Available online at www.sciencedirect.
com
ScienceDirect
Next generation industrial biotechnology based on
extremophilic bacteria
Guo-Qiang Chen1,2,3 and Xiao-Ran Jiang1,2,3
Industrial biotechnology aims to produce bulk chemicals
including polymeric materials and biofuels based on
bioprocessing sustainable agriculture products such as starch,
fatty acids and/or cellulose. However, traditional bioprocesses
require bioreactors made of stainless steel, complicated
sterilization, difficult and expensive separation procedures as
well as well-trained engineers that are able to conduct
bioprocessing under sterile conditions, reducing the
competitiveness of the bio-products. Amid the continuous low
petroleum price, next generation industrial biotechnology
(NGIB) allows bioprocessing to be conducted under unsterile
(open) conditions using ceramic, cement or plastic bioreactors
in a continuous way, it should be an energy, water and
substrate saving technology with convenient operation
procedure. NGIB also requires less capital investment and
reduces demand on highly trained engineers. The foundation
for the simplified NGIB is microorganisms that resist
contaminations by other microbes, one of the examples is rapid
growing halophilic bacteria inoculated under high salt
concentration and alkali pH. They have been engineered to
produce multiple products in various scales.
Addresses
1
MOE Key Lab on Bioinformatics, School of Life Sciences, Tsinghua
University, Beijing 100084, China
2
Center for Nano and Micro-Mechanics, Tsinghua University, Beijing
100084, China
3
Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing
100084, China
Corresponding author: Chen, Guo-Qiang (chengq@mail.tsinghua.edu.
cn)
Current Opinion in Biotechnology 2018, 50:94–100
This review comes from a themed issue on Energy biotechnology
Edited by Akihiko Kondo and Hal Alper
For a complete overview see the Issue and the Editorial
Available online 7th December 2017
https://doi.org/10.1016/j.copbio.2017.11.016
0958-1669/ã 2017 Elsevier Ltd. All rights reserved.
Introduction
Due to the low petroleum price combined with other
new energy sources including solar power, shale gas,
wind power and natural gas hydrate, products of tradi-
tional industrial biotechnology such as small molecular
Current Opinion in Biotechnology 2018, 50:94–100
chemicals, polymeric materials and biofuels cannot com-
pete with those based on petroleum [1,2]. It is therefore
very urgent to develop new technology, namely, the
‘Next Generation Industrial Biotechnology’ or ‘NGIB’,
to overcome the disadvantages of the current industrial
biotechnology [3].
The NGIB should avoid the problems of current indus-
trial biotechnology that reduce the competitiveness
including heavy consumptions of fresh water and energy
(for sterilization, oxygen supply and agitation), frequent
microbial contaminations, batch instead of continuous
production processes, difficulty to recycle the broth water,
difficult and expensive product separation and purifica-
tion, lengthy process development of one product from
one strain (without a platform strain for multiple pro-
ducts), slow growth of production organisms, difficulty to
develop fully automatic production processes, a low sub-
strate to product conversion efficiency, and very expen-
sive capital investment on facilities and equipment to
fight contamination, among others (Table 1) [4–8].
All the difficulties from the current industrial biotechnol-
ogy could be attributed to the production microorganisms
that are fragile to contaminations by other microbes. For
example, common industrial strains, such as Escherichia
coli, Bacillus spp., Corynebacterium glutamicum, Pseudomo-
nas spp. and yeast, are grown under mild conditions at
weak acidic or neutral pH of 5–7 and 30–37  C supple-
mented with yeast extract, the mild condition allow most
microorganisms in the air, water and soils to grow [9].
Production facilities have to be sealed and completely
sterilized to prevent any microbial invasion, this proce-
dure dramatically increases the complexity of the biopro-
cesses, leading to high production cost. Thus, a platform
microorganism with robustness and contamination resis-
tance is very important. Other new properties of the
platform organisms could be added via metabolic engi-
neering or synthetic biology approaches [10].
This paper reviews challenges, opportunities and recent
progresses on developing NGIB to increase biotechnol-
ogy competitiveness.
Challenges
Economic and technological challenges
Except labor cost, the production cost of a bio-product
consists of upstream and downstream parts: the up-
stream part contains substrates, including substrate
pre-treatments, process energy including sterilization,
www.sciencedirect.com
 Next generation industrial biotechnology Chen and Jiang 95

Table 1

Next generation industrial biotechnology (NGIB) compared with current industrial biotechnology.

