Hydrogen production by Escherichia coli using brewery waste: Optimal pretreatment of waste and role of different hydrogenases

Accepted Manuscript Hydrogen production by Escherichia coli using brewery waste: optimal pretreatment of waste and role of different hydrogenases Anna Poladyan, Karen Trchounian, Anait Vassilian, Armen Trchounian PII:

S0960-1481(17)30884-4

DOI:

10.1016/j.renene.2017.09.022

Reference:

RENE 9220

To appear in:

Renewable Energy

Please cite this article as: Anna Poladyan, Karen Trchounian, Anait Vassilian, Armen Trchounian, Hydrogen production by Escherichia coli using brewery waste optimal pretreatment of waste and role of different hydrogenases, Renewable Energy (2017), doi: 10.1016/j.renene.2017.09.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

HIGHLIGHTS •

Brewery spent grains (BSG) dilute acid pretreatment was developed.

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Optimization of conditions for E. coli growth on BSG hydrolysate and H2 production was

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improved.

Hydrogenase (Hyd)-3 and Hyd-4 are responsible for H2 production.

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Mutations in the genes for Hyd-1 and Hyd-2 led to enhanced H2 production; cumulative H2 yield was determined.

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ACCEPTED MANUSCRIPT 1

Hydrogen production by Escherichia coli using brewery waste: optimal

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pretreatment of waste and role of different hydrogenases

3 Anna Poladyana,b, Karen Trchounianb, Anait Vassilianc, Armen Trchouniana,b*

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a

Department of Biochemistry, Microbiology and Biotechnology, bResearch Institute of Biology,

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Department of Ecology and Nature Protection, Biology Faculty, Yerevan State University,1 A.

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Manoukian Str., 0025 Yerevan, Armenia

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*Corresponding author: Prof. Armen Trchounian, Department of Biochemistry, Microbiology and

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Biotechnology Yerevan State University, 1 A. Manoogian Str, 0025 Yerevan, Armenia; Tel. +374-

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60710520; E-mail: [email protected]

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12 ABSTRACT

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Brewery spent grains (BSG), one of the by-products of brewery production, were used for

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Escherichia coli growth and hydrogen (H2) production. The dilute acid and alkali pretreatment

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methods were used to hydrolyze the rough lignocellulose structure, and optimal conditions for the

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BSG hydrolysate (BSGH) preparation were developed. E. coli BW25113 wild type strain and

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hydrogenase (Hyd)-negative mutants with deletions of genes encoding key subunits of Hyd 1-4

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(∆hyaB, ∆hybC, ∆hycE, ∆hyfG), as well as for a ∆hyaB ∆hybC double mutant were investigated

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with regards to growth, acidification of the medium, redox potential kinetics and H2 production

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when using BSGH. Readings of redox Pt electrode dropped to -400±10 mV, with H2 yield of ~0.75

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mmol H2 L-1 at the 3rd h wild type strain growth. Changes in redox Ti-Si electrode readings were

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negligible. H2 production was not observed with defective Hyd-3 and Hyd-4; therefore, Hyd-3 and

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Hyd-4 are responsible for H2 production using BSGH, whereas defective Hyd-1 and Hyd-2 led to a

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~2-fold stimulation of H2 yield. The data were confirmed by determining cumulative H2 yield. These

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findings are useful for development of renewable energy, especially H2 production biotechnology,

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using different organic wastes as sustainable energy feedstocks.

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Keywords: Renewable energy; Brewery spent grains; Escherichia coli; Hydrogen production; Waste

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pretreatment; Hydrogenases

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1. Introduction 1

ACCEPTED MANUSCRIPT There is an urgent need to find alternative sources of ecologically clean, renewable and

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cheap energy to satisfy the demands of modern industrialized civilization and sustainable

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development [1]. Another problem that is worth addressing is generation of different wastes, which

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are produced in huge amount after many industrial and agricultural activities, utilization of which

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could lead both to waste treatment and cost-effective energy production. In this respect, molecular

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hydrogen (H2) is being recognized as an alternative energy source of the future, as it is renewable,

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ecologically advantageous (only water is formed upon H2 oxidation) and, moreover, the energy

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released upon it’s combustion (~142 kJ g-1) is ~3.5 fold greater than for oil [2].

