Journal of Environmental Chemical Engineering 11 (2023) 110998
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Journal of Environmental Chemical Engineering
journal homepage: www.elsevier.com/locate/jece
Current analysis on 1,3-propanediol production from glycerol via pure wild
strain fermentation
Ker Yee Tey a, Jian Ping Tan a, b, *, Swee Keong Yeap c, Ning He b, Nurul Adela Bukhari d,
Yew Woh Hui c, Abdullah Amru Indera Luthfi e, Shareena Fairuz Abdul Manaf f
a
School of Energy and Chemical Engineering, Xiamen University Malaysia, 43900 Sepang, Selangor Darul Ehsan, Malaysia
College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China
China-ASEAN College of Marine Sciences, Xiamen University Malaysia, Jalan Sunsuria, Bandar Sunsuria, 43900 Sepang, Selangor Darul Ehsan, Malaysia
d
Energy and Environment Unit, Engineering & Processing Research Division, Malaysian Palm Oil Board (MPOB), 6, Persiaran Institusi, Bandar Baru Bangi, 43000
Kajang, Selangor, Malaysia
e
Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor Darul Ehsan,
Malaysia
f
School of Chemical Engineering, College of Engineering, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia
b
c
A R T I C L E I N F O
A B S T R A C T
Editor: Yujie Men
Bioproduction of 1,3-propanediol (1,3-PDO), a ubiquitous green chemical, has received great attention due to its
wide range of applications, coupled with the abundance of crude glycerol from biodiesel industry. To
commercialize this product, the bottlenecks of low yield and productivity of 1,3-PDO should be addressed.
Herein, the present review carefully analyses the state-of-the-art performance of a myriad of wild-type 1,3-PDOproducing genera over the past decade. The effects of fermentation modes, aeration conditions, substrate
tolerance ability, and extent of metabolite inhibition on the synthesis of 1,3-PDO by different bacterial strains are
discussed comprehensively to realize the efficient utilization of the industrial wastes in harvesting the high
valued chemical commodity. Among the various cultivation method, continuous mode fermentation possesses
certain robustness over the others in terms of yield and productivity. Strict anaerobes are regarded as the best
microbe than other aerotolerant anaerobes and facultative anaerobes because of the outstanding fermentation
performance at anaerobic conditions. The responses of various microbes to high initial substrate and impurities
in crude glycerol have concluded that the discovery of osmotolerant microbe is crucial. Moreover, the 1,3-PDOproducing bacteria with minimum generation of byproducts along with appreciable tolerance towards the me­
tabolites is a determining factor of commercializing 1,3-PDO bioproduction.
Keywords:
Glycerol
1,3-propanediol
Operation mode
Substrate tolerance
Aeration condition
1. Introduction
Bioenergy, regarded as renewable and eco-friendly in nature, is a
potential resolution to the continuous global energetic crisis that arises
due to the depletion of unsustainable petrochemical resources by
establishing a new supply chain of sustainable fuels and chemicals.
Likewise, bioenergy corresponds to Sustainable Development Goal 7:
Affordable and Clean Energy, which is a common long-term climate goal
around the world. In fact, the rapid industrialization and modernization
in the transportation sector in this world have improved the living
qualities of mankind. Hand in hand with this achievement are the
numerous unresolved and complicated problems such as carbon emis­
sions [67]. Within this context, biodiesel appears as a great substitute for
conventional petroleum-derived fuels with a notable increment in its
production, which helps to alleviate the environmental impacts owing to
the non-polluting and renewable features. Production of biodiesel is
Abbreviations: µ, specific growth rate; 1,3-PDO, 1,3-propanediol; 2,3-BDO, 2,3-butanediol; 3-HPA, 3-hydroxypropionaldehyde; ATP, adenosine triphosphate;
CAGR, compound annual growth rate; COD, Chemical oxygen demand; CO2,, carbon dioxide; D, dilution rate; DCP, dichloro-2-propanol; DHA, dihydroxyacetone;
DHAK, dihydroxyacetone kinase; GDH, glycerol dehydrogenase; GDHt, glycerol dehydratase; glpF, glycerol uptake facilitator protein; H2, hydrogen; MONG, matter
organic non-glycerol; Mt, megatons; N2, nitrogen; NaCl, sodium chloride; NADH, nicotinamide adenine dinucleotide; PDOR, 1,3-propanediol dehydrogenase; Pdu,
propanediol utilization; PTT, polytrimethylene terephthalate.
* Corresponding author at: School of Energy and Chemical Engineering, Xiamen University Malaysia, 43900 Sepang, Selangor Darul Ehsan, Malaysia.
E-mail address: jianping.tan@xmu.edu.my (J.P. Tan).
https://doi.org/10.1016/j.jece.2023.110998
Received 29 May 2023; Received in revised form 31 July 2023; Accepted 10 September 2023
Available online 13 September 2023
2213-3437/© 2023 Elsevier Ltd. All rights reserved.
K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
bound to increase by 0.5%, which is equivalent to 130,000 barrels a day
by 2050 [14]. Unfortunately, the euphoria associated with biodiesel
production has brought disappointment afterwards due to the myriad of
adverse effects. Despite the benefits brought by the clean and non-toxic
fuel, the fact is that the production of methyl ester (biodiesel) from
transesterification of animal fats or vegetable oils with short chain al­
cohols would leave behind a massive amount of raw glycerol (also
known as glycerine) as an inherent residue, approximately 10% of the
total fuel produced by weight [14,95]. Additionally, the abundant crude
glycerol resulting from the explosive growth in biodiesel production
leads to substantial surpluses for the current glycerol market demand.