Traditional industrial biotechnology NGIB How to achieve? References

Heavy consumption of fresh water
Heavy energy consumption
Frequent microbial contaminations
Heavy capital investments
Narrow microbial growth conditions
Batch processes
Hard separation of cells from
medium
Lengthy cell growth processes
One strain for one product
Product either in medium or in cells
Growth on a single carbon source
Complete automation difficult
Low conversion of substrate to
product
Hard to obtain intracellular products
Low extracellular product
excretions
Less dependent on fresh water
Reduced energy consumption
Contamination resistant organisms
Reduced capital investment
Flexible growth conditions
Continuous processes
Reduced separation difficulty
Acceleration of cell growth
A platform strain for multiple
products
Intra-cellular and extracellular
products
Growth on mixed carbon sources
such as kitchen wastes
AI control automation
Convert more substrate to product
Weakening the cell walls
Increase outer membrane leakages
Seawater based or recycling process water [17,42,47]
Open unsterile process and high O2 update [17,52]
Screening robust microorganisms [10,17]
Low cost facilities and equipment [44]
Selection on robust microorganisms [41]
Contamination resistant microorganisms [42]
Morphology engineering for large cell sizes [51]
Synthetic biology to speed up cell growth [52]
Construction of multiple product synthesis [44,45]
pathways in the platform strain
Engineering co-production of more [53,54]
products
Screening or constructing multiple [17,28,42]
substrate consuming organisms
Robust organisms allow large control errors [11]
Removal or weakening competing [17,28]
pathways
Engineering cell wall synthesis mechanism [54]
Weakening the outer membrane structures [45]

AI: artificial intelligence.

agitation, aeration, cooling and heating. Although the
downstream part requires equipment and energy to sep-
arate tiny microbial cells from their growth medium,
extract and purify intracellular or extracellular products.
Any attempt to reduce energy consumption is beneficial
for production cost reduction (Table 1). In addition, most
microbial fermentation processes are prone to be contam-
inated over a long period of culture time, prohibiting the
more efficient continuous processes from being widely
used. A robust and contamination resistant microorganism
is thus important to reduce production cost. This forms
the basis for the NGIB to reduce bio-production cost
(Table 1).
A robust strain resistant to other microbial contamination
is the most important factor for the development of
NGIB. The strain should also be able to rapidly grow.
It should also have molecular engineering methods and
tools for improving performances and for producing mul-
tiple products. As a platform production strain, it should
have its genome information fully available. A strain with
the following properties meets the requirements as a
platform strain for the NGIB (Table 2): (1) rapid growth
at very low or high temperatures, low or high pH and/or
low or high osmotic pressure; (2) rapid growth on some
unusual substrates such as long-chain fatty acids, metha-
nol, cellulose, chitin, rubbers or even gaseous substrates
H2, CO2 or methane; (3) rapid growth in the presence of
high concentrated substrates, toxic chemicals such as
short-chain length alcohols (C1–C4), short-chain length
fatty acids (C1–C6), heavy metals (such as Cu, Zn, Pd,
Hg, among others) or toxic compounds such as unsatu-
rated fatty acids or aldehydes; (4) fast growth in the
absence of sufficient water. A strain with combinations
of any two or three or more features will even be better
than a single property as the property combination will
provide the platform strain even stronger property to
resistant contaminations (Table 2).
Many archaea meet some of the above requirements
except that they can only grow slowly. In addition, they
are very difficult to be genetically engineered. Therefore,
archaea are not suitable platform strains for NGIB.
Although some extremophile bacteria, algae or fungi
satisfy the above requirements, engineering methods
and tools are also available or more easily developed
for improved properties, especially for prokaryotic
bacteria.
The main challenge is now becoming to identify and
develop platform bacterial strains for NGIB with some
combinations of the above properties.
Opportunities
Fortunately, some prokaryotes possess some properties
that meet some of the above requirements, including
acidophiles, alkaliphiles, psychrophiles, thermophiles,
xerophiles, methanotrophs, halophiles and some bacteria
that can utilize gaseous substrates or cellulose, among
others (Table 2). Biocatalysts isolated from these organ-
isms are termed extremozymes that possess extraordinary
properties of salt allowance, thermostability, cold adap-
tivity, among others [11]. A few of these bacteria could be
developed as platform strains for NGIB as described
below.

www.sciencedirect.com Current Opinion in Biotechnology 2018, 50:94–100

96 Energy biotechnology

Table 2

Microbial property preferences as a platform strain for the NGIB.

Growth property preferences Example(s) Typical microorganisms References

 
Low or high temperature (T) T < 25 C or T > 45 C Psychrophiles or thermophiles [1,21]
Low or high pH pH < 5 or pH > 8.5 Acidophiles or alkaliphiles [16,17]
High osmotic pressure (P) NaCl > 30 g/l Halophiles [41,42]
Utilization of unusual substrate(s) Long-chain fatty acids (C > 14), cellulose, Xerophiles or methanotrophs or cellulose or [35,38,39]
chitin, rubbers, methane, methanol, H2, gaseous substrates utilizers
CO2
In the presence of toxic compounds Short-chain length alcohols (ethanol, Xerophiles or various resistant [35,38–40]
propanol, butanol), short-chain length fatty microorganisms
acids (formate, propionate, butanoate),
heavy metals (Cu, Zn) or toxic compounds
(unsaturated fatty acids or aldehydes),
among others
Above combinations High T + high pH Thermophiles: high T + low pH [28,29,41,42]
High T + low pH Halophiles: high pH + high P
High pH + high osmotic P
High pH + unusual substrate(s), among
others

All strains require to be grown rapidly.

