The Additive Free Microwave Hydrolysis of

Lignocellulosic Biomass for Fermentation to High

Value Products

Jiajun Fan,a Fabio Santomauro,b Vitaliy L. Budarin,a Fraeya Whiffin,c Felix Abeln,c Tanakorn

Chantasuban,b Deborah Gore-Lloyd,d Daniel Henk,d Roderick J. Scott,d James Clark *a and

Christopher J. Chuck *b

a. Green Chemistry Centre of Excellence, Department of Chemistry, University of York,

Heslington, York, YO10 5DD, UK.

b. Department of Chemical Engineering, University of Bath, Bath, BA2 7AY, UK.

c. Centre for Doctoral Training in Sustainable Chemical Technologies, Department of

Chemical Engineering, University of Bath, Bath, BA2 7AY, UK.

d. Department of Biology & Biochemistry, University of Bath, Bath, BA2 7AY, UK

* Correspondence should be addressed to james.clark@york.ac.uk or c.chuck@bath.ac.uk

KEYWORDS

Bio-refinery, Integrated Technology, Microwave Hydrolysis, Fermentation

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ABSTRACT

Current biorefineries are predominantly based around single feedstock sources, extensively

hydrolysed using multiple unit operations. The hydrolysate is generally converted to a single

product by one of a few well-characterised organisms. Here, we report on a new approach to the

biorefinery, combining a rapid, microwave heated, one-step depolymerisation process, with a

yeast, Metschnikowia pulcherrima which is able to metabolise an array of oligo- and

monosaccharides. During the investigation it was found that the microwave hydrolysis process

was able to solubilize upto 50% wheat straw biomass by weight, mainly as oligosaccharides

though also containing mixtures of pentose, hexose and anhydro-sugars with concentrations of

up to 2 g L-1. However, a fine balance between elevated monosaccharide yields and the

production of inhibitive compounds had to be struck with optimal microwave hydrolytic

conditions found to be 190 °C. Further testing utilizing several different types of lignocellulosic

biomass demonstrated it was possible to attain ~65% carbon efficiency in the conversion of

hydrolysis products to yeast biomass. The system was scaled to 600 mL using DDGS

successfully solubilizing 66% of the feedstock, producing 33 g L-1 hydrolysate. M. pulcherrima

grew well on this hydrolysate in a controlled stirred tank bioreactor (2L), yielding 8.38 g L -1

yeast biomass, a yeast biomass coefficient of 0.25. This presents an exciting, feedstock agnostic,

pathway to the energy efficient production of a wide variety of commercially valuable chemical

products without the need for extensive pre and post processing technologies.

1. INTRODUCTION

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Biorefineries have the potential to become major sources of renewable chemicals and fuels

though variable feedstocks, multiple unit operations, avoiding cross contamination and poor

carbon efficiencies remain key challenges in the development of this sector.1 Current second

generation processes for the conversion of biomass to chemicals and fuels rely on a mix of

traditional chemical processes (e.g. acid hydrolysis) and biochemical, enzymatic transformations

run as a series of unit operations.2 This can lead to multiple waste streams and reduced product

yields. Applying such processes to future large scale multi-product biorefineries that can replace

existing petrochemical refineries, is also challenging due to the variable biomass feedstocks that

would need to be used for all year round production. For this reason existing biorefineries are

generally based on a single feedstock. Biorefineries will become a significant contributor to

future chemical and fuel supplies, but further work is required to increase the flexibility and

produce more products efficiently. Here we show that the integration of low temperature

microwave chemistry with a novel, robust, yeast fermentation could become the basis of

efficient, feedstock-agnostic, multi-product biorefinery.

We found that the simple additive-free low temperature microwave processing of biomass leads

to a hydrolysate that can be used directly as a substrate for Metschnikowia pulcherrima. The

yeast has little substrate bias and can metabolise the oligosaccharide-rich media while

combatting any microbial invasion by producing an array of antimicrobial compounds.

Remarkably our novel integrated technology works with completely different types of biomass

(agricultural, aquatic and industrial), producing an assortment of compounds.

Our results demonstrate how the integration of two complementary technologies drawn from

different disciplines opens the door to a new generation of multi-product biorefineries that can

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Figure 1. Schematic detailing major unit operations for a conventional second generation

yeast biorefinery and the proposed biorefinery integrated microwave and biological

technologies

operate across the world using different local resources, including wastes, to produce a range of

chemical products with high carbon efficiency and minimal by-products.

