up to 4 mol hydrogen per mole glucose using continuous nitrogen flushing [35].

Table 3. Selected examples of ethanol production from lignocellulosic biomass by thermophilic bacteria. Ethanol yields are reported as mM g−1 substrate degraded along with substrate concentrations and incubation temperatures. Ac—acid; Alk—alkaline; E—enzymatic; and WO—wet oxidation.

Conc. Pre- Ethanol Yields Organisms Substrate References (g L−1) treatment (mM g−1) Thermoanaerobacter strain AK 91 Timothy grass 2.5 Ac/E 7.0 This study Thermoanaerobacter strain AK 91 Rhubarb leaf 2.5 Ac/E 6.3 This study Clostridium thermocellum Paddy straw 8.0 None 6.10–8.00 [34] Thermoanaerobacter mathranii Wheat straw 6.7 WO/E 2.61 [33] Thermoanaerobacter BG1L1 Corn stover 25.0–150.0 WO/E 8.50–9.20 [32] Thermoanaerobacter BG1L1 Wheat straw 30.0–120.0 WO/E 8.50–9.20 [31] Thermoanaerobacter strain J1 Hemp 4.5 Ac/E 4.3 [29]

Fuels 2021, 2 83

(2.5 g L−1) and different L-G, these values shifted depending on the compounds and conditions tested (Figure6B). As for the Whatman paper experiment, ethanol yields on grass hydrolysates were increased to a maximum of 17.4 mM (7.0 mM g dw−1) at the highest L-G phase ratio examined and hydrogen to a maximum of 29.8 mmol/L (1.36 mol mol g dw−1). Similarly, for rhubarb, the highest ethanol concentration obtained was 15.8 mM (6.3 mM g dw−1) and hydrogen of a maximum of 27.2 mmol L−1. The maximum yields of ethanol and hydrogen are comparable with other similar strains. The strain seems to be more sensitive towards high glucose concentrations as compared with Thermoanaerobacter strain J1 [29]. Thermoanaerobacter strain J1 produced 35 mM ethanol from a Whatman paper hydrolysate (4.5 gL−1) but this strain is highly ethanologenic compared to strain AK91. Another strain, Thermoanaerobacterium AK54, that has been investigated for both ethanol and hydrogen production, produced 29.2 mM of ethanol, 18.1 mM of acetate and 37.1 mmol L−1 of hydrogen from grass hydrolysate [30]. Ethanol yields in the current study on grass and rhubarb were maximized by cultivating −1 the strain at low substrate concentration and high pH2 to 7.0 and 6.3 mM g biomass. These yields are 63 and 57% of theoretical yields from complex biomass. Hydrogen yields were also maximized by using low substrate concentrations and L-G ratios, to 11.9 and 10.9 mmol L−1 g−1 biomass. Thus, the strain can be a good choice whether to use it as an ethanol or hydrogen producer from complex biomass. This is to our knowledge the ﬁrst time that rhubarb is used as a potential biofuel feedstock. Maximum theoretical yields of ethanol from pure cellulose is 2 mol ethanol from 1 mol of glucose, or 11.1 mM g−1. However, lower yields are typically observed from lignocellulosic biomass because of the variety of sugars present and because a portion of the sugars are lost in the pre-treatment steps of the biomass. Examples of high ethanol yields from various lignocellulosic biomass are shown in Table3. Thermoanaerobacter species have been shown to produce high ethanol yields from complex biomass in the literature. Thermoanaerobacter strain BG1L1 produces between 8.5–9.2 mM g−1 sugar consumed from wheat straw and corn stover [31,32] in continuous culture. Examples of other high yields from lignocellulose are that of Thermoanaerobacter mathranii on wheat straw [33] and Thermoanarobacter strain J1 on various lignocellulosic biomasses [29]. Other genera, such as Thermoanaerobacterium, are also good ethanol producers when grown on carbohydrate biomass. As an example, Thermoanaeroacterium strain AK17 produces 8.6 mM g−1 cellulose hydrolysate and 5.5 mM g−1 grass hydrolysate at very low initial substrate (2.5 g L−1) concentrations [14] and Clostridium thermocellum on paddy straw [34]. Rhubarb has to our knowledge not been used for bioethanol or biohydrogen production before. Some strains within the genus of Thermoanarobacter have also been shown to be good hydrogen producers, such as T. tengcongensis which can reportedly product up to 4 mol hydrogen per mole glucose using continuous nitrogen ﬂushing [35].

Table 3. Selected examples of ethanol production from lignocellulosic biomass by thermophilic bacteria. Ethanol yields are reported as mM g−1 substrate degraded along with substrate concentrations and incubation temperatures. Ac—acid; Alk—alkaline; E—enzymatic; and WO—wet oxidation.