NGIB based on acidophiles and alkaliphiles
Acidophiles with networked cellular adaptations to regu-
late pH inside the cell, thrive in acidic natural solfataric
fields or sulfuric pools as well as artificial environments,
several extracellular enzymes from acidophiles are known
to be functional at much lower pH than the cytoplasmic
pH [12]. They can produce enzymes like amylases,
proteases, ligases, cellulases, xylanases, alpha-glucosi-
dases, endoglucanases, and esterases stable at low pH
[13]. Acidophiles can be engineered genetically to further
boost their industrial applications. Attempts have been
made to employ thermo-acidophilic microbes for biofuel
production from lignocellulosic biomass to prevent con-
tamination, improve cellulose hydrolysis and accelerate
ethanol evaporation for avoiding end product feedback
inhibition [14]. The enzymes from acidophiles are mainly
used in biopolymer substrate degradation such as starch,
proteins and cellulose [15]. Growth at low pH on biopo-
lymeric substrates increases possibility to prevent other
microbial contamination as few competitors could prolif-
erate on very low pH and biopolymeric substrates at the
same time [16].
Alkaliphile Bacillus marmarensis, for example, has been
studied as a platform for new bioprocesses against micro-
bial contamination challenges. With a newly developed
transformation protocol and genetic tools, along with
optimized RBSs and antisense RNA, B. marmarensis is
able to produce ethanol at titers of 38 g/l and 65% yields
from glucose in unsterile media, and ethanol titers and
yields of 12 g/l and 50%, respectively, from cellobiose and
xylose in unsterile seawater and algal-contaminated
wastewater [17]. Alkaliphiles present a promising
approach for the contamination-resistant biorefining of
a wide range of carbohydrates in unsterilized, non-potable
seawater.
Alkaliphilic Bacillus species and their extracellular
enzymes are intensively studied and applied in industry
because of (1) their ubiquitous presence, (2) non-patho-
genicity, and (3) high production capacity of extracellular
enzymes. Kimura et al. isolated an alkaliphilic Bacillus
strain from soil which grows well in media containing
cholic acid (CA) at 5% (wt/vol) or higher concentrations.
The 7a-hydroxyl and 12a-hydroxyl groups of CA were
efficiently converted to keto-groups, with the conversion
rate for both hydroxyl groups reaching 100% by 72 h of
cultivation [18]. The use of CGTase of alkaliphilic Bacil-
lus sp. strain 38-2 led to the mass production of crystalline
a-cyclodextrin, b-cyclodextrin, g-cyclodextrin at low
cost. The yield of cyclodextrins was 85–90% from amy-
lose and 70–80% from potato starch on a laboratory scale
[19].
NGIB based on psychrophiles and thermophiles
Cold-adapted marine bacteria producing extracellular
hydrolytic enzymes are important for industrial applica-
tions and play a key role in degradation of particulate
organic matter in the natural environment [20]. Selective
conditions allow isolate producers of extracellular hydro-
lytic enzymes that increase the probability of recovering
bacteria able to produce additional extracellular enzymes
for growth at low temperature on polymeric substrates.
Cold-adapted psychrophilic bacteria establish a starting
point for future programs oriented to the prospecting of
less contamination process at low temperature and on
polymeric substrates simultaneously [21]. Enzymes from
psychrophiles have become interesting for industrial
application, partly because of ongoing efforts to decrease
energy consumption. Modern biotech industry requires
biomacromolecules that can function under extreme con-
ditions. Psychrophilic and psychrotolerant microorgan-
isms, their cold-adapted proteins and enzymes have a