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Nowadays it is of interest to produce H2 by using bacteria. Escherichia coli, performing dark

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fermentation resulting in H2 evolution, is a good candidate to use for these biotechnological

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proposes, because it is well characterized, genetically and metabolically easy to manipulate

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bacterium [3]. Moreover, local agricultural and food industry wastes rich in carbohydrate and

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protein can be used for H2 formation by implementing proper bio-process technologies [4-6]. In this

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context, consumption of the mentioned wastes for H2 production provides a low-priced energy

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generation and, which is also important, accompanying waste treatment [7]. Transition of H2

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production by dark fermentation from laboratory to pilot-scale systems is emerging [8-9]. This will

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lead to the strategy in which local energy supply systems are developed; without the need for long-

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distance transportation of resources, products and associated expenses. The resources can include

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cereal distiller’s grains wastes which are as by-product of ethanol fermentation [5], and also brewery

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wastes [6, 7, 11-13].

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A major part of brewery wastes is used in agriculture, particularly as an animal feed, but also

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for production of specialized valuable products such as yeast extract and other compounds.

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Reductions in dairy farming, limits on farm activities due to disease and other factors have restricted

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these opportunities [14]. Thus, brewery waste utilization can be used to release energy, particularly

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H2 [15].

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Beer is one of the most highly consumed beverages throughout the world. During brewing,

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various by-products are generated, the most common of which are spent grains (BSG), spent hops

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and surplus yeast; the latter are generated from the main raw materials [15-17]. BSG is the by-

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product of the mashing process; which is one of the initial steps of brewery production necessary to

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solubilize the malt and cereal grains and to extract of the wort (water with extracted biomass) [15-

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17]. The worldwide annual production of BSG has been estimated to be approximately 38.6×106

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tons [16]. 2

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protein content; the most abundant monosaccharides found in BSG are xylose, glucose and

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arabinose [15, 17, 18], as well as minerals, many vitamins and amino acids. It is very important to

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pretreat BSG correctly. Various pretreatment methods are now available to fractionate, solubilize,

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hydrolyze and separate cellulose, hemicellulose, and lignin components [12, 19-21]. The first step of

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pretreatment starts with mechanical curing that can be followed by chemical, physicochemical,

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thermal, biological and other procedures to further destroy the tough structure [21]. The next step is

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conversion of monomeric carbohydrates into H2 by using appropriate microorganisms. There are

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some studies reporting the possibility of producing biogas from different brewery wastes [4, 6, 17].

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It was shown that brewery waste can be used for H2 production via dark- and photo-fermentation by

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bacteria [4, 13, 18, 22] including Bacillus sp., Enterobacter sp. or Rhodobacter sphaeroides which

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could be considered as efficient bacteria towards industrially relevant H2 production. However,

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mechanisms and optimal conditions for H2 production using brewery and other food industry wastes

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should be studied, making possible genetic and metabolic engineering of bacteria to enhance

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worldwide H2 production.

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It is well known that E. coli during utilization of different carbon sources (glucose or

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glycerol) also efficiently produces H2 [2, 3]. H2 production from distiller’s grains wastes was shown

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for E. coli and R. sphaeroides and the optimal pretreatment conditions were investigated [5].

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In E. coli H2 is produced from formate decomposition, the end product of both glycerol and

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glucose fermentation via action of membrane-associated [Ni-Fe]-hydrogenase (Hyd) enzymes [23,

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24]. In E. coli four Hyd enzymes (Hyd 1-4) encoded by the hya, hyb, hyc and hyf operons,

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respectively, are involved in H2 metabolism: they are responsible for both H2 production and

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oxidation [23, 24]. Moreover, the reaction directions and activity of the Hyd enzymes are

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determined by the substrate of fermentation (glucose or glycerol), pH of the medium, oxidation-

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reduction potential (ORP) and other factors [2, 3, 23, 25-27]. Consequently, optimal activity of Hyd-

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1 was shown under anaerobic conditions, at low, acidic pH or upon formate supplementation while

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Hyd-2 was active under more reducing conditions (less ORP) or at alkaline pH. Formate

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dehydrogenase H (FDH-H) together with Hyd-3 or Hyd-4 compose the formate hydrogenlyase

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(FHL) 1 and 2 pathways during glucose anaerobic utilization at acidic and alkaline pHs, respectively