Conc. Ethanol Yields Organisms Substrate Pre-Treatment References (g L−1) (mM g−1) Thermoanaerobacter strain AK 91 Timothy grass 2.5 Ac/E 7.0 This study Thermoanaerobacter strain AK 91 Rhubarb leaf 2.5 Ac/E 6.3 This study Clostridium thermocellum Paddy straw 8.0 None 6.10–8.00 [34] Thermoanaerobacter mathranii Wheat straw 6.7 WO/E 2.61 [33] Thermoanaerobacter BG1L1 Corn stover 25.0–150.0 WO/E 8.50–9.20 [32] Thermoanaerobacter BG1L1 Wheat straw 30.0–120.0 WO/E 8.50–9.20 [31] Thermoanaerobacter strain J1 Hemp 4.5 Ac/E 4.3 [29] Thermoanaerobacterium strain AK17 Grass 2.5 Ac/Alk/E 5.5 [14] Fuels 2021, 2, FOR PEER REVIEW 15

Fuels 2021, 2 84 Thermoanaerobacterium strain Grass 2.5 Ac/Alk/E 5.5 [14] AK17

3.7. NMR Studies To conclusively demonstrate n-propanol n-propanol production production from from exogenously exogenously added added propi- pro- 13 pionateonate to to glucose glucose containing containing culture, culture, 13CC1-labeled1-labeled propionate propionate was was used used as as a a model model compound.pound. AsAs hashas been been demonstrated demonstrated for for other otherThermoanaerobacter Thermoanaerobacterstrains, strains, strain strain AK91 AK91 also also has thehas abilitythe ability to convert to convert carboxylic carboxylic acids acids to their to correspondingtheir corresponding primary primary alcohols alcohols as evidenced as evi- 13 bydenced the appearance by the appearance of a peak of at a 63.8 peak ppm at 63.8 which ppm can which be attributed can be attributed to C1-labelled to 13C propanol1-labelled (Figurepropanol7). (Figure 7).

13 Figure 7. NMR spectrum of glucose (20 mM) degradation with13 C1 1-propionate by Thermoanaerobacter strain AK91. Figure 7. NMR spectrum of glucose (20 mM) degradation with C1 1-propionate by Thermoanaerobacter strain AK91.

While this strain produces less of the alcoholsalcohols from carboxylic acids than other Ther- moanaerobacter strains investigated to date, thisthis physiologicalphysiological strategystrategy maymay bebe usefuluseful forfor dealing withwith carboxyliccarboxylic acids acids liberated liberated during during the the pre-treatment pre-treatment of lignocellulosic of lignocellulosic biomass. bio- Inmass. the caseIn the of case biomasses of biomasses containing containing dicarboxylic dicarboxylic acids suchacids as such oxalate, as oxalate, the production the produc- of ation diol of such a diol as such ethylene as ethylene glycol mayglycol present may present a potentially a potentially useful useful route toroute generating to generating these commoditythese commodity chemicals chemicals as a co-product as a co-product during duri biofuelng biofuel production production in addition in addition to primary to pri- alcoholsmary alcohols such as such propanol as propanol and butanol and butanol being usefulbeing useful biofuels biofuels in their in own their right. own right.

AuthorAuthor Contributions:Contributions:Both Both authors authors have have read read and and agreed agreed to theto the published published version version of the of manuscript the manu- withscript individual with individual contributions contributions as follows: as follows: Conceptualization, Conceptualization, J.O.; methodology, J.O.; methodology, S.M.S. and S.M.S. J.O.; and investigation,J.O.; investigation, S.M.S. resources,S.M.S. resources, J.O.; data J.O; curation, data curation, J.O. and J.O S.M.S.; and S.M.S.; writing—original writing—original draft preparation, draft prep- S.M.S.; writing—review and editing, S.M.S. and J.O.; visualization, S.M.S. and J.O.; supervision, J.O.; project administration, J.O.; funding acquisition, J.O. All authors have read and agreed to the published version of the manuscript.

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Funding: The authors gratefully acknowledge funding from Landsvirkjun (project NÝR-25-2018) and the Research Fund of the University of Akureyri (R1817). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors would like to gratefully acknowledge Edda Kamilla Örnólfsdóttir for providing rhubarb leaves for analysis and Sigríður Jónsdóttir (University of Iceland) for her aid in obtaining NMR spectra. Finally, Eva María Ingvadóttir (University of Akureyri) is thanked for her thoughtful review of our work. Conﬂicts of Interest: There are no conﬂict of interest.

