Industrial fermentation of renewable diesel fuels

Available online at www.sciencedirect.com

Industrial fermentation of renewable diesel fuels

Patrick J Westfall and Timothy S Gardner

In commodity chemicals, cost drives everything. A working

class family of four drives up to the gas pumps and faces a

choice of a renewable diesel or petroleum diesel. Renewable

diesel costs $0.50 more per gallon. Which fuel do they pick?

Petroleum diesel will be the winner every time, unless the

renewable fuel can achieve cost and performance parity with

petrol. Nascent producers of advanced biofuels, including

Amyris, LS9, Neste and Solazyme, aim to deliver renewable

diesel fuels that not only meet the cost challenge, but also

exceed the storage, transport, engine performance and

emissions properties of petroleum diesel.

Address

Amyris, Inc, 5885 Hollis Street, Suite 100, Emeryville, CA 94608, United

States

Corresponding author: Gardner, Timothy S ([email protected])

Current Opinion in Biotechnology 2011, 22:344–350

This review comes from a themed issue on

Energy biotechnology

Edited by Peter Durre and Tom Richard

Available online 24th May 2011

0958-1669/$ – see front matter

# 2011 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.copbio.2011.04.023

IntroductionThe most commonly known biomass-derived diesel fuel is

‘biodiesel.’ Biodiesel is a specific class of diesel composed

of fatty acid methyl esters (FAMEs) of various molecular

weights. With its simple, mature production technology –and a cadre of government manufacturing and blending

incentives – biodiesel became the first biomass-derived

diesel to reach industrial scale. Current US production

capacity is 2.8B gallons/year [1]. Biodiesel also enjoys

excellent emissions properties and provides significant

greenhouse gas (GHG) reductions. But its downsides,

including lower energy density, oxidative in stability,

and cold-temperature viscosity limit applications and range

of operating conditions [2]. Thus, biodiesel is not con-

sidered a ‘drop-in’ fuel – meaning it is not fully inter-

changeable with petroleum diesel in the existing fuel

distribution and diesel engine infrastructure. Moreover,

high feedstock prices (driven by non-fuel demands for

animal and plant oils) have kept the cost of production

high, limiting market size [1,2]. After the enactment of


tariffs in Europe to protect local biodiesel producers in

2009, US production dropped to 475–554 million gallons,

only 17–20% of US production capacity [1,3].

A new crop of advanced biofuel companies is attempting

to overcome the limitations of biodiesel with drop-in

renewable diesel fuels. Amyris, for example, has met

the performance properties of the ASTM D975 Table

1a diesel standard and has received EPA certification for

blends up to 35% with petroleum diesel. Similarly, Sola-

zyme has met ASTM, EN and military diesel specifica-

tion properties [4]. Here we review the landscape of

renewable diesel fuels, competing technologies for the

fermentation of diesel from biomass, and the challenges

of scale-up.

Properties of biomass-derived dieselmoleculesCurrently, there are three classes of biomass-derived

molecules that have qualified for use in diesel engines:

FAMEs (biodiesel), alkanes/olefin mixtures, and farne-

sane (an isoprenoid). Fatty acid ethyl esters (FAEEs) are

also sometimes grouped into the biodiesel category [5��].Alkanes/olefin mixtures produced via the hydrotreatment

of triglycerides have been termed ‘renewable diesel’ [1].

Here also classify farnesane and all biomass-derived

alkanes/olefins mixtures as renewable diesels, while we

classify FAMEs and FAEEs as biodiesel. In all cases

these classifications are based on the fuel molecule,

irrespective of the process used to make them.

Farnesane, a C15 isoprenoid, is the only single-molecule

diesel fuel available. By contrast, alkane/olefin fuels,

FAMEs and FAEEs are mixtures of linear carbon chains

ranging from 8 to 22 carbons [2,5��,6], but are usually

dominated by a narrower range of chain lengths. Soy

biodiesel, for example, is composed of C16–C18 methyl

esters [7]. Petroleum diesel predominantly contains a

mixture of C10 through C19 hydrocarbons, including

approximately 64% aliphatic hydrocarbons, 1–2% olefinic

hydrocarbons, and 35% aromatic hydrocarbons [8]. The

differences in the molecular structure and chain length

composition of diesel fuels give rise to a spectrum of

combustion and emissions properties.