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[23, 28]. E. coli might be also considered as a good candidate for industrial H2 production using

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brewery wastes. For development of H2 production technology by bacteria it is essential to

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investigate and control the conditions of Hyd enzymes activities using appropriate mutant strains

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during the utilization of different organic wastes, including those from breweries. The present study aimed to develop optimal pretreatment methods of BSG; to design optimal

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conditions for growth and H2 production of E. coli upon utilization of BSG hydrolysate (BSGH); to

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reveal the responsible Hyd enzymes for H2 production under certain optimized conditions with the

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help of E. coli different Hyd mutants; as well as to enhance H2 production. Thus, BSG pretreatment

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conditions were designed to be effective for E. coli bacterial growth and H2 production and

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conditions; utilization of BSGH were also optimized. The role of Hyd 3 and Hyd 4 in H2 production

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was determined; and improved H2 production was observed in E. coli single and double mutants

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lacking Hyd 1 and Hyd 2.

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These findings are novel and might have application with benefits for producing applied and

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renewable energy when brewery waste is used. Utilizing different wastes for energy production are

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of significance to supply renewable and sustainable energy and to maintain clean environment.

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2. Materials and methods

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2.1. Bacteria, the growth conditions for bacteria; growth yield and rate determinations The E. coli wild type parental strain (WT) and different Hyd-negative mutant strains have been

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kindly provided by Prof. T. Wood (Pennsylvania State University, University Park, PA, USA) [29]

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and are described in Table 1.

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Bacteria were cultivated for 72 h in anaerobic conditions, at 37 °C. The batch culture was

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performed in 150 mL glass vessels covered by plastic press-caps and composed of growth peptone

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medium (PM) or BSGH or minimal salt medium (MSM). Bacterial subculture inoculums were

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grown in PM under the same anaerobic conditions. PM was composed of 20 g L-1 peptone, 2 g L-1

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K2HPO4, 5 g L-1 NaCl, pH 6.5; 10 g L-1, whereas, MSM contained 8.004 g L-1 K2HPO4, 3.128 g L-1

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KH2PO4, 1.056 g L-1 NH4SO4, 0.048 g L-1 MgSO4, 0.009 g L-1 FeSO4

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used to measure the pH by HJ1131B pH-meter (Hanna Instruments, Portugal). The pH was settled

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using 0.1 M NaOH or KOH or 0.1 N HCl.

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[26, 27]. A pH-selective electrode was

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BSG were supplied by “Kilikia” beer factory (Yerevan, Armenia). BSG was treated by using

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dilute acid and alkali hydrolysis methods in steam sterilizer for 1 h, 121 ºC [19, 20], and BSGH was

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obtained.

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Spectro UV-VIS Auto spectrophotometer (Labomed, Los Angeles CA, USA), by determining the

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optical density (OD) values of the bacterial culture absorbance at 600 nm. The specific growth rate

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(µ) was stated as lg2/doubling time, which was counted when the logarithm of OD was increased

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linearly with time, as described [26]. Finally, bacterial growth yield was estimated by determining

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bacterial culture dry weight (CDW) and expressing in g L-1, as done before [26].

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2.2. H2 production determination

The pair of ORP platinum (Pt), (EPB-1, GSEEE; or PT42BNC, HANNA Instruments, Portugal) and

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titanium-silicate (Ti-Si), (EO-02, GSEEE, Gomel, Belarus) electrodes were used to determine the

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ORP of bacterial culture [26, 30, 31]. Compared to the Ti-Si electrode, the Pt electrode is sensitive

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to H2 or O2 and its readings drop down to negative values (~400 mV) in the presence of medium H2.

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The difference between the readings of the two mentioned electrodes permits to estimate the H2

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production under particular conditions, as earlier reported [26, 30, 31]. The H2 production yield was

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calculated and expressed in mmol H2 L-1, as described [26, 30, 31]. H2 determination using the

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electrochemical method described [2, 3, 23] gives more accurate results for cumulative H2 yield in

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liquids.

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Cumulative H2 yield measurements were performed in 500 mL glass vessels under permanently

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stirring conditions; the gases bubbled (H2 and CO2) were treated by 1 M NaOH solution (to

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eliminate CO2 from gas mixture); and H2 gas was collected and estimated by the water displacement

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volume [5, 23].