References 1. Salameh, T.; Tawalbeh, M.; Al-Shannag, M.; Saidan, M.; Melhem, K.B.; Alkasrawi, M. Energy saving in the process of bioethanol production from renewable paper mill sludge. Energy 2020, 196, 117085. [CrossRef] 2. Alkasrawi, M.; Rudolf, A.; Lidén, G.; Zacchi, G. Influence of strain and cultivation procedure on the performance of simultaneous saccharification and fermentation of steam pretreated spruce. Enzym. Microb. Technol. 2006, 38, 279–286. [CrossRef] 3. Alkasrawi, M.; Abu Jrai, A.; Al-Muhtaseb, A.H. Simultaneous saccharification and fermentation process for ethanol production from steam-pretreated softwood: Recirculation of condensate streams. Chem. Eng. J. 2013, 225, 574–579. [CrossRef] 4. Taylor, M.P.; Eley, K.L.; Martin, S.; Tuffin, M.I.; Burton, S.G.; Cowan, D.A. Thermophilic ethanologenesis: Future prospects for second-generation bioethanol production. Trends Biotechnol. 2009, 27, 398–405. [CrossRef][PubMed] 5. Onuki, S.; Koziel, J.A.; Jenks, W.S.; Cai, L.; Grewell, D.; van Leeuwen, J.H. Taking ethanol quality beyond fuel grade: A review. J. Inst. Brew. 2016, 122, 588–598. [CrossRef] 6. Scully, S.M.; Orlygsson, J. Recent Advances in Second Generation Ethanol Production by Thermophilic Bacteria. Energies 2015, 8, 1–30. [CrossRef] 7. Robertson, G.P.; Hamilton, S.K.; Barham, B.L.; Dale, B.E.; Izaurralde, R.C.; Jackson, R.D.; Landis, D.A.; Swinton, S.M.; Thelen, K.D.; Tiedje, J.M. Cellulosic biofuel contributions to a sustainable energy future: Choices and outcomes. Science 2017, 356, eaal2324. [CrossRef] 8. Sánchez, Ó.J.; Cardona, C.A. Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresour. Technol. 2008, 99, 5270–5295. [CrossRef][PubMed] 9. Modig, T. Kinetics and inhibition effects of furfural and hydroxymethyl furfural on enzymes in yeast. Biochem. J. 2002, 363, 769–776. [CrossRef][PubMed] 10. Pucher, G.W.; Wakeman, A.J.; Vickery, H.B. The Organic Acids of Rhubarb (Rheum hybridium). III: The Behavior of the Organic Acids During Culture of Excised Leaves. J. Biol. Chem. 1938, 126, 43–54. [CrossRef] 11. USDA. USDA Database for Oxalic Acid Content of Selected Vegetables; U.S. Department of Agriculture: Washington, DC, USA, 1984; ISBN 978-1-61583-987-2. 12. Ahring, B.K.; Jensen, K.; Nielsen, P.; Bjerre, A.B.; Schmidt, A.S. Pretreament of wheat straw and conversion of xylose and xylan to ethanol by thermophilic anaerobic bacteria. Bioresour. Technol. 1997, 58, 107–113. [CrossRef] 13. Ponnusamy, V.K.; Nguyen, D.D.; Dharmaraja, J.; Shobana, S.; Banu, J.R.; Saratale, R.G.; Chang, S.W.; Kumar, G. A review on lignin structure, pretreatments, fermentation reactions and biorefinery potential. Bioresour. Technol. 2019, 271, 462–472. [CrossRef] [PubMed] 14. Almarsdottir, A.R.; Sigurbjornsdottir, M.A.; Orlygsson, J. Effect of various factors on ethanol yields from lignocellulosic biomass by Thermoanaerobacterium AK 17. Biotechnol. Bioeng. 2012, 109, 686–694. [CrossRef][PubMed] 15. Brynjarsdottir, H.; Wawiernia, B.; Orlygsson, J. Ethanol production from sugars and complex biomass by Thermoanaerobacter AK5: The effect of electron-scavenging systems on end-product formation. Energy Fuels 2012, 26, 4568–4574. [CrossRef] 16. Brynjarsdottir, H.; Scully, S.M.; Orlygsson, J. Production of biohydrogen from sugars and lignocellulosic biomass using Ther- moanaerobacter GHL15. Int. J. Hydrog. Energy 2013, 38.[CrossRef] 17. Vipotnik, Z.; Jessen, J.E.; Scully, S.M.; Orlygsson, J. Effect of culture conditions on hydrogen production by Thermoanaerobacter strain AK68. Int. J. Hydrog. Energy 2016, 41.[CrossRef] 18. AOAC. Official Methods of Analysis; Association of Official Analytical Chemists: Washington, DC, USA, 2000. 19. Orlygsson, J.; Baldursson, S.R.B. Phylogenetic and physiological studies of four hydrogen-producing thermoanareobes. Icel. Agric. Sci. 2007, 20, 93–105. 20. Scully, S.M.; Brown, A.; Ross, A.B.; Orlygsson, J. Biotransformation of organic acids to their corresponding alcohols by Ther- moanaerobacter pseudoethanolicus. Anaerobe 2019, 57, 28–31. [CrossRef][PubMed] 21. Scully, S.M.; Iloranta, P.; Myllymaki, P.; Orlygsson, J. Branched-chain alcohol formation by thermophilic bacteria within the genera of Thermoanaerobacter and Caldanaerobacter. Extremophiles 2015, 19, 809–818. [CrossRef] Fuels 2021, 2 86