Currently, three of the key energetic demands in processing biomass are the energy required to

make enzymes, to provide heating and electrical power for the pre-treatment stages and the

milling of the feedstock. While estimates differ widely, enzyme production requires as little as

10% of the total energy, where milling could be as low as 1%, instead the majority of the energy

is needed for heating and electrical power for the pre-treatment stage.3-5 At present this is

generally delivered by conventional electrical heating, though MW heating has been estimated to

need significantly less power. For example, MW heating was shown to lead to a 5-7x increased

rate of sucrose hydrolysis and a lowering of the activation energy.6

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Similarly in the acid pre-treatment of sugar bagasse, MW heating was shown to give rise to 4x

higher sugar production in less than 20% of the time.7 From an economic perspective, enzymes

are one of the largest single costs of a second generation process, estimated to be as high as 20%

of the total cost involved in ethanol production from softwood for example.8 Commercialised

processes for the production/extraction of sugars, such as that of Virdia;9 a cold acid solvent

extraction of cellulosic feedstock, and Renmatix; two step ‘hemi hydrolysis’10 and ‘supercritical

hydrolysis’11 demonstrate the feasibility of upcoming technologies in the bio-refining sector, but

still utilise multi-unit operations and employ additives and harsh processing conditions.

Therefore, by removing or reducing the need for enzymes and by using a more efficient

microwave heating source the cost of biomass processing could be reduced substantially.

Microwaves (MW) are a very energy efficient method of heating, especially of aqueous

solutions, due to the high polarity of water. Furthermore, MW processing is selective, rapid and

highly controllable.12 Recently, we reported a method for the hydrothermal microwave

depolymerisation of lignocellulosic materials, that releases substantial quantities of sugar,

without any enzymes or other additives.13 Under MW treatment, the maximum glucose yield at

220°C was nearly 50 times higher than that under the same conventional hydrolysis conditions

when using microcrystalline cellulose.13 This process could potentially be more commercially

viable, due to lower energy input and reduced process units including no pre-enzymatic

treatment (fig. 1).

High temperatures used in biomass processing can cause the degradation of released sugars into

furfural, 5-hydroxymethylfurfural (5-HMF) and organic acids. The lignin can also degrade

partially into a range of monomeric aromatic compounds including vanillin, vanillic acid, 4-

hydroxybenzoic acid (4-HBA), syringaldehyde, and coniferyl alcohol.14 MW processing gives

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better control over the reaction kinetics. By reducing the temperature, inhibitor production can be

limited, though at the expense of decreasing the monosaccharide concentration and increasing

oligosaccharide concentration. Therefore, it is important to find a balance between the MW pre-

treatment and a fermentation process capable of handling the initial feedstock and subsequent

hydrolysate enabling the industrial application of the combined technologies.

Traditional microbial bioprocessing, based around a limited pool of well-known organisms,

appears to be reaching a limit in terms of the economically achievable products and the complex

processing required to release simple sugars from recalcitrant biomass. Recent comparative

genomics studies have shown a huge variety of Saccharomyces and non-Saccharomyces yeast

with untapped potential to be used in future industrial biotechnology.15,16 In these publications,

the authors highlighted the available complexity, robustness and the vast product potential

available in this natural biota. To expand the potential of future biorefineries, it is these yeasts

that must be further developed, including aligning these positive traits with the feedstock

processing side. To this end, we selected the little known wine yeast Metschnikowia pulcherrima

to integrate with the microwave processing stage to demonstrate this concept.

2. MATERIALS AND METHODS

2.1 Materials

All chemicals were purchased from Sigma Aldrich unless otherwise stated and used without

purification. Spring wheat straw was obtained from a farm local to Bath, UK and stored at 18°C

in a sealed plastic container in the dark. Samples of straw were reduced in size by cutting with

scissors to >1 cm in size pieces, then grinding in a food blender for 5 minutes, the fraction that

could pass through a 1.2 mm sieve was retained. DDGS and DRAFF, were sourced from a UK

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agricultural firm, seaweed was harvested from the South West UK coast in August, washed in

deionised water and freeze dried. Defatted rapeseed meal was sourced from a UK chemical

company and used without any further purification.

2.2 Methods

2.2.1 Microwave processing of biomass

The depolymerisation/MW hydrolysis of wheat straw was undertaken using a CEM MARS 6

microwave rector. For this, 2g ± 0.005g of biomass was weighed into a 75ml PTFE vial

(provided by CEM) and 40ml of distilled water added in order to make a sample to water ratio of

1:20. To this mixture, a PTFE magnetic stirrer bar was added. Experiments were subsequently

performed to ascertain the optimum conditions for the production of fermentable mixtures from

the fixture by systematically testing differing final temperatures, ranging between 180-240°C

with a maximum applied microwave power of 1800W. The remaining five types of biomass,

including rapeseed meal, DRAFF, DDGS, Ascophyllum nodosum and Laminaria saccharina

were processed under the same conditions at 190 °C, with a hold time of 0 mins, and a total

irradiance time of 15 mins.