All renewable diesels meet or exceed the engine per-

formance properties of petroleum diesel in cetane, cloud

point, energy density and, when additized, lubricity (see

Table 1). On the contrary, biodiesel performs well in

cetane and lubricity, but underperforms on cloud point

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http://dx.doi.org/10.1016/j.copbio.2011.04.023

Industrial fermentation of biomass-based diesel fuels Westfall and Gardner 345

Table 1

Diesel fuel properties

Diesel molecule Synthesis method Cetane Cloud Point (C) Energy Density

(MJ/kg)

Lubricity

(mm)

Oxygen (%) Stability

Farnesane Fermentation 58 �40 43 400 0 Good

Alkanes via hydrotreatment

or Fischer-Tropsch [15]

Fermentation

or Chemical

70–90 �20 to 20 44 520 0 Good

FAMEs [1,3] Fermentation

or Chemical

47–59 0 38 250 11 Marginal

Alkanes/aromatics

from Petrol [15]

Distillation 40 �5 43 <520* 0 Good

*Ultra-low sulfur diesel (<15 ppm sulfur) requires additives to achieve less than 510 mm lubricity.

(limiting its use at low temperatures), in energy density,

and instability owing to the presence of oxygen and

unsaturated bonds in the FAMEs.

Renewable diesels and biodiesel outperform petroleum

diesel in all categories of emissions, including particu-

lates, unburned hydrocarbons, NOx, SOx, and carbon

monoxide [9,10]. The improvements stem partly from

the narrower molecular size distributions, low or zero

aromatic content, and low or zero sulfur content of renew-

able diesel and biodiesel fuels. The exception to this is

some biodiesels, which have elevated NOx emissions [9].

NOx’s react with unburned hydrocarbons to form ozone

[11]. However, biodiesel also produces the lowest

unburned hydrocarbons of any fuel owing to its low vapor

pressure [2], thereby helping to minimize ozone for-

mation.

GHG reduction properties of biomass-derived diesel

fuels are more difficult to assess. GHG estimates depend

on life cycle analysis models that integrate assessments of

agricultural energy inputs, feedstock type, production

processes, fuel distribution, and combustion. Default

GHG reductions estimated by the European Union are

19–56% for biodiesel and 26–65% for hydrotreated renew-

able diesels derived from agricultural feedstocks (as

opposed to waste oils) [14]. Other renewable diesel fuels

(such as fermented alkanes, farnesane) are still largely in a

development and demonstration phase, so their GHG

savings and costs of production have not yet been certified

by regulatory agencies. Nevertheless, independent, third-

party life cycle analysis shows excellent GHG reductions

for renewable diesels. Amyris’s No Compromise1 diesel

from sugar cane, for example, provides an estimated

reduction >90%. These numbers do not include still-

controversial indirect land use penalties applied to bio-

mass-derived fuels [15].

Like the GHG reduction figure, the costs of production of

various biomass derived diesels are difficult to assess.

Most companies publicly claim target production costs

around $0.60/L ($2/gallon) with matured technology, a

cost that would enable profitable sales into a volatile

diesel market without subsidies. But most technologies


are not yet mature and most companies are not yet

producing at full scale (>10 M liter/year).

Fermentation of biomass-derived diesel fuelsMultiple fermentation technologies are being developed

for the production of renewable diesels (Table 2). All

technologies begin with the fermentation of biomass-

derived sugars to acid, alcohol, triglyceride or olefin

intermediates. The intermediates are then transformed

via a chemical finishing process into the diesel fuel

molecules. The chemical processing complexity varies

significantly from technology to technology. Amyris’s

farnesane, for example, is produced though a simple

process of distillation and hydrogenation of farnesene

(the fermentation product). On the other end of the

complexity spectrum, Terrabon’s alkane diesel is pro-

duced through a multistep chemical transformation of

mixed carboxylic acids including thermal conversion to

ketones, hydrogenation to alcohols, condensation, oligi-

merization and distillation [12].