The market value for pure glycerol is at around 1135 US$/ton [12], but
the price of crude glycerol is as low as about 156 US$/ton to 174 US
$/ton [14]. As a matter of fact, there is a downfall of pure glycerol price
in the Asia Pacific region and the reason behind is the ongoing geopo­
litical conflicts, steady demand and existing inventories, and price
depreciation of palm oil and other edible oils [12]. On the contrary,
glycerol is the byproduct of the routes of saponification as well as hy­
drolysis in oleochemical plants [105]. The dilemma has led to a
considerable reduction in the market price of waste glycerol, which in
turn, gives way to the rise of alternative technologies utilizing the crude
glycerol.
Typically, biodiesel-based glycerine is categorized based on its purity
of the glycerol content where crude glycerol has 60–80% of glycerol,
while pure or refined glycerol has 99.1–99.8% of purity [44]. Crude
glycerol has a high chemical oxygen demand (COD) and a high
contamination of impurities such as alcohol, alkalis, fatty acids, and
catalysts; thereby either proper treatment or disposal of these wastes is
inevitable [94,109]. The purification steps of crude glycerol, such as
distillation and membrane filtration to obtain pure glycerol to be used in
food, pharmaceutical, or cosmetic industries are not economically ad­
vantageous owing to the high cost induced, despite the existence of
various well-developed purification methods. Thus, there is a need for
another consumer market for biodiesel crude glycerol to emerge, which
has the characteristics of higher product volume demand, lower quality
standards, and lower costs [14]. As such, the global burgeoning interest
is to utilize the vast amount of crude glycerol which cannot be simply
incinerated or disposed of directly in order to follow the pace of expo­
nential increase of demand and production of biodiesel. As a result, the
price and supply of crude glycerol will be stabilized and, at the same
time environment impact associated with the accumulation of this
underutilized material will be diminished.
Therefore, biodiesel that is nowadays regarded as grey fuel instead of
green fuel should be re-evaluated on the management of its raw glycerol
byproducts. Numerous alternative uses of crude glycerol have been
investigated and proposed, such as combustion [47], composting [5],
poultry-feeding [58], and thermochemical/biological conversions to
value-added commodity [62]. To date, glycerol has a plethora of in­
dustrial usages, which is over two thousand different applications,
depending on the chemical and physical characteristics, for example,
elasticity, flexibility, high miscibility, high viscosity, low volatility,
materials compatibility, non-toxicity, softness, solubility, and stability
[14]. The value-added products converted from fermentable crude
glycerol are for instance, 1,3-propanediol (1,3-PDO), acrolein, butanol,
citric acid, ethanol, hydrogen, polyunsaturated fatty acid, propionic
acid, single cell oil, biopolymers like PHA, and PHB and others [14,105].
Among those, valorization of this waste stream into diversified chem­
icals via biological mean is of increasing interest and has aroused
worldwide attention, given the possible boost in economic, environ­
mental and sustainable development in society. As there is a quest to
find alternatives to managing waste, fermentation seems to be one of the
best approaches which simultaneously combat the issues of waste
disposal and natural resources scarcity.
Due to the simple metabolic pathway compared to glucose metabolic
pathway, crude bio-glycerol or refined glycerine can serve as a feedstock
in microbial fermentation to be upgraded into valuable products for a
wide variety of applications. Owing to the reducing nature of carbon
atoms in glycerol (presence of hydroxyl group), its fermentation pro­
duces twice the amount of reducing equivalents than that produced from
other fermentable sugars (e.g. glucose, sucrose, and xylose) [43,108].
Besides, almost double of reducing equivalents known as nicotinamide
adenine dinucleotide (NADH) are produced from glycerol metabolism
than glucose metabolism, eventually resulted in higher yields of reduced
compounds [47]. Theoretically, glycerol can be valorized biologically
into a number of highly valued and widely used compounds including 1,
3-PDO, 2,3-butanediol (2,3-BDO), 3-hydroxypropionaldehyde, acetic
acid, acrolein, amino acids, butyric acid, butanol, citric acid, dihy­
droxyacetone (DHA), ethanol, glyceric acid, hydrogen, lactic acid, for­
mic acid, pigments, polyhydroxyalkanoates, propionic acid, succinic
acid, dichloro-2-propanol (DCP), and others [15,109]. Of these, 1,3-PDO
production from glycerol fermentation by a natural producer is the main
research focus due to the high demand for this chemical to be used as the
precursors in a multitude of applications. For instance, a newly identi­
fied C. butyricum SCUT343–4 isolated from deep mountain soil [59] and
a recently discovered K. pneumoniae KKU5 (Kp KKU5) [98] isolated from
soil near biodiesel plant were discovered with the ability in utilizing
glycerol to produce 1,3-PDO, contributing to the continuous develop­
ment of the biotechnology industry.