After the MW process, the solid and liquid were separated by filtration. The hydrolysate was

then used for the fermentation process directly, and the solid residue was oven dried at 105 °C

for 24 hours with the mass subsequently determined.

The scale up was undertaken using a Milestone SynthWAVE microwave rector.For this, 30 g ±

0.05 g of biomass was weighed into a 1 L PTFE vial (provided by Milestone) and 600ml of

distilled water added in order to make a sample to water ratio of 1:20. The reactor uses a PTFE

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stirrer to mix the material during the microwave reaction. Based on the small scale CEM MARS

6 results, the 190 C, 15mins ramping with no holding microwave conditions were used to

process DRAFF and DDGS. After the MW process, the solid and liquid were separated and

processed in the same way as described above.

2.2.2 Yeast cultures

M. pulcherrima (NCYC 373) was obtained from National Collection of Yeast Cultures (Norfolk,

UK) and stored on sterile YPD agar plate at 4°C. New strains used in the zone inhibition

experiments were isolated locally from wild growing fruits. In brief, collected fruits were

incubated in SMB media (30 g L-1 Tryptic Soy Broth, 25 g L-1 Malt Extract; pH5) for 1 hour at

25°C with 200rpm. 300µl of the media was serially diluted prior to 100µl being plated on to malt

extract agar containing chloramphenicol. Plates were incubated for 4 days at 25°C. Yeasts that

grew were identified via PCR and Sanger sequencing of the variable ITS1 and 2 regions. All

strains are available on request. All strains of M. pulcherrima were re-plated every two months

to ensure the cultures remained viable and uncontaminated.

Unless otherwise stated, the control medium was yeast minimal medium, YMM (H2KPO4, 7.00 g

L-1, Na2HPO4, 2.50 g L-1, MgSO4.7H2O, 0.188 g L-1, MgCl2.6H2O, 1.08 g L-1, ZnSO4.7H2O,

0.0200 g L-1, (NH4)2SO4, 0.0625 g L-1, NH4Cl, 0.354 g L-1, Glucose/glycerol, 30.0 g L-1, Yeast

extract, 1.00 g L-1, CaCl2.2H2O, 0.150 g L-1). The medium was prepared in deionised water in a

Duran bottle (without adding calcium chloride), the pH was then adjusted to 5 using

concentrated hydrochloric acid, then the calcium chloride added, then the Duran bottle was

autoclaved at 121°C, 20 mins.

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Cultivation was carried out under sterile conditions unless otherwise stated. Media and non-

irradiated fermentation vessels were autoclaved at 121 °C for 20 mins prior to use and handled

using aseptic techniques in a laminar flow hood. Innocula were prepared in sterilised YPD (10 g

L-1 yeast extract, 20 g L-1 peptone, 20 g L-1 glucose) from a single colony of M. pulcherrima,

incubated at 25 °C for 24 hours, then diluted to an OD600nm of 0.6 with sterile YPD. Where

YMS (30 g L-1 yeast extract, 5 g L-1 mannitol, 5 g L-1 sorbose) was used, the same method was

applied. Sterile vessels were charged with minimal medium to the volume specified in a 1:5

media to air ratio. Cultivation was carried out in the dark in incubators set to 25 °C ± 1 °C

shaken at 180 RPM.

Optical density at 600 nm was measured after diluting a 10-50 µl sample in DI water by at least a

factor of 10, using a UV-vis spectrophotometer (Perkin-Elmer), blanked to DI water for control

medium or to the hydrolysate prior to inoculation at the same dilution.

To test the effect of inhibitors on M. pulcherrima, 96-well plates were charged with 200 µl 6

replicates of medium, in non-sterile conditions, sterilised by UV light in laminar flowhood for 1

hour, then inoculated with 5 µl YPD inoculum. The lid was sealed with parafilm, an OD reading

taken, then the plate was incubated at the specified temperature, and shaken at 180 rpm (except

for the fermentation carried out at 22 °C). OD was measured once or twice per day at 600 nm

using a microplate reader (either by Versamax, Molecular devices or a Modulus II by Turner

BioSystems).