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H2 production upon E. coli growth was visualized by the presence of gas bubbles in the test

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Durham tubes and also was validated by the chemical assay based on the decolorizing of KMnO4

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solution in H2SO4 with H2 [26, 30, 31].

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2.3.Reagents used in the study and data processing

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Glycerol and peptone (Carl Roths GmbH, Germany) were used, and other reagents were of

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analytical grade.

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The average data are expressed from three experiments done; the standard errors were

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considered and Student criteria (P) were used to confirm the difference in average data between

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different series of experiments, as defined before [25-27]; the difference was considered valid when

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P<0.05. 5

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3. Results and discussion 3.1. Pretreatment of BSG and its optimization for E. coli growth and H2 production

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It is very important for bacterial growth and H2 production to pretreat BSG correctly prior to use.

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During the few past years a large number of pretreatment methods have been developed, including

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dilute acid or alkali treatment, ammonia exposure, ionic liquid pretreatment and others [12, 19-21].

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So, the initial step of our investigation was directed to hydrolyze the lignocelluloses containing

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material. For this proposes different acids and alkalis were applied: 1-10% BSG were dissolved in

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0.5 to 3% acid (HCl, H2SO4) or alkali (NaOH, KOH) and treated, as described (see Materials and

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methods). After filtration the next step was adjustment of pH of the BSG hydrolysate (BSGH) from

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1.5 to 7.5. The latter was done by using various alkalis (NaOH, KOH) and with potassium salt

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(K2HPO4); the pH value was reached to pH 7.5 by using HCl. Finally, appropriate dilutions of

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BSGH in a broad range, from 1 to 20, were applied.

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Given these observations, the optimal conditions for bacterial growth and H2 production

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were found to be 4% (w/v) of BSG treated with 0.7% (v/v) sulfuric acid in a steam sterilizer, at

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121°C, the pH was adjusted to pH 7.5, and 2.5-fold dilutions of BSGH were more optimal for E.

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coli WT strain growth and H2 production (see Fig. 1).

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Next, E. coli BW25113 WT strain growth, ORP kinetics and H2 production were

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investigated. The results were compared with the data of E. coli grown on PM, with 10 g L-1

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glycerol supplementation (see Materials and methods). It should be noted that glycerol being by-

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product of various industries, including bio-diesel production, is currently very cheap; and it was

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shown that it can be fermented by E. coli [3]. Medium acidification was observed upon E. coli WT

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strain growth on both BSGH and PM with glycerol: after 6 h growth the pH dropped from 7.5 to 5.5

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and to 6.9, respectively (Fig. 2b). Bacterial biomass yield was ~1.8 fold less after 24 h growth on

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BGSH compared with that on PM under glycerol fermentation (Fig. 3a). There was no difference

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between bacterial specific growth rates (µ) values (0.63±0.02 h-1) in both cases (Fig. 3b). It was

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reported that pretreatment of lignocellulosic biomass may produce degradation products with an

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inhibitory effect on the fermentation process which might affect bacterial growth [12, 19-21]. On the

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other hand, lower biomass yield might be regarded due to medium rapid acidification (Fig. 2b) or

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limitations of fermentation substrates.

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moreover, ORP is parameter linked to the activity of Hyd enzymes and H2 production [32].

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Readings of the Pt electrode dropped from positive values down to negative ones, -400±10 mV

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value, with a H2 production yield of 0.74±0.02 mmol H2 L-1 at the 3rd h of growth, corresponding to

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the beginning of the log growth phase of E. coli BW25113 fermenting BSGH (see Fig. 2a). Upon

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bacterial glycerol fermentation in PMH2 production was observed at the end of the log growth phase

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with the H2 production yield of 0.75±0.03 mmol H2 L-1. In contrast, the drop in Ti-Si electrode

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readings was negligible and remained positive when compared to glycerol fermentation in PM (see

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Fig. 2a). H2 was not produced after 72 h growth (data not shown).

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BSGH used in the study is a typical hemicellulosic hydrolysate which contains a mixture of

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xylose, arabinose, glucose, formic acid, acetic acid, etc. [15, 17, 18], all of which can be fermented

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by E. coli [2, 3, 28, 33, 34]. Thus utilization of these substrates might lead to bacterial biomass

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formation, medium acidification and H2 production. Future studies are required to enhance

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bacterial biomass and to prevent rapid drop of medium pH upon E. coli growth on BSGH.