The fermentation approaches are equally varied. Amyr-

is’s farnesene is fermented via the mevalonate or deox-

yxulose phosphate metabolic pathways. Both pathways

form farnesyl pyrophosphate, an intermediate that is

transformed to farnesene via the farnesane synthase

enzyme. These reactions can be carried out in E. colior yeast, the two favorite workhorses for renewable diesel

production. Amyris has focused its energies on deploying

these pathways in yeast based on its superior properties in

industrial-scale fermentations.

Alkane diesel is being pursued by multiple companies.

Solzyme uses microalgae to ferment sugars into triglycer-

ides and then employs UOP’s hydotreatment technology

to reduce the triglycerides to fully saturated alkanes. LS9

has focused on the direct fermentation of alkenes and

alkanes, bypassing triglyceride formation altogether. The

company has developed multiple approaches to this in-

cluding the formation of alkenes via reduction/decarbo-

nylation of fatty acyl-ACP [13��], and the production of

fatty alcohols via a thioesterase/fatty acid reductase path-

way [5��]. In addition, LS9 has developed an approach for

the direct fermentation of fatty acid ethyl esters, an


346

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Table 2

Production processes for Renewable Diesel

Diesel molecule Common Name Trade Name Feedstock Fermentation route Fermentation Product Chemical Process/finishing Companies

Fermentation processes

Farnesane Renewable Diesel No Compromise1

Diesel

Sugars Mevalonate/terpene

synthases

farnesene hydrogenation Amyris

Alkanes Renewable Diesel UltraCleanTM

Diesel

Sugars fatty-ACP/decarbonylase/

reductase

C13–C17 alkenes/alkanes hydrogenation LS9

Alkanes Renewable Diesel UltraCleanTM

Diesel

Sugars fatty-ACP/thioesterase Fatty alcohols hydrogenation LS9

Alkanes Renewable Diesel Soladiesel1,

Green Crude

Sugars fat synthesis Triglycerides (vegetable oils

or animal fats)

hydrotreatment Solzyme,

Sapphire

Alkanes Renewable Diesel MixAlco Diesel biomass mixed acid fermentation

after lime pretreatment

mixed acids dewatering, thermal

conversion, hydrotreatment

and oligimerization

Terrabon

FAEEs Biodiesel Sugars acyl-CoA ligase/ethanol

transesterase

FAEEs none LS9

FAMEs Biodiesel Green Crude Sugars fat synthesis Triglycerides (vegetable oils

or animal fats)

Transesterification

with methanol

Sapphire,

Solazyme, LS9

Chemical processes

Alkanes Renewable Diesel Ecofining, NExBTL Triglycerides

(vegetable oils

or animal fats)

– – hydrotreatment UOP, Neste

Alkanes Renewable Diesel Bioforming biomass – – aqueous phase reforming +

catalytic condensation,

deoxygenation, dehydration

and saturation

Virent

Alkanes FT Diesel biomass – – Fischer-Tropsch (syngas)

FAMEs Biodiesel Triglycerides

(vegetable oils

or animal fats)

– – Transesterification

with methanol

many

Various Renewable Diesel BioOil biomass – – Pyrolysis/hydrotreatment Dynamotive

Petrol Diesel Diesel, Petrodiesel Petroleum – – Distillation many

Cu

rren

t O

pin

ion

in B

iote

ch

no

log

y 2

011,

22:3

44

–350

w

ww

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irect.c

om


alternative biodiesel molecule, via an acyl-CoA ligase/

ethanol transesterase pathway [5��].

From the lab to industrial scale fermentationsGoing from proof-of-concept at the laboratory bench to a

scaled-up industrial process can be difficult owing to the

physical constraints of large-scale infrastructure and cost

considerations. The section that follows highlights some

of these challenges dealing with the laboratory to com-

mercial-scale transition (Figure 1).