Table 1 Concentrations of inhibitors used to test the tolerance of M. pulcherrima

Inhibitor Low value Medium value High value

(mM) (g L-1) (mM) (g L-1) (mM) (g L-1)

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Furfural 1 0.10 10 0.96 60 5.76

5-HMF 1 0.13 10 1.26 60 7.57

Acetic acid 10 0.60 60 3.60 200 12.01

Formic acid 10 0.46 60 2.76 200 9.21

Levulinic acid 10 1.16 60 6.97 200 23.22

Vanillin 1 0.15 10 1.52 30 4.56

Vanillic acid 1 0.17 10 1.68 30 5.04

4-HBA 1 0.14 10 1.38 30 4.14

Syringaldehyde

1 0.18 10 1.82 30 5.47

Coniferyl alcohol

1 0.18 10 1.80 30 5.40

The OD was analysed one well at a time. Firstly the change in optical density (ΔOD) after time

(t) was found, and then normalised to the average ΔOD for the control medium ΔOD(control) to

give norm ΔODt=x

Equation 1) ∆OD=〖OD〗_(t=x)-〖OD〗_(t=0)

Equation 2) norm∆OD= ∆OD/(∆〖OD〗_((control)) )

Inhibitor concentrations were selected based on the typical range of values found in

lignocellulose hydrolysates,17-20 This included a low, medium and high value for each inhibitor

(table 1). Microplates were charged with media contaminated by inhibitors and fermentation was

carried out as described above.

For the zone of inhibition experiments, M. pulcherrima strains were streaked on to Malt Extract

Agar plates and incubated for two days at 25 °C. From each plate, individual colonies were

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picked using 10 µl pipette tips and placed into 10 µl of sterile Milli-Q water. Lactococcus lactis.

was grown overnight in 5mls Tryptic Soy Broth and 200 µl of this was spread evenly on to

Tryptic Soy Agar plates. When the surface had dried, 2 µl of each yeast suspension was dropped

onto the plate which was then incubated at 25 °C for 48 hours. Zones of inhibition, defined as the

distance extending from the edges of the M. pulcherrima colonies to the beginning of the

bacterial lawn, were measured manually and expressed in millimetres and plates were

photographed using a smart phone camera/GelDoc.

In the fermentation of microwave hydrolysate, 20ml of the hydrolysate produced from the

microwave process was directly inoculated without further supplementation with 200 µl of an

inoculum of M. pulcherrima grown at 20 °C, 180 rpm in YMS media (yeast extract: 30 g L-1,

mannitol: 5 g L-1; L-sorbose: 5 g L-1) for 48h. The cultures were incubated at 20 °C for 96h with

an agitation of 180 rpm. The cultures were then centrifuged at 6000 rpm for 10 mins at room

temperature, the supernatant was discarded and the pellets were freeze-dried and weighed prior

to lipid analysis.

For the production of 2PE, a synthetic grape juice (SGJ) media was adapted from the literature,

given by Chantasuban et al,21 to replicate grape must in wine fermentation. In short, the media

concentrations used were tartaric acid (7 g L-1), malic acid (10 g L-1), (NH4)2HPO4 (3.75 g L-1),

KH2PO4 (0.67 g L-1) MgSO4 *7H2O (1.5 g L-1), NaCl (0.15 g L-1), FeSO4 * 7H2O (0.021 g L-1),

ZnSO4 *7H2O (0.0075 g L-1), CaCl2 (0.15 g /L-1). To this either glucose/fructose (70 g L-1 / 30 g

L-1) or glucose/xylose (70 g L-1 / 30 g L-1) were added. Limited nitrogen and micronutrients were

added to imitate the natural wine must, the C/N ratio was set at 550:1. To study the effect of the

limited nitrogen to the M. pulcherrima fermentation, a modified synthetic grape juice media

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(MSGJ media) which had an increased nitrogen content (C/N ratio of 100:1) was also used. The

yeast was cultured as given above, the pH controlled to 4 at 180 rpm over 7 days.

For the larger scale fermentation, M. pulcherrima was cultured in a 2 L Fermac 320 Bioreactor

Fermenter (Electrolab Biolab Ltd.). Temperature, pH and dissolved oxygen (DO) were

controlled through the controller system using probes and could be kept constant. The threshold

for automatic control was pH±0.05, temperature ±0.1°C and DO ±1 unit. The bioreactor jar was

sterilised prior to fermentation through autoclaving at 121°C, 15 minutes. Aeration was set at 2

L/min, with the working volume 600 mL in the bioreactor. The conditions of fermentation were

set at pH 5, 20°C with an agitation speed of 180 rpm. The bioreactor jar and lid were autoclaved

prior to fermentation. The DDGS and DRAFF hydrolysates were used as received from the

microwave process, with no additional nutrients. The yeast biomass dry weight was calculated

gravimetrically from a 10 mL sample.

2.2.3 Product characterization

Lipid content for all cultures was assessed though a modified procedure given by Pan et al.22 in

which dried yeast biomass of known mass (0.02-0.1g) was heated at 50 °C in 4M HCl for 60

mins, cooled, 1:1 chloroform:methanol added and stirred overnight. The chloroform layer was

carefully removed by pipette into tared vials, the solvent removed and the vials re-weighed.