Despite the 1,3-PDO bioproduction has received extensive reviews,
the glycerol fermentation via a myriad of wild strain was neglected. The
previous published review discussed on the metabolic engineering,
evolutionary engineering, synthetic biology strategies, mutagenesis,
construction of a microbial consortium system in such biological pro­
duction [1,30,96,99,130,131]. However, the investigations of the
operation mode, aeration condition, substrate tolerance, formation of
byproducts by pure strain cultivation that greatly affect the 1,3-PDO
yield and productivity are not elucidated in detail. These key aspects
which are of significant interest and critical in this biotechnology field
are equally important during the commercialization of 1,3-PDO bio­
production. Therefore, current review focuses on these key aspects that
govern the performance of 1,3-PDO producing native bacteria.
2. Global market of 1,3-propanediol
1,3-propanediol (C3H8O2), which is frequently termed by its abbre­
viation 1,3-PDO is a potential C3 dihydroxy compound that is colorless
and miscible in polar solvents such as water, alcohols, and ethers [113].
1,3-PDO consists of two hydroxyl groups located at the first and third
carbon atoms. It has other names, such as trimethylene glycol or 1,
3-dihydroxypropane. This short chain diol is a class of specialty com­
pounds that known for more than a century, which is either produced by
the bio-based or petrochemical-based processes. In other words, 1,
3-PDO can be produced either from a biological route (fermentation)
or through a chemical process [54,111]. In recent years, sustainability
and environmental topics gradually become the main concern around
the globe and thus, the preference of consumers toward bio-based
products has pushed the market opportunities toward the green
technology.
Fig. 1 illustrates the projection of a 1,3-PDO market value in US$
billion/metric tons and its volume (mega tons) from the year 2022–2032
([71].Mr [27], Reportlinker [86], Shandong Richnow Chemical Co.,
2022, [93], Inner Mongolia Pulis Chemical Co., 2022). The price of 1,
3-PDO in 2022 was estimated at US$ 1700/metric ton. In the year
2022, the 1,3-PDO market has been valued at US$ 0.68 billion. The
global market of 1,3-PDO has boomed in the past few years and based on
the estimation, it is expected to increase at a compound annual growth
rate (CAGR) of around 10.63% during the forecast period (2022–2032),
which is equivalent to an estimated growth of US$ 1.18 billion from
2022 to 2032. The volume of 1,3-PDO in 2022 is around 0.40 Mt and an
increase of 0.69 Mt is anticipated after 10 years, reaching 1.09 Mt in
2032. According to the global market analysis, 1,3-PDO seems to be a
valuable commodity with great potential in bringing revenue for various
2
K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
Fig. 1. Global 1,3-propanediol market – value (US$ million) and volume (kilo tons), 2022–2032.
industries as well as improving product variety and quality in multiple
end-use industries, ensuring the economic sustainability of the produc­
tion process.
1,3-PDO has a wide spectrum of industrial applications. Polymers,
cosmetics & personal care, hygiene and domestic cleaning, engine
coolants, and heat transfer fluid are few dominant products formulated
by this platform chemical as displayed in Fig. 2 [21,71]. Among the
various application, 1,3-PDO is often utilized as the monomer to pro­
duce polyethers, polyurethanes, and polyesters. The main driver of 1,
3-PDO production is polytrimethylene terephthalate (PTT) which can
be employed as the precursor for the production of textiles and carpet
fibers with improved softness and stain resistance. PTT is an aromatic
polyester with unique properties of high elastic recoveries, high resil­
iency, high bulk, and soft hand. Also, the high reactivity and thermal
stability of 1,3-PDO enables it a suitable candidate to be applied in the
textile and polymer industry [79]. 1,3-PDO which has a reductive nature
undergoes a polycondensation reaction with terephthalic acid, resulting
in the creation of the novel PTT [111]. This economical and biode­
gradable polymer can be a perfect substitute for the present
pollution-causing synthetic plastics, and subsequently, able to tackle the
ecological imbalances triggered by toxic pollutants.
Table 1 lists the three main bio-based 1,3-PDO producing companies
and their detailed information. DuPont Tate & Lyle Bioproducts, a joint
venture of DuPont Tate & Lyle is the main key player in the 1,3-PDO
industry. This company, which is located in Loudon, Tennessee, US is
the world largest bio-based 1,3-PDO producer that dominate the market
of North America region. 1,3-PDO (Bio-PDO™) is produced commer­
cially from corn syrup as a feedstock by genetically modified Escherichia
coli (E. coli) under trademark of Susterra™ (industrial grade) and Zemea
™ (pharmaceutical grade) [16]. Therefore, the most prominent region
with great concern in the global 1,3-PDO market is located in North
America as DuPont Tate & Lyle Bioproducts leads the major producers
Fig. 2. Applications of 1,3-PDO.
3
K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
Co.Ltd, Tokyo Chemical Industry Co. Ltd and others [16]. However, it
should be noted that the developing countries especially those in South
East Asia (e.g., Indonesia, Malaysia, Vietnam, and the Philippines) lack
of awareness on the importance of 1,3-PDO as well as the future po­
tential of 1,3-PDO microbial production.
Table 1
Bio-based 1,3-PDO producing companies.