Choosing the correct organismThe host organism for diesel production is a choice that

could ultimately determine the success or failure of a

biofuel platform. Each organism has certain pros and cons

that are both vigorously touted and aggressively

defended. Many renewable diesel platforms have been

Figure 1

LaboratoryScale-up

IndustrialScale-up

Current Opinion in Biotechnology

Crucial decision points when transitioning from laboratory to industrial

scale.


developed in bacteria such as E. coli owing to their rapid

doubling time, ease of genetic manipulation and detailed

understanding of their metabolism – all of which make

bacteria appear to be ideal candidates for both metabolic

engineering and large scale production of petroleum

replacements [13��,16��,17,18]. However, drawbacks like

susceptibility to contamination [19], incompatibility with

existing ethanol fermentation infrastructure, downstream

waste processing and the general public’s perception of E.coli are problems that must be addressed in order for

renewable diesel generated by this type of organism to

succeed.

S. cerevisiae or other similar fungal systems are another

popular choice for a developmental platform for pro-

duction of renewable diesel [20�,21,22]. Like bacterial

systems, yeast are amenable to common molecular

biology techniques, have robust and well studied genetics

and have a long, successful history in biotech. S. cerevisiaeis also tolerant of low pH, resistant to osmotic stress and

immune to viral contamination – characteristics that make

for more robust fermentations. In addition, yeast has a

well established reputation as an innocuous organism

because of it prevalence in the already established brew-

ing and fermentation industries.

A third group of potential biofuel producing organisms,

microalgae, have a unique set of properties that make

them candidates for industrial production of renewable

diesel. Selected for their ability to convert sunlight and

CO2 directly into storage lipids such as fatty acids (up to

70% of their dry cell weight), algae would appear to be

attractive platform to generate petroleum replacements.

However, microalgae’s limited access to traditional mol-

ecular biology manipulations [23�] along with the chal-

lenges in developing non-traditional cultivation processes

such as dark fermentations or large scale open water pond

cultivation means that algal based renewable diesel pro-

duction faces difficulties that must be dealt with before

they would be able to effectively compete with traditional

fermentation based processes or petroleum derived fuels

[24,25�,26].

ProductivityWith tens of millions of dollars invested into a typical

commercial-scale fermentation facility, efficient capital

utilization is a high priority. This is facilitated by building

biomass to high densities quickly. A typical fermentation

may require more than 50 cell divisions from single cell to

peak operating densities. To accelerate this process, it is

desirable to incorporate a biphasic switch-like system that

allows biomass to build in a low-production or non-pro-

duction state, but then trigger production such that all

growth is limited but production of product is allowed

[27]. In a laboratory setting this is often accomplished by

incorporating a regulatable induction system, such as

IPTG in E. coli or galactose in S. cerevisiae, to massively


348 Energy biotechnology

upregulate transcription of key genes and induce pro-

duction of the end product after significant biomass has

accumulated. At scale these induction systems would not

be cost-competitive and alternative routes to limit growth

while allowing production are necessary. Some successful

scalable solutions for controlling the biomass build vs

product formation stages include: carbon or nitrogen

restriction [28�,29] or changing the amount of O2 available

to cells [30,31].

ContaminationUse of antibiotics to control contamination during fer-

mentation is not uncommon, but has come under

increased scrutiny in recent years, especially in the etha-

nol industry [32,33]. Despite the fact that S. cerevisiaerapidly consumes the available sugar source to produce

ethanol that allows it to outcompete other organisms for

the same carbon source, low level contamination of bac-

teria and other competing, non-ethanol fermenting wild

yeast species can decrease yields over time. In general,

the open fermentation systems currently used for ethanol

production are not compatible with organisms designed to

synthesize hydrocarbon molecules because the hydrocar-

bons generally will not be able to suppress the growth of

competing microbes. Likewise, bacterial cultures would

be susceptible to phage and other competing microor-

ganisms, and open pond algal systems would face similar

difficulties. Dealing with contamination will typically

require the use of sterile, closed bioreactor systems

similar to those used in the food and pharmaceutical

industries, and can significantly increase infrastructure

costs.