Glucose, xylose, cellobiose, arabinose, acetic acid, formic acid, levulinic acid and arabitol

content were determined by Shimadzu 10AVP HPLC system (Shimadzu corp., Japan) fitted with

a pump (LC-10AD), an auto injector (SIL-10AD), a system controller (SCL-107A). Filtered

(0.22 μm, Millipore, UK) hydrolysate samples (10 μl) were injected without dilution onto a 300

x 7.8 mm Aminex HPX-87H column (BioRad, CA, USA) at 65°C fitted with RID-10A detector.

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Isocratic elution took place was over a 25 min period at 0.6 ml/min using 0.2 μm-filtered and

degassed 5 mM sulfuric acid. 2-phenylethanol content was determined using an identical HPLC

system with a UV detector, filtered samples diluted by a factor of 20 and 10 μl injected onto a

Dionex column eluted at 40:60 water:acetonitrile at 0.4 ml min-1.

CHN elemental analysis was carried out in duplicate at University of York.

Cell counts were by flow cytometry (Guava EasyCyte) or with a microscope and

haemocytometer.

Oligosaccharide detection was carried out using HPLC on a Hewlett Packard Series 1100 with

Evaporative Light-Scattering Detector with an Alltech 3300 Hi-Plex Na, 10 um, 300x7.7mm

with a Hi-Plex Na, 10 um, 50 x 7.7 mm guard column. Mobile phase was 100% water, flow rate

of 0.3 mL min-1. This HPLC system was also used in the determination of rhamnose,

levoglucosan, sucrose, fructose, mannose and galactose, using a normal phase Luna NH2 (5µm)

column (Phenomenex, CA, USA) and Hi-Plex Pb (Agilent, CA, USA) both with acetonitrile and

water, whereas furfural and HMF concentrations were found on reversed phase C18 column.

2-phenylethanol was determined on a HPLC system (Shimadzu 10AVP HPLC system with LC-

10AD pump, auto injector (SIL-10AD) with Hypersil C-18 reverse phase column (Thermo Inc.)

and UV detector at 216nm. The LC eluent was acetonitrile/water 60:40, flowing at 0.8 mL min-1.

Samples were diluted in range of 0-35 mg L-1 and filtered through 0.22 µm filter prior to

analysis.

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3. RESULTS AND DISCUSSION

180 190 210 230 2500%

5%

10%

15%

20%

25%

30%

35%

40%

Temperature of hydrolysis (°C)

Frac

tion

solu

bilis

ed (%

)

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Figure 2. a) Solubilisation of wheat straw at a wt loading of 1:20 over a range of

temperatures. b) Yield of mono- and disaccharides and inhibitor compounds from

microwave hydrolysis of wheat straw at temperatures ranging from 180 °C to 250 °C, b)

Cell dry mass of M. pulcherrima grown at 25°C for seven days on microwave hydrolysed

wheat straw

Samples of wheat straw that underwent microwave hydrolysis of temperatures between 180 °C

and 250 °C demonstrated significant changes in total solubilized material in the hydrolysate with

the with up to 40% of the biomass solubilized at 250 °C (fig. 2a), double that observed at 190 °C.

Interestingly it was at 210 °C that the maximum yield of sugars was achieved, however this was

accompanied with a maxima of inhibitors. Beyond 210 °C sugar/inhibitor yield declined

substantially as they were decomposed further to gas molecules such CO, CO2 and water. From

Fig. 2c it could be seen that the yeast biomass did not directly correlate to the yield of sugars or

solubilized material, but rather exhibited behavior indicating that higher inhibitor yield was

negatively impacting its growth. This data demonstrates the importance of developing an

approach requiring the integration of microwave and biological technology. It was not sufficient

to only ascertain maximum solubilized biomass, or yields of monosaccharides from the

microwave process, without a greater understanding of M. pulcherrima tolerance to inhibitors

and ability to metabolise oligosaccharides.

In order to establish the tolerance of M. pulcherrima to non-detoxified lignocellulosic

hydrolysates, pure inhibitor compounds were added to a minimal media (YMM) containing 30 g

L-1 glucose. Typical low, medium and high concentrations of inhibitors were used, in accordance

with previous published reports.14,17-20 M. pulcherrima has excellent tolerance to furfural, 5-HMF

and organic acids, generally in excess of common yeasts (fig. 3a). Growth of M. pulcherrima

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was not significantly inhibited by most compounds tested, except at unrealistically high

concentrations. For example, furfural and 5-HMF substantially affect the growth of most

oleaginous yeasts at concentrations of 10 mM or lower.23 The organic acids (acetic, formic and

levulinic) at 10 or 60 mM concentrations increased the growth of M. pulcherrima, but were

inhibitory at 200 mM. Of the aromatic alcohols, acids and aldehydes produced from the

decomposition of lignin, vanillic acid and 4-hydroxybenzoic acid (4-HBA) had little effect on

growth, though vanillin and syringaldehyde were inhibitory at medium concentrations.