Aspect
DuPont Tate & Lyle
Bioproducts
Zhangjiagang Glory
Biomaterial
METEX
NØØVISTA
Operational
date
Trademark
November 2006
January 2013
March 2021
Bio-PDO™
(Susterra™
propanediol,
Zemea™
propanediol)
77
Bioglory®1,3propanediol
METEX
Propanediol,
TILAMAR® PDO
with
NØØVISTA™
25
Annual
output (kt)
Feedstock
Corn syrup (glucose)
Strain
Genetically modified
Escherichia coli (E.
coli)
Loudon, Tennessee,
US
Aerobic
Location
Aeration
condition
Applications
References
Susterra ®
(polyurethanes, heattransfer fluids,
unsaturated polyester
resins)
Zemea® (cosmetics &
personal care, food
and flavors, laundry
& household
cleaning,
pharmaceutical &
dietary supplements)
[16,21,101]
Plans to increase
capacity from 20 to
65
Palm-oil-biodieselbased glycerol
Likely bacteria
(Klebsiella spp.)
Jiangsu, China
N/A
3. Chemical and biological synthesis method of 1,3-PDO
The conventional chemical processes of 1,3-PDO production are
through the hydration of petroleum acrolein (Degussa-Dupont routes) or
hydroformylation of ethylene oxide (Shell route), both in the presence of
catalyst [62]. However, the use of non-renewable raw materials origi­
nated from fossil resources, severe operating conditions (elevated tem­
perature and pressure), use of special buffering agents, expensive
catalysts, generation of toxic intermediates, and high equipment costs
have hindered the economic feasibility of the process [82,111,115].
Moreover, the shortcomings of the low selectivity, low titer, low yield,
and high energy consumption have encouraged paradigm shifts from
conventional chemical processes to white biotechnology of 1,3-PDO
production [67]. Presently, microbial production of 1,3-PDO has
drawn more attention owing to the remarkable benefits, including
renewable and environmentally benign, abundant cost-effective
renewable substrate and lower energy consumption. Moreover, the
ambient operating condition of biotransformation, which offers lower or
no energy input makes the bioprocess appealing. According to the life
cycle analysis conducted by DuPont Tate & Lyle, the greenhouse gas
emissions and nonrenewable energy consumption of bio-based 1,3-PDO
are 47% and 49% less than petroleum-based 1,3-PDO, respectively [22].
The glycerol fermentation to 1,3-PDO, a biocatalyst-mediated
transformation reaction by a mixed culture containing Clostridium pas­
teurianum was discovered in 1881. At present, the biological route to
synthesize 1,3-PDO is by metabolizing pure, pretreated or crude glycerol
in a reductive pathway using native bacterium under different condi­
tions such as aerobic, microaerophilic, facultative anaerobic or strict
anaerobic. There is a myriad of naturally occurring anaerobic or facul­
tative anaerobic prokaryotic microorganisms with the capability to
break down glycerol into 1,3-PDO, such as the genera Citrobacter (e.g.,
C. beijerinckii, and C. freundii) [29,32], Clostridium (e.g.,
C. acetobutylicum, C. butyricum, C. diolis, and C. pasteurianum) [28,34,51,
107], Enterobacter (e.g., E. cloacae) [97], Klebsiella (e.g., K. oxytoca, and
K. pneumoniae) [88,128], and Lactobacilli (e.g., L. brevis, L. diolivorans,
and L. reuteri) [18,85,110]. The abovementioned microbes are by-far
popular natural producers of 1,3-PDO. However, their feasibility is
mostly proven only in lab-scale glycerol fermentation and the potential
in commercial production of 1,3-PDO still remain ambiguous.
The glycerol dismutation consists of two parallel branches, namely
the oxidative and reductive branches (also known as the propanediol
utilization (Pdu) pathway). At here, the GDHt in all bacteria except
C. butyricum are cobalamin (vitamin B12) dependent. In the parallel
oxidative pathway, glycerol is dehydrogenated by NAD+-dependent
glycerol dehydrogenase (GDH) to dihydroxyacetone (DHA), followed by
phosphorylation through adenosine triphosphate (ATP)-dependent
dihydroxyacetone kinase (DHAK) before entering the glycolytic
pathway to form pyruvate. The oxidative branch provides energy and
reducing power (NADH2) for cell growth and 1,3-PDO syntheses, at the
same time converts pyruvate to form a series of coproducts, such as 2,3BDO, 3-hydroxypropionic (3-HP), acetate, butanol, butyrate, carbon
dioxide (CO2), ethanol, hydrogen (H2), lactate, succinate and others
depending on the bacterial species [43,70,114].
The research papers on 1,3-PDO production from natural bio­
catalysts were reviewed from the year 2010–2022. From the pie chart
shown in Fig. 3, it is obvious that the most studied bacterial species is
Clostridium spp., which contributes approximately 41%, followed by
Klebsiella spp. (37%), Citrobacter spp. (12%), Lactobacillus spp. (3%), and
others (7%) consisting of the genera belonging to Enterobacter, Hafnia,
Halanerobium, Kluyvera, Pantoea, and Shmwellia. Thus, Clostridium spp.
Non-GMO
rapeseed plant
Likely bacteria
(Clostridium
spp.)