22. Lee, Y.-E.; Jain, M.K.; Lee, C.; Zeikus, J.G. Taxonomic Distinction of Saccharolytic Thermophilic Anaerobes: Description of Thermoanaerobacterium xylanolyticum gen. nov., sp. nov., and Thermoanaerobacterium saccharolyticum gen. nov., sp. nov.; Reclassiﬁcation of Thermoanaerobium. Int. J. Syst. Bacteriol. 1993, 43, 41–51. [CrossRef] 23. Wagner, I.D.; Wiegel, J. Diversity of thermophilic anaerobes. Ann. N. Y. Acad. Sci. 2008, 1125, 1–43. [CrossRef] 24. Wagner, I.D.; Zhao, W.; Zhang, C.L.; Romanek, C.S.; Rohde, M.; Wiegel, J. Thermoanaerobacter uzonensis sp. nov., an anaerobic thermophilic bacterium isolated from a hot spring within the Uzon Caldera, Kamchatka, Far East Russia. Int. J. Syst. Evol. Microbiol. 2008, 58, 2565–2573. [CrossRef] 25. Tomás, A.F.; Karakashev, D.; Angelidaki, I. Thermoanaerobacter pentosaceus sp. nov., an anaerobic, extreme thermophilic, high ethanol-yielding bacterium isolated from household waste. Int. J. Syst. Evol. Microbiol. 2013, 63, 2396–2404. [CrossRef] 26. Parte, A.C. LPSN—List of prokaryotic names with standing in nomenclature. Nucleic Acids Res. 2014, 42, D613–D616. [CrossRef] [PubMed] 27. Hitschler, L.; Kuntz, M.; Langschied, F.; Basen, M. Thermoanaerobacter species differ in their potential to reduce organic acids to their corresponding alcohols. Appl. Microbiol. Biotechnol. 2018, 102, 8465–8476. [CrossRef][PubMed] 28. Scully, S.M.; Orlygsson, J. Biotransformation of carboxylic acids to alcohols: Characterization of Thermoanaerobacter strain AK152 and 1-propanol production via propionate reduction. Microorganisms 2020, 8, 945. [CrossRef] 29. Jessen, J.E.J.; Orlygsson, J. Production of ethanol from sugars and lignocellulosic biomass by Thermoanaerobacter J1 isolated from a hot spring in Iceland. J. Biomed. Biotechnol. 2012, 1869–1882. [CrossRef] 30. Sigurbjornsdottir, M.A.; Orlygsson, J. Combined hydrogen and ethanol production from sugars and lignocellulosic biomass by Thermoanaerobacterium AK54, isolated from hot spring. Appl. Energy 2012, 97, 785–791. [CrossRef] 31. Georgieva, T.I.; Mikkelsen, M.J.; Ahring, B.K. Ethanol production from wet-exploded wheat straw hydrolysate by thermophilic anaerobic bacterium Thermoanaerobacter BG1L1 in a continuous immobilized reactor. Appl. Biochem. Biotechnol. 2008, 145, 99–110. [CrossRef] 32. Georgieva, T.I.; Ahring, B.K. Evaluation of continuous ethanol fermentation of dilute-acid corn stover hydrolysate using thermophilic anaerobic bacterium Thermoanaerobacter BG1L1. Appl. Microbiol. Biotechnol. 2007, 77, 61–68. [CrossRef][PubMed] 33. Klinke, H.; Thomsen, A.; Ahring, B. Potential inhibitors from wet oxidation of wheat straw and their effect on growth and ethanol production by Thermoanaerobacter mathranii. Appl. Microbiol. Biotechnol. 2001, 57, 631–638. [CrossRef][PubMed] 34. Rani, K.S.; Swamy, M.V.; Seenayya, G. Increased ethanol production by metabolic modulation of cellulose fermentation in Clostridium thermocellum. Biotechnol. Lett. 1997, 19, 819–823. [CrossRef] 35. Soboh, B.; Linder, D.; Hedderich, R. A multisubunit membrane-bound [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase in the fermenting bacterium Thermoanaerobacter tengcongensis. Microbiology 2004, 2451–2463. [CrossRef]