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Figure 3 a) Growth of M. pulcherrima over 7 days with a low, medium and high loading of

inhibitors b) HPLC chromatograph demonstrating the wide mono- and oligosaccharide

uptake from depolymerised rapeseed meal (20 g L-1, 190 °C, 0 hold time) before (black) and

after fermentation (red), over 6 days, 20°C, 180 rpm. c) Natural variation between 10

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alternative strains of M. pulcherrima in the production of an antibiotic zone of inhibition on

a lawn of Lactococcus lactis bacteria.

M. pulcherrima can also be cultured on partially depolymerised feedstocks, and is able to

metabolise a range of oligosaccharides (fig. 3b) predominantly in the DP3 – 8 range, through the

production of multiple cellulases.24 This allows a balance to be struck between sugar

concentration and inhibitor formation in the microwave process. M. pulcherrima thrives at low

acidity (pH 3-4), high saccharide concentration (>100 g L-1) and produces a range of

antimicrobial compounds such as pulcherriminic acid and 2-phenylethanol. Due to these traits,

M. pulcherrima has been demonstrated to be highly effective at resisting competition from other

invasive microorganisms.25,26 This antimicrobial activity is a key trait, and one that would reduce

costs substantially allowing less stringent sterile control in the fermentation, of the processing of

the initial feedstock and would de-risk the processing stage substantially. We have previously

demonstrated the industrial applicability, culturing M. pulcherrima axenically under non-sterile

conditions in open, stirred tank 500L bioreactors.27 The effect of M. pulcherrima on common

bacteria is highly positive, with the yeast being able to kill off mixed bacterial contaminations in

whey permeate and outcompeting Bacillus subtilis, Acetobacillus spp. and Escherichia coli

under optimal conditions. In figure 3c, the effectiveness of alternative strains of M. pulcherrima

at inhibiting Lactococcus lactis is shown.

When microwave hydrolysates were fermented the optimal processing temperature was found to

be 190 °C for wheat straw (fig. 2b), producing 1.1 g L-1 of total mono- and disaccharide sugars,

and subsequently 2 g L-1 yeast biomass was produced. This demonstrated that inhibitor

concentration played a critical role in the ability of M. pulcherrima to grow, with increases

beyond minimal levels causing substantial decline in growth. This highlights the importance of

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undertaking a combined approach when investigating/designing pre-processing technologies for

biological applications. As optimum hydrolysis temperature was established these conditions

Figure 4 a) Efficiency of the microwave process giving mass % hydrolysate from the

original biomass and the % carbon retained in the hydrolysate compared to the original

biomass. b) Total biomass yields for each fermentation given as a concentration and as a

mass percentage of the original hydrolysate from the microwave process.

were subsequently used with all biomass feedstocks. The highest hydrolysate yields were

observed for biomass from industrial processes, such as DDGS, rapeseed meal and DRAFF

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producing 52%, 38% and 33% (expressed as % weight of the raw material, not just the

fermentable sugars) hydrolysate respectively (fig. 4a).

The seaweeds also demonstrated a high conversion, with 46% and 62% of the A. nodosum and L.

saccharina solubilised respectively. This is presumably due to the lack of lignin. Wheat straw,

which had only been mechanically ground and therefore retained the fibrous structure, produced

a lower amount of hydrolysate (18%). The % hydrolysate correlated strongly with the carbon

efficiency demonstrating that the hydrolysate consisted mainly of organic components. Despite

the lack of monosaccharides or additional nutrients, reasonable yeast biomass yields were

obtained for all the feedstocks investigated. The yeast biomass co-efficient (the conversion of

hydrolysate to yeast biomass) was highly variable however, demonstrating that while a number

of compounds in the hydrolysate were solubilised, not all of these could be fermented by the

yeast (fig. 4b). For example, despite 52% of the DDGS being hydrolysed only 1.5 g L-1 of yeast

was produced, in contrast while only 18% of the wheat straw was hydrolysed, this produced the

highest yeast biomass co-efficient, 0.33, demonstrating this hydrolysate was a highly fermentable

material. The highest production of yeast biomass was from the seaweed L. saccharina with a

yield of 5.5 g L-1, resulting in a yeast biomass co-efficient of 0.18.