Current Opinion in Biotechnology 2018, 50:94–100 www.sciencedirect.com


lot of biotechnological applications [22]. Among cold-
active enzymes such as lipase, aspartate trans-carbamy-
lase, Ca+Zn2+ protease, malate dehydrogenase, triose-
phosphate isomerase, DNA ligase, xylanase, citrate
synthase, metalloprotease, polygalacturonase, cellulases
and xylanase, chitinase, endo-arabinanase, and pectinase,
proteases constitute an important group which have high
catalytic efficiencies at lower temperatures [23–26]. Sev-
eral food processing applications would also benefit from
the availability of low temperature enzymes [27].
Metabolic engineering of extremely thermophilic micro-
organisms to produce bio-based fuels and chemicals provide
pathways and physiological features resident in extreme
thermophiles for improved results. Recent advances have
provided genetic tools for these microorganisms to serve as
metabolic engineering hosts. Beyond providing a higher
temperature alternative to mesophilic platforms, exploita-
tion of high temperature microorganisms grants new oppor-
tunities for contamination free bioprocessing [28].
Because of the relatively small genomes of thermophiles,
they were among the first organisms to be sequenced,
thereby opening up the application of systems biology-
based methods to study their unique physiological, met-
abolic and biotechnological features. Thermophiles bac-
terial genera Caldicellulosiruptor, Thermotoga and Thermus,
are among the most studied thermophiles to date.
Genetic tools for many of these organisms are available
recently, providing the opportunity to move beyond basic
discovery and manipulation to biotechnologically rele-
vant applications of metabolic engineering. The growth at
high temperature provides possibilities for contamination
free bioprocessing [29].
Thermophiles are often highly resistant to harsh condi-
tions such as chemical denaturing agents, wide pH flexi-
bility, and/or non-aqueous solvents. Also, thermozymes
from the thermophiles have impressive performances and
broad applicability [15]. Examples of such enzymes are
cellulases, xylanases, pectinases, chitinases, amylases,
pullulanases, proteases, lipases, glucose isomerases, alco-
hol dehydrogenases, and esterases [30–32]. The xylanases
from Thermus brockianus and Thermotoga thermarum are
suitable candidates for industrial applications due to a
high optimal reaction temperature of 95  C, heat stability
and high specific activity towards soluble and insoluble
xylans [33]. A hyperthermostable b-mannosidase from
the Archaeon Pyrococcus furiosus with transglycosylation
activity and an optimal reaction temperature of 100  C is a
promising candidate for applications in food and pharma-
ceutical industry [34].
NGIB based on xerophiles, methanotrophs and gaseous
substrates utilizers
Few studies have been conducted into the diversity of
xerophilic bacteria in arid areas, which are often related to
www.sciencedirect.com
Next generation industrial biotechnology Chen and Jiang 97
relatively high temperatures, salt concentrations, and
radiation even though they hold promises for many
industrial applications. Members of Streptomyces, Micro-
rnonospora, Saccharothrix, Streptosporangium, Cellulomonas,
Amycolatopsis, Geodermatophilus, Lechevalieria, Nocardia,
and Actinomadura are reported from arid habitats. Mole-
cules with protection activity against drying such as ectoin
and hydroxyectoin with potential application in industry
and agriculture have also been identified from xerophilic
bacteria. Xerophilic bacteria could be a source to develop
contamination free bioprocessing [35]. Moreover, a num-
ber of hydrolytic enzymes such as amylases, xylanases and
cellulase from thermotolerant Actinobacteria can main-
tain their enzymatic activity, even at high temperatures
(50–65  C) [36]. Thermo stable enzymes derived from
such strains can be explored for potential application in
industry for enzymatic digestion purposes at higher tem-
peratures [37].
A significant amount of methane is removed by metha-
notrophic bacteria. Some methanotrophic bacteria have
been found applications in environmental bioengineer-
ing, namely methane removal and biodegradation of toxic
compounds, including removal of methane produced
from landfills and coal mines. Bioreactors using methano-
trophs in bioremediation have been developed in recent
years [38].
Alcohols direct synthesis from syngas (CO + H2) com-
prises a more environmentally-friendly, versatile and
economical alternative. Research efforts in this reaction,
initiated in the 1930s, have fluctuated along with the oil