Carling Saint
Avold, France
N/A
Coating,
polyurethanes,
deicing fluids,
unsaturated
polyester resins,
printing ink,
cosmetics
ingredients
Biobased
polymer,
functional
fluids,
cosmetics &
personal care
[42,61,127]
[23,24,26,81]
around the world with its industrially established bioprocess. METEX
NØØVISTA is another potential player in the Europe market of
manufacturing 1,3-PDO fermentatively. Metabolic Explorer and the
French Société de Projets Industriels successful commissioned the
METEX NØØVISTA production unit and the marketing of 1,3-PDO, the
first made-in-Europe non-GMO cosmetic grade 1,3-PDO alongside with
butyric acid (BA) [25]. In 2021, the scale-up of 1,3-propanediol (1,
3-PDO)-BA technology and its production in cosmetics and industrial
applications was announced by METEX NØØVISTA, proving the surge in
demand for 1,3-PDO [27]. Several patent applications of Metabolic
Explorer, which describes the production of 1,3-PDO from glycerol using
only Clostridium acetobutylicum or co-culture of Clostridium sphenoides
and Clostridium sporogones, suggesting the possible strains employed by
the process [23].
Other than two companies mentioned earlier, Zhangjiagang Glory
Biomaterial which is located in China appears as another potential
candidate of bioproducing 1,3-PDO. Zhangjiagang Glory Biomaterial
was established for a bio-based 1,3-PDO programme, which started the
first production line in January 2013 and then expanded its annual
production to 65 kt. After that, the company become the manufacturer
covering a complete industrial chain of 1,3-PDO, PTT chips, and PTT
fiber. Due to the limitation of information regarding the 1,3-PDO pro­
duction process, the possible bacteria used is Klebsiella variicola ac­
cording to the patent application of Zhangjiagang Glory Biomaterial
[42]. According to Table 1, three of the companies utilized different
kinds of feedstock based on the raw material’s availability in their re­
gion. Unlike DuPont Tate & Lyle Bioproducts who genetically modified
E. coli to produce 1,3-PDO from glucose, the other two companies are
likely to utilize wild Klebsiella spp. or Clostridium spp. in their produc­
tion. Apart from that, there are several prominent players with great
potential in mass production of 1,3-PDO biologically based in Europe (e.
g., Germany) and Asia-Pacific (e.g., China, Japan, India), such as Merck
KGgA, Zouping Mingxing Chemical Co.Ltd, Zouping Mingxing Chemical
4
K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
increased 1,3-PDO yield via overflow metabolism toward reductive
pathways, which induced by concentrated initial glycerol; (3) native
strains have better stability [56]. The 1,3-PDO generation capability of
different microorganisms under anaerobic glycerol fermentation at
various operating modes is summarized in Table 3.
4.1. Batch operation
Batch strategy is a one-time adding of microbes and medium at the
start of cultivation and hence, it is a closed system. The research papers
were selected by the criterion of operation mode, aeration rate, substrate
type, and culture type for better visualization of the fermentation per­
formance for each native strain. Under the similar operating condition
(batch operation, anaerobic condition, glycerol as the exclusive carbon
source, and natural strain), it is noticed that the yield ranges from
0.25 mol/mol to 0.71 mol/mol whereas the range of productivity is
between 0.5 g/L.h and 3.31 g/L.h as displayed in Fig. 4. The highest 1,3PDO yield from anaerobic batch fermentation using only 81% w/w
crude glycerol by the strain of C. butyricum NRRL B-23495 is 0.71 mol/
mol [74]. The achieved 1,3-PDO yield is close to the maximum theo­
retical yield of C. butyricum [125]. Besides, it can be observed that the
C. butyricum generally produced a higher yield of 1,3-PDO
(0.59–0.71 mol/mol) than the other strains under batch operation and
anaerobiosis.
On the other hand, L. reuteri FXZ014 obtained the highest produc­
tivity (3.31 g/L.h) after pure glycerol fermentation [124] than the other
microbes as illustrated in Fig. 4. However, its 1,3-PDO yield of
0.39 mol/mol is comparatively lower than other microorganisms. This
recent study applied whole-cell biotransformation (WCB) in the
biosynthesis of 1,3-PDO where the growth process of L. reuteri FXZ014
and production of 1,3-PDO were separated into two stages of the pro­
cess. The use of resting cells had led to an elevation in volumetric pro­
ductivity, proven by the high productivity of as much as 3.31 g/L.h
despite the conduct of biotransformation of glycerol in typical batch
fermentation [67].
K. pneumoniae GLC29 [15], C. butyricum JKT37 [107], C. butyricum
DSP1 [104], C. perfringens GYL [40], and C. butyricum NCIMB 8082 [72]
are identified as the strains that give excellent results in both yield and
productivity of 1,3-PDO during batch operation, based on the bar chart
(Fig. 4). According to Tee et al. [107], no lag phase observed in the rapid
growth of C. butyricum JKT37 enables high 1,3-PDO productivity.
Membrane module was applied in increasing biomass with two times of
concentrated C. butyricum DSP1, which helps to ameliorate kinetic
properties, especially productivity (2.70 g/L.h) and titer (41.22 g/L.h)
[104]. C. perfringens GYL has a fast growth speed and can grow on
100 g/L of crude glycerol; it gave 39.3 g/L of 1,3-PDO with excellent
yield and productivity [40]. From the study conducted by Martins et al.
[72], C. butyricum NCIMB 8082 was able to produce 32.18 g/L of 1,
3-PDO with 2.38 g/L.h of productivity in batch condition. In short, the
fermentation performance of strains at batch conditions were validated,
which allows a further investigation in other cultivation methods for
yield and productivity enhancement.