With the harder to hydrolyse biomass, such as wheat straw, some of the original cellulose is not

converted and is recovered in the solid fraction at the end of the reaction. To increase the yield of

the resulting yeast biomass, these solids can be simply run through the microwave system

again.28 For example, the solid remaining from the MW hydrolysis of wheat straw was re-

hydrolysed at 190°C under the same conditions. By repeating this 8 times, we were able to

substantially increase the yield of yeast biomass yield to 6.1 g L-1 demonstrating the potential for

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(semi-)continuous pre-processing of the original biomass to maximise downstream product

yields.

To demonstrate this process on the larger scale both DRAFF and DDGS were depolymerized

using the Milestone SynthWAVE MW on the 1L scale. Using this system both DRAFF and

DDGS performed similarly with 32.8 g L-1 and 33.2 g L-1 hydrolysate produced respectively, this

was 66% of the original starting biomass. The majority of solubilized biomass was produced on

the first depolymerisation with recycling the solids from the process giving little further

advantage. While both biomass sources give similar levels of solubilized biomass, the majority

of hydrolysate from DDGS was large DP7+ oligomers and proteinous biomass whereas with

DRAFF there was a larger proportion of DP3-DP6 species. Hardly any furans were observed

under these conditions.

To demonstrate the suitability of the fermentation on the larger scale, M. pulcherrima was

cultured in a 2L controlled stirred tank bioreactor, directly on the hydrolysate produced from the

MW process. The yeast grew extremely well on the DDGS hydrolysate reaching stationary phase

after 48 hours and producing 8.38 g L-1 yeast biomass overall, a yeast biomass co-efficient of

0.25. While the hydrolysate from the DRAFF contained more accessible oligosaccharides, the

yeast co-efficient was lower at 0.19, with 6.08 g L-1 yeast produced over 96 hours.

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Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 50

5

10

15

20

25

30

a) DDGS

Other solubilised biomass

DP3 - DP6 saccharides

DP1 - DP2 saccharides

Organic acids

Furans

Hyd

roly

sate

com

posi

tion

(g L

-1)

Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 50

5

10

15

20

25

30

b) DRAFF

Other solubilised biomass

DP3 - DP6 saccharides

DP1 - DP2 saccharides

Organic acids

Furans

Hyd

roly

sate

com

posi

tion

(g L

-1)

0 24 48 72 96 1200

1

2

3

4

5

6

7

8

9

c) Fermentation DDGS

Time (h)

Yeas

t bio

mas

s (g

L-1

)

Fig 5. a) solubilized products from the microwave depolymerisation of DDGS at 190 °C

with no hold time, the solids were separated and microwaved under the same conditions for

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a further 4 cycles. B) solubilized products from the microwave depolymerisation of DRAFF

at 190 °C with no hold time. C) yeast biomass produced in a controlled, stirred tank

bioreactor using the hydrolysate from the microwave processing with no additional

nutrients, pH 5 at 20 °C.

One of the key products from M. pulcherrima is a lipid similar to terrestrial edible oils (table 2).

Lipid production was found to be in the oleaginous range for all feedstocks examined. For

example, at 20°C up to 38% dry cell weight lipid was produced from M. pulcherrima cultured on

rapeseed meal hydrolysate with additional waste glycerol, though up to 60% of the cell biomass

was produced when cultured on MW hydrolysed straw. However, there are potentially a large

range of other compounds that the yeast can produce. M. pulcherrima is known to contain a

range of suitable metabolic pathways that could lead to complementary products, within a future

biorefinery. While these pathways would need to be upgraded with metabolic engineering the

potential is clear, and as such it is a highly suitable platform organism (fig. 6).

In particular we were able to make the important chemical 2-phenylethanol (2PE) under low

nitrogen conditions coupled with high sugar loadings. On glucose and xylose (2:1 wt%) a yield

of over 1 g L-1 was obtained, which was higher than any other batch de-novo 2PE fermentation to

date.21,29 2PE is a fragrance currently produced from petrochemicals or from extraction from rose

petals. Approximately 10,000 tonnes a year are produced, though if fully exploited the aromatic

functionality of a microbial 2PE could also be harnessed to produce bio-styrene or other useful

aromatic platform molecules. Moderate cost, high volume opportunities for bio-aromatic

compounds are rare. 2PE was also observed on fermenting the biomass depolymerised in the

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Table 2 Lipid and 2-phenylethanol production from M. pulcherrima when cultured on

model feedstocks and on microwave depolymerised waste feedstocks

Feedstock Lipid (% weight cell)

2PE (mg L-1)