price and have considerably increased in the last decade
due to the interest to exploit shale gas and renewable
resources to obtain the gaseous feedstock [39]. Steel
industry contributes considerably to GHG emissions.
Fermentation of carbon monoxide (CO) rich off gases
using acetogenic bacteria can be used to produce ethanol,
acetate, and 2,3-butanediol by an integrated biorefinery
[40]. Since only syngas is the only substrates, only gaseous
substrates utilizers can grow. No contamination is
expected in such a process.
NGIB based on halophiles
Halophilic microorganisms usually inhabit on hypersaline
environments or in the sea. They are sources for discovery
bioactive compounds including antioxidants, sunscreen
and antibiotic compounds and compatible solutes that are
useful as stabilizers for biomolecules or stress-protective
agents [41]. Recently, alkaphlic halophiles have been
successfully used to develop seawater based fermentation
processes for production of polyhydroxyalkanoates (PHA)
under open and continuous processes [42]. Genetic engi-
neering tools are now available to add new features to the
halophiles, namely, Halomonas spp. [43]. Halomonas spp.
has been engineered for multiple product production
[44]. It appears that Halomonas spp. can be developed
Current Opinion in Biotechnology 2018, 50:94–100
98 Energy biotechnology
into suitable platform for NGIB. Since most halophilic
bacteria are both alkaliphilic and halophilic, they provide
double barriers for microbial contaminations to happen.
Halomonas bluephagenesis strain TD (short as H. bluepha-
genesis) has been successfully developed as a platform for
NGIB to produce multiple products in a pilot scale of
1000 L fermentor vessel [45]. H. bluephagenesis was
isolated from Xinjiang/China with its whole genome
sequenced in 2011 [46]. The strain was used to produce
polyhydroxybutyrate (PHB) under open unsterile and
continuous condition in seawater with a cell density of
80 g/l containing 80% PHB in the cell dry weight [42]. H.
bluephagenesis and another similar strain Halomonas cam-
paniensis LS21 are able to grow in seawater under open
unsterile and continuous conditions without contamina-
tion for weeks to months [42,47]. The results demon-
strated that Halomonas spp. are platforms or chassis for
further molecular engineering studies. Technology
including conjugation procedure, plasmids, chromosome
engineering and CRISPR/Cas9 have been developed for
H. bluephagenesis [48–50], allowing possible property
improvements and multiple product production by the
platform strain. In addition, a very strong and sensitive
T7-like promoter has also been specially made available
for the H. bluephagenesis platform strain [51]. Since
sterilization is avoided, energy to turn water into high
Figure 1
Supernatant Recycle
Seawater
Filtration
motor
pH
Halophiles
Transparent
plastic reactor
Cell growth
Continuous Open Unsterile Fermentation
Water and energy saving H. bluephagenesis based NGIB has been successfully developed operated in an open and continuous way with
convenient downstream processing means.
Current Opinion in Biotechnology 2018, 50:94–100
temperature and high pressure steam is saved [44]. In
addition, promoter Pvgb of a bacterial hemoglobin gene vgb
is cloned and engineered to allow induction of a product
production, in this case, PHB, at low aeration intensity,
thus saving energy for air compressors [52].
Synthetic biology and metabolic engineering approaches
have been applied to develop recombinants H. bluepha-
genesis for production of poly-3-hydroxybutyrate (PHB)
with ultrahigh 92% content in the cells [43], copolymers
of 3-hydroxybutyarte and 3-hydroxyvalerate (PHBV)
[49], copolymers of 3-hydroxybutyarte and 43-hydroxy-
butyarte (P34HB) [45], a biosurfactant protein PhaP
[53] and 5-aminolevulinic acid (ALA) [54].
The wild type and engineered H. bluephagenesis have been
successfully scaled up from lab 1 L fermentor to 1000 L
industrial fermentor for polyhydroxyalkanoates (PHA)
production without problem [45]. H. bluephagenesis seems
to be an industrially suitable strain. H. bluephagenesis can be
engineered to increase its sizes or elongate its shapes to
allow easy separation from the broth via gravity separation
or convenient filtration [51]. Thus, a water and energy
saving H. bluephagenesis based NGIB has been successfully
developed operated in an open and continuous way with
convenient downstream processing means (Figure 1).
Extracellular
Product Recovery
motor
pH
Induced morphology
change for gravity
Halophiles precipitation
Biomass
Intracellular Product Recovery
Product formation
Current Opinion in Biotechnology
www.sciencedirect.com