Fig. 3. Bacterial strains studied for 1,3-PDO production from glycerol
fermentation.
and Klebsiella spp. have been studied intensively in glycerol fermenta­
tion to produce 1,3-PDO due to their relatively outstanding product
concentration, molar yield, and production rate [7,63]. The perfor­
mance of microbial strain is normally evaluated by product concentra­
tion, yield, and productivity where the final product concentration, as
well as the composition of fermentation solution, determines the
complication of separation process. Especially, the separation of salts,
protein, polysaccharide and other components is the most important
part of the downstream separation. Besides, yield will affect the cost of
substrate whereas productivity aids in reducing the capital cost of
equipment and subsequent operation expenses. 1,3-PDO yield is defined
as the amount of 1,3-PDO produced from glycerol (mol PDO/mol glyc­
erol) [13]. From the estimation of the glycerol fermentation pathway
analysis, the maximum theoretical yield of 1,3-PDO by C. butyricum
[125] and K. pneumoniae [73] is 0.72 mol/mol, given that only acetic
acid is produced as a byproduct and no hydrogen is released. However,
the 1,3-PDO maximum theoretical yield can reach up to 0.875 mol/mol,
provided that all acetyl-CoA enters TCA cycle instead of the acetic acid
pathway under an ideal anaerobic condition [68].
The fermentation performance of genetically modified E. coli on the
glucose of commercial DuPont Tate & Lyle industrial process are 135 g/
L PDO, 0.62 mol/mol glucose, and 3.5 g/L.h [69,83]. Several strains,
which have comparable fermentation performance with DuPont Tate &
Lyle are listed in Table 2. However, numerous factors will determine the
final 1,3-PDO production capacity as dissimilar bacteria perform
differently under respective conditions, which will be discussed in the
following section.
4. Operation modes
The method of cultivating the microorganisms and dosing of sub­
strates is critically affecting various biokinetics of the fermentation
profile for every biological process. Batch, fed-batch, repeated batch,
repeated fed-batch, and continuous are some of the reported approaches
on the production of 1,3-PDO. Anaerobic glycerol fermentation by wildtype strain is selected as the basis in the following analysis of operation
modes because of the following reasons: (1) glycerol is a reduced sub­
strate where its anaerobic oxidation can increase cellular NADH/NAD+
ratio and shift metabolic fluxes; (2) anaerobic condition allows an
Table 2
Glycerol conversion by microorganisms to 1,3-PDO.
Bacterial strain
Substrate
Fermentation mode
1,3-PDO titer (g/L)
Yield (mol/mol)
Productivity (g/L.h)
Reference
C. butyricum DL07
C. butyricum DL07
C. butyricum AKR102a
C. butyricum SCUT343–4
C. butyricum DL07
K. pneumoniae HSL4
Pure glycerol
78% crude glycerol
Pure glycerol
95% pure glycerol
Pure glycerol
Glycerol
Fed-batch; anaerobic
Fed-batch; anaerobic
Fed-batch; anaerobic
Repeated fed-batch; anaerobic
Sequential fed-batch; anaerobic
Fed-batch; aerobic
104.80
94.20
93.70
86.00
85.00
80.08
0.65
0.63
0.63
0.63
0.63
0.53
3.38
3.04
3.30
4.20
6.77
2.22
[112]
[112]
[115]
[59]
[112]
[129]
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K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
Table 3
Production of 1,3-PDO from glycerol fermentation of different native bacteria under anaerobic condition at various operation modes.
Operation mode
Bacterial strain
Substrate
1,3-PDO titer
(g/L)
Yield (mol/
mol)
Productivity (g/
L.h)
References
Batch
C. acetobutylicum ATCC 4259
99% pure glycerol
N/A
0.25
N/A
C. beijerinckii NRRL B593
C. butyricum DSM 10702
54.35% w/v waste glycerol
Pretreated glycerol by ion
exchange resin
85.6% w/w crude glycerol
80.61 wt% pretreated glycerol
Crude glycerol
81% w/w crude glycerol
95% glycerol
81% w/w crude glycerol
Crude glycerol
Glycerol
80% crude glycerol
10.00
41.40
0.60
0.68
0.50
0.99
(Ferreira, F. et al.,
2012)
[37]
[66]
41.22
10.79
32.18
32.30
42.80
35.10
26.00
23.30
4.350
0.62
0.64
0.63
0.71
0.65
0.69
0.61
0.61
0.28
2.75
2.16