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3.2. Production of H2 by the E. coli wild type strain BW25113 and Hyd-negative mutants upon

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fermentation of BSGH

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As was mentioned above, H2 is produced from formate decomposition upon glucose or glycerol

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fermentation [2, 3, 28], and Hyd enzymes are involved in this process [23, 24]. To understand the

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role and participation of individual Hyd enzymes both in bacterial growth and H2 production upon

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utilization of BSGH, the growth and H2 production of E. coli ∆hyaB, ∆hybC, ∆hycE, ∆hyfG mutants

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with deletions of genes for different key subunits of Hyd-1 to 4, respectively, and a ∆hyaB ∆hybC

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double mutant with deletions of genes for key subunits of both Hyd1 and Hyd 2 were studied (Table

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1).

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All of the mutants were able to grow on BSGH: after 24 h of growth the biomass yield

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values were around 0.3 (g CDW) L-1, close to the BW25113 value, and again, ~1.8-fold less

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compared with the data of the WT strain grown on PM with glycerol (see Fig. 3a). µ was more or

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less similar each strain, even ~1.1-fold stimulated in the ∆hyaB ∆hybC double mutant (see Fig. 3b).

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Medium pH dropped from 7.5 to 5.5 upon bacterial all strains at 6 h growth on BSGH (data not

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shown).

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From the beginning of the lag growth phase of the WT strain and all mutants a drop of the Pt

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electrode readings from positive to negative values (down to ~ -100 to -500 mV) was detected (Fig. 7

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4): the H2 production yield was stimulated ~2 fold (~1.45 mmol H2 L-1) in the ∆hyaB, ∆hybC single

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and the ∆hyaB ∆hybC double mutants (see Table 1) compared with the WT strain. In contract, H2

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production was absent in the ∆hycE and ∆hyfG mutants (see Table 1) during log growth phase (see

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Fig. 4): the Pt electrode readings dropped to ~ -200 mV and -100 mV, respectively. As was mentioned above, activity of the Hyd enzymes and their operation direction (H2

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producing or oxidizing mode) depended on the substrate of fermentation, medium pH, etc. [23, 24].

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So, our results point out the key roles of Hyd-3 and Hyd-4 in H2 production and verify the role of

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the Hyd-1 and Hyd-2 in reverse, H2 oxidizing mode upon BSGH utilization by E. coli. The results

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for Hyd-3 are in accordance with the previous findings obtained, when it was shown that Hyd-3 is

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the major in H2 production by E. coli in the presence of formate, however Hyd-4 activity is inhibited

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by high concentration of glucose mainly at pH 7.5 [23,28,35].

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3.3. Cumulative H2 production measurements during growth of the E. coli BW 25113 and ∆hyaB ∆hybC double mutant under different conditions

Cumulative H2 production was measured upon bacterial anaerobic growth in 500 mL medium upon

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permanent stirring conditions (to remove H2 gas from the growth medium) (Fig. 5, Table 2). As was

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mentioned above, H2 and CO2 are produced during growth of E. coli on different fermentation

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substrates [23]. Thus, CO2 was eliminated from the gas mixture by 1 M NaOH solution; and H2 gas

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was collected and estimated by water displacement volume (see Materials and methods).

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For standard comparison, glucose was used as a reference for comparing the yield of H2

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obtained from BSGH to the yield of glucose. Moreover, the influence of the medium composition

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on the yield of H2 was also tested where the bacteria were grown in PM and minimal salt medium

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(MSM) with 0.2% glucose as fermentation substrate. Thus, cumulative H2 yield reached up to 170±5

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mL and 39±3 mL for growth of the WT strain on PM and MSM, respectively (Fig. 5) at the 11th h of

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growth on 0.2% glucose (1 g glucose was present in 500 mL PM). It should be noted, that upon

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bacterial growth on MSM the lag phase of growth was prolonged and noticeable H2 production was

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observed after 7 h of growth. With growth of the WT strain in 500 mL of BSGH (with 8 g BSG)

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cumulative H2 yield reached up to 26±3 mL, but stimulated ~ 1.7-fold (46±2 mL) in the ∆hyaB

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∆hybC double mutant (Fig. 5). Data of cumulative H2 production per bacterial CDW are presented

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in Table 2: thus, growth of the WT strain on rich (PM) medium with glucose led to ~3 and ~1.7-fold

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higher amount of H2 yield compared to growth of the WT strain and mutant on BSGH, respectively.