Future prospects
Alkalihalophilic Halomonas spp. have been exploited for
NGIB. It is expected that extreme acidophiles combined
with thermophilic property will be a promising chassis for
bulk chemical production from biopolymeric substrates
such as starch, cellulose or fats, as a low pH and a high
temperature will accelerate degradation of the substrates
for easy microbial consumption. At the same time, the
engineering of multiple substrate consumption pathways
allow growth on mixed substrates, this will further reduce
production costs. Other suitable organisms for NGIB like
xerophiles have not yet been exploited. Xerophiles grown
on reduced water activity such as high concentrated
substrates (molasses, concentrated glucose or other
sugars) also help save fresh water and reduced process
complexity. Of course, all of these efforts also require
molecular biology tools available. It is expected that more
extremophilic bacteria and their related enzymes will
become either chassis or catalysis for NGIB to produce
more competitive bulk products. Since NGIB does not
require high temperature and high pressure steam for
sterilization, bioreactor constructions become less expen-
sive as plastics, cerements and/or ceramics can all be used
to build bioreactors. Since sterilization is avoided, fer-
mentation operation can become much more simple,
highly trained microbial fermentation engineers may
not be needed. As the extremophiles are more robust,
they are more stable under changing dynamic growth
conditions, automation can become more realistic. NGIB
based fully automatic bioprocessing plants can be
expected.
Conflict of interest
The authors declare no financial or commercial conflict of
interest.
Acknowledgements
This research was financially supported by a grant from Ministry of Sciences
and Technology (Grant No. 2016YFB0302504), and grants from National
Natural Science Foundation of China (Grant No. 31430003). Tsinghua
President Fund also supported this project (Grant No. 2015THZ10).
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
 of outstanding interest
1. Noda S, Kondo A: Recent advances in microbial production of
aromatic chemicals and derivatives. Trends Biotechnol 2017,
35:785-796.
2. Nagarajan D, Lee DJ, Kondo A, Chang JS: Recent insights into
biohydrogen production by microalgae — from biophotolysis
to dark fermentation. Biores Technol 2017, 227:373-387.
3. Wang Y, Yin J, Chen GQ: Microbial polyhydroxyalkanoates,
challenges and opportunities. Curr Opin Biotechnol 2014, 30:59-
65.
4. Yang JE, Choi YJ, Lee SJ, Kang K-H, Lee H, Oh YH, Lee SH,
Park SJ, Lee SY: Metabolic engineering of Escherichia coli
for biosynthesis of poly (3-hydroxybutyrate-co-3-
www.sciencedirect.com
Next generation industrial biotechnology Chen and Jiang 99
hydroxyvalerate) from glucose. Appl Microbiol Biotechnol 2014,
98:95-104.
5. Park SJ, Kim TW, Kim MK, Lee SY, Lim SC: Advanced bacterial
polyhydroxyalkanoates: towards a versatile and sustainable
platform for unnatural tailor-made polyesters. Biotechnol Adv
2012, 30:1196-1206.
6. Crook N, Alper HS: Classical strain improvement. In
Engineering Complex Phenotypes in Industrial Strains. Edited by
Patnaik R. John Wiley & Sons, Inc.; 2012 http://dx.doi.org/
10.1002/9781118433034.ch1.
7. Tyo KE, Fischer CR, Simeon F, Stephanopoulos G: Analysis of
polyhydroxybutyrate flux limitations by systematic genetic
and metabolic perturbations. Metab Eng 2010, 12:187-195.
8. Sun J, Alper HS: Metabolic engineering of strains: from
industrial-scale to lab-scale chemical production. J Ind
Microbiol Biot 2015, 42:423-436.
9. Fukuda H, Kondo A, Tamalampudi S: Bioenergy: sustainable
fuels from biomass by yeast and fungal whole-cell
biocatalysts. Biochem Eng J 2009, 44:2-12.
10. Li T, Chen XB, Chen JC, Wu Q, Chen GQ: Open and continuous
fermentation: products, conditions and bioprocess economy.
Biotechnol J 2014, 9:1503-1511.
11. Dumorne K, Cordova DC, Astorga-Elo M, Renganathan P:
Extremozymes: a potential source for industrial applications. J
Microbiol Biotechnol 2017, 27:649-659.
12. Golyshina OV, Hai T, Reva ON, Lemak S, Yakunin AF,
Goesmann A, Nechitaylo TY, Lacono V, Smedile F, Slesarev A:
Metabolic and evolutionary patterns in the extremely
acidophilic archaeon Ferroplasma acidiphilum YT. Sci Rep
2017, 7:3682.
13. Xian L, Wang F, Luo X, Feng YL, Feng JX: Purification and
characterization of a highly efficient calcium-independent
a-amylase from Talaromyces pinophilus 1-95. PLoS One 2015,
10:e0121531.