2.38
N/A
1.78
N/A
0.72
N/A
N/A
81% w/w crude glycerol
Crude glycerol
56% m/v crude glycerol
Glycerol
82.8% w/w crude glycerol
Pure glycerol
54.35% w/v waste glycerol
10.10
39.30
2.40
13.51
23.80
19.09
11.00
0.58
0.58
0.50
0.41
0.46
0.51
0.61
1.01
2.67
N/A
N/A
0.99
1.57
1.34
[104]
[106]
[72]
[74]
[59]
[9]
[52]
[20]
(Ferreira, T.F. et al.,
2012)
[74]
[40]
[55]
[116]
[90]
[60]
[37]
Glycerol
Glycerol
Pure glycerol
65.8% w/w crude glycerol
Crude glycerol (after removal of
floating layer)
Pure glycerol
0.885 g/g crude glycerol
85.6% w/w crude glycerol
Crude glycerol
95% glycerol
75% w/w crude glycerol
Pure glycerol
79.4% w/w crude glycerol
81% w/w crude glycerol
50% w/w diluted crude glycerol
80% w/w crude glycerol
Crude glycerol
Crude glycerol
82.8% w/w crude glycerol
80% w/w crude glycerol
Refined glycerol
Crude glycerol
0.885 g/g crude glycerol
Glycerol
81% w/w crude glycerol
54.35% w/v crude glycerol
54.35% w/w crude glycerol
Glycerol
20.40
N/A
9.94
13.84
61.5
0.62
0.37
0.39
0.53
0.64
2.92
0.53
3.31
1.15
5.00
[15]
[119]
[124]
[87]
[115]
104.80
36.10
71.00
29.83
59.15
67.90
63.50
37.70
68.10
> 55.00
40.00
50.10
62.72
36.86
71.1
9.00
18.00
24.30
33.80
14.20
N/A
N/A
19.70
0.65
0.59
0.65
0.46
0.64
0.67
N/A
0.67
0.48
0.63
0.68
0.40
0.73
0.23
0.67
N/A
0.66
0.69
0.70
0.69
0.78
N/A
0.59
3.38
0.72
1.00
2.55
2.11
3.50
1.35
0.69
1.62
5.20
2.00
0.90
1.74
0.77
1.51
0.91
6.40
1.2
16.90
1.41
1.27
2.70
N/A
[112]
[66]
[103]
[72]
[59]
[11]
[51]
[70]
[76]
[49]
[40]
[75]
[121]
[90]
[48]
[18]
[32]
[65]
[100]
[10]
[36]
[6]
[31]
Crude glycerol
2.50
N/A
4.80
[39]
54.35% w/w crude glycerol
Pure glycerol
85.6% w/w crude glycerol
Raw glycerol
Glycerol
Crude glycerol
81.8% w/w crude glycerol
95% glycerol
N/A
28.30
62.00
42.39
67.80
20.1
24.99
86.00
N/A
0.51
0.53
0.59
0.73
0.64
0.58
0.63
3.60
6.80
1.68
2.14
1.04
1.26
6.29
4.20
[6]
[19]
[102]
[126]
[53]
[122]
[17]
[59]
80% w/w crude glycerol
Glycerol
Pure glycerol
64.00
66.00
85.00
N/A
0.61
0.63
1.61
3.43
6.78
[48]
[120]
Wang et al.[112]
C. butyricum DSP1
C. butyricum JKT37
C. butyricum NCIMB 8082
C. butyricum NRRL B-23495
C. butyricum SCUT343–4
C. butyricum VPI 1718
C. diolis DSM 15410
C. freundii AD119
C. freundii ATCC 8090
Fed-batch
Continuous
Repeated batch
Repeated fedbatch
Sequential fedbatch
C. freundii FMCC-B 294 (VK-19)
C. perfringens GYL
H. saccharolyticum DSM 6643
K. oxytoca NRRL-B199
K. pneumoniae BLh-1
K. pneumoniae DSMZ 2026
K. pneumoniae GenBank no:
27FHM063413
K. pneumoniae GLC29
K. pneumoniae HE1
L. reuteri FXZ014
S. blattae ATCC 33430
C. butyricum AKR102a
C. butyricum DL07
C. butyricum DSM 10702
C. butyricum DSP1
C. butyricum NCIMB 8082
C. butyricum SCUT343–4
C. butyricum VPI 1718
C. diolis DSM15410
C. freundii FMCC-8
C. freundii FMCC-B 294 (VK-19)
C. pasteurianum K1
C. perfringens GYL
K. oxytoca FMCC-197
K. pneumoniae ATCC 8724
K. pneumoniae BLh-1
K. pneumoniae DSM 4799
L. reuteri RPRB3007
C. beijerinckii B-593 (immobilized)
C. butyricum DSM 10702
C. butyricum DSM 5431 (immobilized)
C. butyricum VPI 1718
C. freundii DSM 15979 (immobilized)
C. freundii DSM 15979
K. pneumoniae GenBank no.
27FHM063413 (immobilized)
K. pneumoniae GenBank no.
27FHM063413 (immobilized)
P. agglomerans DSM 30077 (immobilized)
C. butyricum DSM 4278 (immobilized)
C. butyricum DSP1
C. butyricum H304
C. diolis DSM 15410
K. pneumoniae ATCC 8724 (immobilized)
K. pneumoniae BLh-1 (immobilized)
C. butyricum SCUT343–4 (immobilized)
K. pneumoniae DSM 4799 (immobilized)
K. pneumoniae LX3
C. butyricum DL07
4.2. Fed-batch operation
process. From Fig. 5(a) and (b), the 1,3-PDO yields of different strains
range from 0.398 mol/mol to 0.73 mol/mol whilst all bacterial strains
have productivities of values between 0.67 g/L.h and 5.2 g/L.h. Among
the bacterial strains that undergo fed-batch fermentation, the highest
Fed-batch fermentation is a semi-closed system with a systematic
feeding of substrate and supplements throughout the cultivation
6
K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
Fig. 4. Yield and productivity of different native strains under batch fermentation. Data are available from Table 3.