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higher H2 production levels, nevertheless, glucose and peptone are much more expensive substrates

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for sustainable, long-term H2 production than BSG [37]. Moreover, the results demonstrate that

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upon utilization of BSGH by the bacterium MW1000, with abolished H2 oxidation properties,

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improved levels of H2 accumulation can be achieved. Significant amounts of BSG are annually

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produced from breweries [16]. Thus, providing wet BSG with the cheapest (low-value) price to local

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consumers, like biogas stations or farmers (for use as cattle feed, etc.) will be a good solution for the

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breweries to eliminate BSG, since this is a cheap “fuel” alternative that avoids the energy spent

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needed for drying of BSG. However, the costs of transporting BSG are still significant and should be

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considered [36]. Usually, on average 16 USD per ton of wet BSG is the transport cost over a

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distance of 5 miles (∼8 km) [16, 36]. So, comparing the prices for glucose [37] and BSG [16, 36]

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and yields of H2 produced by the WT strain and the ∆hyaB ∆hybC double mutant under different

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conditions suggests the possibility of BSG application for low-priced H2 generation (see Table 2).

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4. Conclusions and significance

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Taken together, our results demonstrated that use of BSG consisting of cellulose, hemicellulose and

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lignin needs a treatment process to release the substrates to achieve bacterial growth and sufficient

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H2 production by E. coli. Thus, the optimal treatment of BSG has been developed and E. coli growth

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and H2 production have been shown using BSGH as substrate. Moreover, the responsible Hyd

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enzymes for H2 production – Hyd-3 and Hyd-4 were determined for bacterial growth on BSGH. The

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mutations in E. coli genes encoding Hyd-1 and Hyd-2 resulted in enhanced H2 production.

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These findings on bio-hydrogen production by bioconversion of organic waste are novel and

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of great interest for renewable energy supply. They should lead to development of a strategy for

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sustainable and renewable energy production from available and cheap wastes like BSG. In addition,

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they have ecological and economic advantages. Therefore, the results might lead to evaluation of

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new approaches to produce H2 simultaneously with waste treatment and offer the opportunity to

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effectively obtain H2 by bacteria.

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Conflict of interest

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The authors have no conflict of interests.

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Acknowledgement 9

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This work was supported by Armenian National Science and Education Fund (USA) Research

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grants to KT (NS-Biotech-4184) and AP (NS-Biotech-4376), Research grants of the State

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Committee of Science, Ministry of Education and Science of Armenia, to AT (#15T-1F123 and

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#AB16-37).

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SC

355

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Transforming biomass conversion with ionic liquids: process intensification and the

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[37] https:// www.ers.usda.gov

AC C

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SC

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Table 1. E. coli strains characteristics used in the study

422 Strains

Genotype

Protein’s absent subunit

Reference

BW 25113

laclq rrnBT14 ∆lacZW116

WT parental strain

[29]

RI PT

hsdR514 ∆araBADAH33 ∆rha BADLD78 BW 25113 ∆hyaB::Kan

Large subunit of Hyd-1

[29]

JW 2962*

BW 25113 ∆hybC::Kan

Large subunit of Hyd-2

[29]

JW 2917*

BW 25113 ∆hycE::Kan

Large subunit of Hyd-3

[29]

JW 2472*

BW 25113 ∆hyfG::Kan

Large subunit of Hyd-4

[29]

MW1000*

BW25113 ∆hyaB ∆hybC::Kan

Large subunits of Hyd-1

[29]

M AN U

SC

JW 0955*

and Hyd-2

423

EP

TE D

*Resistant to kanamycin

AC C

424

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ACCEPTED MANUSCRIPT 425

Table 2. Cumulative H2 yield of E. coli wild type parental strain BW25113 (WT) and the ∆hyaB

426

∆hybC mutant MW1000 upon growth in different conditions.

427 H2, mL (g

Estimated

peculiarities*

CDW)-1

price (USD)**

WT grown on BSGH

100±3

Mutant strain

175±5

grown on BSGH WT grown on PM with

290±5

WT grown on MSM

160±5

M AN U

with glucose 428

432

4

** Prices are calculated for 1000 L of H2 yield considering only the prices for glucose [37] and BSG [16, 36].

TE D

431

2.20

(see Materials and methods); for E. coli strains, see Table 1.