14. Sun SN, Sun SL, Cao XF, Sun RC: The role of pretreatment in
improving the enzymatic hydrolysis of lignocellulosic
materials. Biores Technol 2016, 199:49.
15. Elleuche S, Schäfers C, Blank S, Schröder C, Antranikian G:
Exploration of extremophiles for high temperature
biotechnological processes. Curr Opin Microbiol 2015, 25:113.
16. Sharma A, Kawarabayasi Y, Satyanarayana T: Acidophilic
bacteria and archaea: acid stable biocatalysts and their
potential applications. Extremophiles 2012, 16:1-19.
17. Wernick DG, Pontrelli SP, Pollock AW, Liao JC: Sustainable
 biorefining in wastewater by engineered extreme alkaliphile
Bacillus marmarensis. Sci Rep 2016, 6:20224.
This paper present engineering of the unexplored, extreme alkaliphile
Bacillus marmarensis as a platform for new bioprocesses. With a newly
developed transformation protocol and genetic tools, along with opti-
mized RBSs and antisense RNA, B. marmarensis produce ethanol at titers
of 38 g/l and 65% yields from glucose in unsterilized media.
18. Jisha VN, Smitha RB, Pradeep S, Sreedevi S, Unni KN, Sajith S,
Priji P, Josh MS, Benjamin S: Versatility of microbial proteases.
Adv Enzyme Res 2013, 1:39-51.
19. Vester JK, Glaring MA, Stougaard P: Discovery of novel enzymes
with industrial potential from a cold and alkaline environment
by a combination of functional metagenomics and culturing.
Microb Cell Fact 2014, 13:72.
20. Lima RN, Porto AL: Recent advances in marine enzymes for
biotechnological processes. Adv Food Nutr Res 2016, 78:153.
21. Tropeano M, Vazquez S, Coria S, Turjanski A, Cicero D,
Bercovich A, Mac Cormack W: Extracellular hydrolytic enzyme
production by proteolytic bacteria from the Antarctic. Polish
Polar Res 2013, 34:253-267.
22. Wang Y, Gao C, Zheng Z, Liu FM, Zang JY, Miao JL:
Immobilization of cold-active cellulase from Antarctic
bacterium and its use for kelp cellulose ethanol fermentation.
Bioresources 2015:10.
Current Opinion in Biotechnology 2018, 50:94–100
100 Energy biotechnology
23. Jeon JH, Kim JT, Kim YJ, Kim HK, Lee HS, Kang SG, Kim SJ,
Lee JH: Cloning and characterization of a new cold-active
lipase from a deep-sea sediment metagenome. Appl Microbiol
Biotechnol 2009, 81:865-874.
24. Madhavan A, Sindhu R, Parameswaran B, Sukumaran RK,
Pandey A: Metagenome analysis: a powerful tool for enzyme
bioprospecting. Appl Biochem Biotech 2017, 56:1-16.
25. Michetti D, Brandsdal BO, Bon D, Isaksen GV, Tiberti M,
Papaleo E: A comparative study of cold- and warm-adapted
endonucleases A using sequence analyses and molecular
dynamics simulations. PLoS One 2017, 12:e0169586.
26. Joshi S, Satyanarayana T: Biotechnology of cold-active
proteases. Biology 2013, 2:755-783.
27. Khan M, Sathya TA: Extremozymes from metagenome:
potential applications in food processing. Crit Rev Food Sci
2017 http://dx.doi.org/10.1080/10408398.2017.1296408.
28. Straub CT, Zeldes BM, Schut GJ, Adams MWW, Kelly RM:
 Extremely thermophilic energy metabolisms: biotechnological
prospects. Curr Opin Biotechnol 2017, 45:104-112.
The paper summarizes recent developments in extreme thermophile
biology as they relate to new horizons for energy biotechnology.
29. Counts JA, Zeldes BM, Lee LL, Straub CT, Adams MWW,
Kelly RM: Physiological, metabolic and biotechnological
features of extremely thermophilic microorganisms. Wires Syst
Boil Med 2017, 9:e1377.
30. Saul D, Morgan HW, Bergquist PL: Selected enzymes from
extreme thermophiles with applications in biotechnology. Curr
Biotechnol 2014, 3:45-59.
31. Elleuche S, Schröder C, Sahm K, Antranikian G:
Extremozymes — biocatalysts with unique properties from
extremophilic microorganisms. Curr Opin Biotechnol 2014,
29:116-123.
32. Chai YY, Rahman RN, Illias RM, Goh KM: Cloning and
characterization of two new thermostable and alkalitolerant
a-amylases from the Anoxybacillus species that produce high
levels of maltose. J Ind Microbiol Biotechnol 2012, 39:731-741.
33. Blank S, Schröder C, Schirrmacher G, Reisinger C, Antranikian G:
Biochemical characterization of a recombinant xylanase from
Thermus brockianus, suitable for biofuel production. JSM
Biotechnol Bioeng 2014, 2:1027.
34. Park SH, Park KH, Oh BC, Alli I, Lee BH: Expression and
characterization of an extremely thermostable b-glycosidase
(mannosidase) from the hyperthermophilic archaeon
Pyrococcus furiosus DSM3638. Nat Biotechnol 2011, 28:639-
648.
35. Mohammadipanah F, Wink J: Actinobacteria from arid and
desert habitats: diversity and biological activity. Frontiers
Microbiol 2016, 6:1541.
36. Stutzenberger F: Selective adsorption of endoglucanases from