Glucose 35% 698

Glucose + xylose

37% 1015

Rapeseed meal

20% 9

Rapeseed meal with glycerol

39% 185

DDGS 20% 0.1

DRAFF 14% 1.6

L. saccharina 37% 47

Wheat straw 60% 11

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Figure 6 Product and product classes available from strains of M. pulcherrima when

cultured on waste feedstocks.

microwave (table 2). Though this was lower than from pure sugars, this opens up the possibility

of continually producing 2PE as a co-product alongside other lower value compounds such as the

lipid. M. pulcherrima is known to produce a range of terpenols,30 especially when glycerol is

used as a feedstock. Previously, when cultured on glycerol, we demonstrated that M.

pulcherrima produces sterols, mainly ergosterol. While sterols are an interesting pharmaceutical

precursor, they can be hard to extract and purify from lipids effectively. However, the

manipulation of sterol pathways in yeast allows access to terpene based molecules, such as

farnesene a precursor to biojet fuel,31 or larger terpenes for use in lubricating oils,

pharmaceuticals or personal care products.32 In other yeasts, such as S. cerevisiae, farnesene has

been made as an extracellular product, ejected from the cell, aiding in the separation.

A range of short chain chiral sugars and chemical compounds have also been reported to be

produced from the yeast, and anaerobic conditions these include isoamyl alcohol, butanol,

isobutanol, hexanol, ethyl hexanoate, ethyl octanoate,30 and arabitol under aerobic conditions and

low pH. The alcohols and esters would be suitable as fuels, while the chiral sugars and small

chain oxygenates could plausibly be converted into an entire range of polymers and bulk

chemicals. The highest production of these compounds was found to be arabitol, with up to 11 g

L-1 produced from a mixture of glucose and xylose at pH 3.

Separation of products is a critical issue in the design of future biorefineries.33 The flexibility in

which products are made makes M. pulcherrima a potential chemical platform for future

biorefineries, especially with enhanced metabolic engineering. To that end, much of the genetic

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structure underpinning the production of these molecules can be identified using comparative

genomics with well characterized yeasts within the same CUG-ser alternative codon group. Core

gene families and networks are easily identified and can be targeted for genetic manipulation.

The relative ease of high throughput genome sequencing allows access to both the conserved

genes within the yeast and identification of genes that are novel or divergent compared to other

yeasts. Combined with high throughput phenotypic characterisation, comparison within and

across isolates reveals natural genetic and phenotypic variation in the production of target

chemicals and in growth parameters across industrially relevant conditions such as temperature,

dissolved oxygen content, and inhibitors. The presence of highly divergent traits across small

genetic distances supports the notion that beneficial traits can be combined across strains and that

alleles can be edited to other natural variant forms or subjected to adaptive laboratory evolution

to result in improved phenotypes.

4. CONCLUSIONS

Using the proposed microwave biomass pre-processing system to produce solubilised

carbohydrates allows us to ferment more of the lignocellulosic waste resources, while reducing

the cost of production. Using the microwave system, up to 40% of the total wheat straw biomass

was solubilized at 250 °C in just 15 minutes. The majority of this material was oligosaccharide

and proteinous products, though up to 2 g L-1 of mono- and disaccharides were produced as well.

The yeast M. pulcherrima produces an array of cellulases and as such can metabolise a portion of

the solubilsed oligosaccharides, as such on hydrolysate produced at 190 °C, a yeast coefficient of

between 0.05-0.33 was achieved on the hydrolysate depending on the biomass source. The

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system was scaled to 1L total volume in a SynthWAVE microwave, with both DDGS and

DRAFF feedstocks. At this scale both feedstocks produced approximately 33 g L -1 solubilised

material, a total 66% of the original weight, with the vast majority being produced on the first

cycle. The hydrolysates were then fermented in a stirred tank controlled bioreactor (2L total

volume), producing 8.38 g L-1 and 6.08 g L-1 yeast biomass respectively, a coefficient of 0.25 and

0.19.

Creating products through a zero waste biorefinery reduces waste to landfill and has

multiple social benefits by displacing edible feedstocks from non-food production. This is a

significant improvement on current systems. This innovative tailored approach could transform

the biorefinery industry through inexpensive biomass processing and robust bioprocessing to

produce desirable products cheaply, especially if coupled with further synthetic biological

transformation of the yeast.

ACKNOWEDGMENTS

This research has been funded by the Industrial Biotechnology Catalyst (Innovate UK, BBSRC,

EPSRC) to support the translation, development and commercialisation of innovative Industrial

Biotechnology processes (EP/N013522/1), and H2020-MSCA-CO-FUND-2014, #665992,

MSCA FIRE: Fellows with Industrial Research Enhancement.

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