Feedstock and fermentation conditionsProof-of-concept production of renewable diesel often

uses idealized laboratory conditions that minimize the

stress on the cells and maximize the cellular output, but

are not necessarily practical at scale. Growth and pro-

duction medium used in the laboratory is sterile, consists

of defined components, has a defined carbon source and is

optimized for maximum growth and reproducibility. For

large scale fermentation these parameters are more diffi-

cult to control. For example, in order to achieve optimal

cost targets, multiple sources of feedstocks (i.e. sugarcane

syrup, corn syrup, sugar beet syrup, etc.) may be utilized

but the lot-to-lot variations in both sugar content and

nutrients and may impact final yields. Temperature, pH

and cellular stress responses also play an important part in

determining the success of a production platform [34].

Higher temperature fermentations, for example, can be

desirable because they increase heat transfer rates from

the culture broth and enable higher productivities,

especially during the summer months – thus increasing

capital efficiency. On the contrary, lower temperature

fermentations can decrease the risk of bacterial contami-

nation. But temperature also impacts protein folding and

the rate at which a reaction can take place. Proteins that


may be fully functional at 28–30C may have a significant

change in activity at 34–40C. Thus identifying a tempera-

ture that meets fermentation and strain physiological

constraints presents a significant strain development chal-

lenge. Osmotic stress, pH and nutrient variations also may

play a much bigger role at the 600,000 L scale where

levels of each of these factors can rapidly change depend-

ing on a cell’s position in the bioreactor [35]. If these

considerations are not accounted for at the laboratory

scale, then major problems could arise when scaling-up.

GMM certificationRecent advances in genomic sequencing, DNA synthesis

and synthetic biology practices have given unprece-

dented access to diverse genomes allowing for identifi-

cation of novel enzymes with more desirable properties

than those native to the host organism. Although the

function of any one enzyme is independent of its host,

care must be taken to reassure the public that horizontal

gene transfer cannot take place.

Likewise, common laboratory genetic tools must be used

with caution, and in many cases not utilized at all, in order

to ensure that Genetically Modified Organism (GMO) or

Genetically Modified Microorganisms (GMM) certifica-

tion at the government level is granted. For example,

commonly used drug resistance markers such as ampi-

cillin or kanamycin, utilized for targeted gene replace-

ment or plasmid retention, may not be applicable at large

scale because of concerns of drug resistant isolates being

released into the environment. At the very least, more

costly downstream waste processing operations may be

required to ensure that production organisms are properly

destroyed.

Plasmid based genetic engineering may also be proble-

matic at scale. Not only do plasmids introduce a degree of

potential genetic instability, but they also provide a

potential route to horizontal gene transfer to related wild

species. Similarly, ensuring that genetically engineered

species are unable to cross with related wild species is

another concern that should be given proper attention in

order to ensure compliance with government regulations.

Separations, purification and quality controlSome form of continuous fermentation process is desir-

able in order to ensure the minimal amount of bioreactor

downtime and to maximize productivity. Not only is

maintaining productivity after biomass build key to a

continuous fermentation process, but removal of product

during fermentation may also be necessary in order to

mitigate inhibitory or toxic effects of the product [36,37�].Ideally, the product is excreted from the cell and could be

harvested without having to also destroy the production

host, thus minimizing the need to continuously build

more biomass [38]. In addition, it would be preferable

that the isolated product be free of cellular debris and



compounds that may interfere with downstream proces-

sing steps such as distillation or chemical modification

[39].

ConclusionThe challenges of carrying a fermentation process from the

lab scale to full scale are manifold, pressing the extremes of

cell physiology and stretching the skills of strain and

fermentation engineers. Challenges include the achieve-

ment of yields near the chemical transformation limit,

extreme product titers, high productivities, high tempera-

tures, cost restrictions on production inducers and vitamin

supplements, feedstock variations and toxins, free-radical

stress, low pH, contaminant load and product toxicity.

Many young companies like Amyris, LS9, Sapphire, Sola-

zyme and Terrabon are stepping up to these challenges,

and the race to be the first company to deliver on the

promise of advanced renewable diesel has begun.

AcknowledgementsWe would like to thank Jordan Thaeler and Christine Ring for theirdiscussions and comments on the paper.

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http://dx.doi.org/10.1007/s10295-010-0885-4

http://dx.doi.org/10.1007/s10295-010-0885-4

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