Thermomonospora curvata on protein-extracted lucerne
fibres. Lett Appl Microbiol 2010, 5:1-4.
37. Ilayaraja S, Rajkumar J, Swarnakumar SN, Sivakumar K,
Thangaradjou T, Kannan L: Isolation of two thermophilic
actinobacterial strains mud volcano of the Baratang Island,
India. Afr J Microbiol Res 2014, 8:40-45.
38. Jiang H, Chen Y, Jiang PX, Zhang C, Smith TJ, Murrell JC, Xing XH:
Methanotrophs: multifunctional bacteria with promising
applications in environmental bioengineering. Biochem Eng J
2010, 49:277-288.
39. Luk HT, Mondelli C, Ferre DC, Stewart JA, Perez-Ramirez J:
Status and prospects in higher alcohols synthesis from
syngas. Chem Soc Rev 2017, 46:1358-1426.
40. Molitor B, Richter H, Martin ME, Jensen RO, Juminaga A,
Mihalcea C, Angenent LT: Carbon recovery by fermentation of
CO-rich off gases — turning steel mills into biorefineries.
Biores Technol 2016, 215:386-396.
Current Opinion in Biotechnology 2018, 50:94–100
41. Waditee-Sirisattha R, Kageyama H, Takabe T: Halophilic
microorganism resources and their applications in industrial
and environmental biotechnology. AIMS Microbiol 2016, 2:42-
54.
42. Tan D, Xue YS, Gulsimay, Chen GQ: Unsterile and continuous
production of polyhydroxybutyrate by Halomonas TD01. Biores
Technol 2011, 102:8130-8136.
43. Fu XZ, Tan D, Aibaidula G, Wu Q, Chen JC, Chen GQ:
Development of Halomonas TD01 as a host for open
production of chemicals. Metab Eng 2014, 23:78-91.
44. Yin J, Chen JC, Wu Q, Chen GQ: Halomonas spp., a rising star
 for industrial biotechnology. Biotechnol Adv 2015, 33:1433-
1442.
This paper summarizes the production of bioplastics polyhydroxyalk-
anoates (PHA), ectoines, enzymes, and bio-surfactants via halophiles.
And the advances in molecular manipulation for halophilic
microorganisms.
45. Chen XB, Jin Y, Ye JW, Zhang HQ, Che XM, Ma YM, Li MY,
 Chen GQ: Engineering Halomonas bluephagenesis TD01 for
unsterile production of poly(3-hydroxybutyrate-co-4-
hydroxybutyrate). Biores Technol 2017, 244:534-541.
By integrating orfZ gene encoding 4HB-CoA transferase into the chro-
mosome, Halomonas bluephagenesis TD01 was engineered to produce
70–80 g/l cell biomass with >60% P(3HB-co-16 mol% 4HB) in fermen-
tors. And the non-sterile fermentations were scaled up from 1, 7 to 1000-L
with similar results.
46. Cai L, Tan D, Aibaidula G, Dong XR, Chen JC, Tian WD, Chen GQ:
Comparative genomics study of PHA and ectoine relevant
genes from Halomonas sp. TD01 revealed extensive horizontal
gene transfer events and co-evolutionary relationships.
Microb Cell Fact 2011, 10:88.
47. Yue HT, Chen XB, Ling C, Chen YL, Deng HT, Chen GQ: A sea
water based open and continuous process for
polyhydroxyalkanoates production by Halomonas
campaniensis LS21. Biotechnol Biofuels 2014, 7:108.
48. Tan D, Wu Q, Chen JC, Chen GQ: Engineering Halomonas TD01
for low cost production of polyhydroxyalkanoates. Metab Eng
2014, 26:34-47.
49. Yin J, Fu XZ, Wu Q, Chen JC, Chen GQ: Development of an
enhanced chromosomal expression system based on porin
synthesis operon in Halomonas TD01. Appl Microbiol
Biotechnol 2014, 98:8987-8997.
50. Tao W, Lv L, Li D, Chen JC, Chen GQ: CRISPRi engineering
Halomonas for polyhydroxyalkanoates (PHA) synthesis.
Microb Cell Fact 2017, 16:48.
51. Zhao H, Zhang HQM, Li T, Lou CB, Ouyang Q, Chen GQ: Novel T7-
 like expression systems used for Halomonas. Metab Eng 2017,
39:128-140.
In this paper, novel T7-like expression systems were developed for non-
model bacteria including Halomonas and Pseudomonas
entomophila. Ultrahigh PHB accumulation (92% of CDW) was achieved
in Halomonas TD01 using T7-like MmP1.
52. Wu H, Chen JC, Chen GQ: Engineering the growth pattern and
cell morphology for enhanced PHB production by Escherichia
coli. Appl Microbiol Biotechnol 2016, 100:9907-9916.
53. Lan LH, Zhao H, Chen JC, Chen GQ: Engineering Halomonas
 spp. as a low-cost production host for production of bio-
surfactant protein PhaP. Biotechnol J 2016, 11:1595-1604.
In this paper, Halomonas strain TD is used to produce a protein named
PHA phasin or PhaP which has a potential to be developed into a bio-
surfactant.
54. Li T, Guo YY, Qiao GQ, Wu Q, Chen GQ: Microbial synthesis of 5-
 aminolevulinic acid (ALA) and its co-production with
polyhydroxybutyrate (PHB). ACS Syn Biol 2016, 5:1264-1275.
This paper reviews the recent strategies developed in PHA co-production
with other compounds, discusses the challenges and prospective during
the scale up of the co-production strategies.
www.sciencedirect.com