EP

430

0.7

*Data on the 11th h of bacterial growth are presented; PM is peptone medium; MSM is minimal salt medium

AC C

429

1.28

SC

glucose

RI PT

Growth

15

ACCEPTED MANUSCRIPT 433

LEGENDS TO FIGURES

434 435

Fig.1. Main steps of H2 production by E. coli bacteria using BSGH. See the text.

436 Fig. 2. ORP (a) and pH (b) changes during growth of E. coli BW25113 on peptone medium (PM)

438

with glycerol and BSGH. Bacteria were grown anaerobically, at pH 7.5, with 10 g L-1 glycerol

439

added when necessary. WT is wild type parental strain. ORP was determined by platinum (Pt) and

440

titanium-silicate (Ti-Si) electrodes and expressed in mV (vs Ag/AgCl (saturated by KCl)).

RI PT

437

441

Fig. 3. Growth characteristics of E. coli BW25113 and the Hyd-negative mutants. Bacteria were

443

grown anaerobically, at pH 7.5. a) Cell dry weight (CDW) (g L-1); b) specific growth rate (µ) during

444

growth on BSGH and PM with 10 g L-1 glycerol addition. WT is wild type parental strain; BSGH is

445

brewery spent grains hydrolysate; PM is peptone medium. For strains used, see Table 1.

M AN U

SC

442

446 447

Fig. 4. The ORP kinetics of by E. coli BW25113 and the Hyd-negative mutants during utilization of

448

BSGH. BSGH is brewery spent grains hydrolysate. For the others, see the legends to Figs. 2 and 3.

449

Fig. 5. Kinetics of cumulative H2 yield during growth of E. coli BW25113 and the ∆hyaB ∆hybC

451

double mutant under different conditions. In all cases, bacteria were grown in 500 mL growth

452

medium: MSM is minimal salt medium; PM is peptone medium; BSGH is brewery spent grains

453

hydrolysate. WT is wild type parental strain and M is the ∆hyaB ∆hybC double mutant. G is

454

glucose. For strains used, see Table 1.

457

EP

456

AC C

455

TE D

450

16

ACCEPTED MANUSCRIPT 458 459

Fig. 1.

460 H2 E. coli addition

Hydrolysis

EP

TE D

M AN U

SC

Brewery spent grains

RI PT

Optimization

AC C

461 462 463 464 465

17

ACCEPTED MANUSCRIPT 466

Fig. 2. ORP, mV 300 200 100 0 -100 -200 -300 -400 -500 -600

3

4

5

Ti-Si, WT, BSG hydrolisate

Pt, WT, BSG hydrolisate

pH

7 6.5

4.5

1

2

3

Time, h

4

5

EP

4

TE D

6 5

b)

AC C

WT, Peptone + glycerol

470 471 472

48

Pt, WT, Peptone +glycerol

7.5

5.5

24

Ti-Si,WT, peptone+glycerol

a)

8

6

RI PT

2

SC

1

M AN U

467 468 469

Time, h

18

6

24

48

72

WT, BSG hydrolisate

ACCEPTED MANUSCRIPT 473 474 475

Fig. 3. a) CDW, g L-1

476 477

b) µ, h-1 0.8 0.7 0.6

TE D

0.5

M AN U

SC

RI PT

0.6 0.5 0.4 0.3 0.2 0.1 0

0.4 0.3 0.2 0.1

478

AC C

EP

0

19

ACCEPTED MANUSCRIPT 479 480

Fig. 4.

100 Time, h 0 1

2

3

4

5

6

7

RI PT

ORP, mV

200

WT

-100

SC

hyaB hybC

-200

M AN U

hycE

-300 -400

EP

-600

AC C

481

TE D

-500

20

hyfG hyaBhybC

ACCEPTED MANUSCRIPT

Fig. 5.

200

H2, ml

RI PT

482 483 484

180 160

SC

140 120

M AN U

100 80 60 40 20

TE D

0 2

3

4

5

6

7

8

21

WT, MSM+G M, BSGH WT, PM+G

9

10 11

Time, h

AC C

485

1

EP

0

WT, BSGH