Fig. 5. Yield (a) and productivity (b) of different native strains under fed-batch fermentation. Data are available from Table 3.
yield of 0.73 mol/mol is obtained by K. pneumoniae ATCC 8724 [121];
on the other hand, C. pasteurianum K1 has the highest productivity of
5.2 g/L.h [49]. In an optimization study conducted by Yang et al. [121],
the fed-batch glycerol fermentation with a continuous feed strategy
coupled with increased working volume and switch culture pH control
by K. pneumoniae ATCC 8724 had produced 62.72 g/L and
0.73 mol/mol of 1,3-PDO at the productivity of 1.74 g/L.h.
C. pasteurianum K1 and C. butyricum AKR102a are the two strains
with exceptional yield and productivity by the means of fed-batch
fermentation. In a study conducted by Kaeding et al. [49], the 1,
3-PDO production in 1 m3 miniplant scale starting from substrate pre­
treatment, fed-batch fermentation by a newly isolated C. pasteurianum
K1, until downstream processing was in success; over 55 g/L of 1,3-PDO
was produced with 0.63 mol/mol of yield. Apart from C. pasteurianum
K1, the non-sterile 200-L fed-batch fermentation of C. butyricum
AKR102a on crude glycerol had resulted in a 1,3-PDO titer of 61.5 g/L, a
yield of 0.641 mol/mol, and maximum productivity of 5 g/L.h. In brief,
there are noticeable improvements in 1,3-PDO productivity for
fed-batch operation compared to batchwise cultivation.
4.3. Continuous operation
Although a majority of literature deals with glycerol fermentation by
microbes under batch or fed-batch conditions, a limited number of
publications report on bacterial glycerol metabolism using the contin­
uous mode of operation. Continuous fermentation is an open system
where the addition of nutrients and removal of culture broth (cells and
metabolites) are carried out simultaneously within fixed volume. Ac­
cording to Fig. 6(a) and (b), the yield and productivity of various strains
are in between 0.59 and 0.78 mol/mol and 1.2–16.9 g/L.h, respectively.
The highest 1,3-PDO molar yield of 0.78 mol/mol was achieved by
Güngörmüşler [36] where modified continuous biofilm reactor (MCBR)
was utilized in immobilizing C. freundii DSM 15979 on ceramic supports
to ferment biodiesel-derived glycerol. C. butyricum DSM 5431 produced
1,3-PDO with the second highest yield of 0.70 mol/mol and at excellent
productivity as high as 16.9 g/L.h [100]. The reason why C. butyricum
DSM 5431 can surpass the other strains in terms of yield and produc­
tivity due to the use of a moving bed bioreactor coupled with BCN-009
as the cell carriers [100]. This is the first report on the use of a moving
bed bioreactor for glycerol fermentation to synthesize 1,3-PDO, and this
configuration helps to enhance liquid mixing, which in turn improves
7
K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
Fig. 6. Yield (a) and productivity (b) of different native strains under continuous fermentation. Data are available from Table 3.
growth conditions in the media bed. Moreover, the continuous operation
of the moving bed bioreactor can provide an appropriate environment
for C. butyricum DSM 5431 and maintain effective biomass retention.
Besides, the continuous production of 1,3-PDO using immobilized
cells of C. beijerrinckii NRRL B-593 by glass rushing rings in a packed bed
bioreactor successfully achieved a high molar yield of 0.66 mol/mol and
6.4 g/L.h. The continuous operation at a shorter hydraulic retention
time (HRT), which is 2 h enables the highest productivity and vice versa
for the productivity obtained when applied HRT increases. At HRT of 8 h
under the same fermentation condition, C. beijerrinckii NRRL B-593
managed to consume 100% of glycerol and thus, giving 0.77 mol/mol of
1,3-PDO [32]. In summary, continuous cultivation represents a more
practical situation of glycerol fermentation by microbes to produce 1,
3-PDO, which gives better insight into this bioprocess
commercialization.
4.4. Repeated batch operation
Repeated batch is the hybrid feeding strategy by removing a signif­
icant portion of the bioreactor working volume at specified intervals and
replacing it with a fresh fermentation medium, where the process is
repeated for a few cycles. Repeated batches which utilize a portion of
metabolically active biomass as inoculum can reduce the time used for
the whole operation due to the elimination of the stage of inoculum
growth. This fermentation formula helps to improve the kinetic pa­
rameters of 1,3-PDO production because the microbes can adapt well to
the carbon source [102]. Based on the bar charts (Fig. 7(a) and (b)), the
molar yield and productivity of various strains are in the range of
Fig. 7. Yield (a) and productivity (b) of different native strains under repeated batch fermentation. Data are available from Table 3.
8
K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
0.51–0.73 mol/mol and 1.04–6.8 g/L.h, respectively. The attempt of a
repeated batch mode of bioreactor operation (empty-and-fill protocol)
for 1,3-PDO production using C. diolis DSM 15410 was first reported by
Kaur and his coworkers [53]. As depicted in Fig. 7(a) and (b), C. diolis
DSM 15410 produces 1,3-PDO with the highest yield and lowest pro­
ductivity, which are 0.73 mol/mol and 1.04 g/L.h respectively [53]. On
the other hand, pure glycerol fermentation using immobilized
C. butyricum DSM 4278 had resulted in the highest productivity (6.8 g/L.
h) but the lowest yield (0.51 mol/mol) under the repeated batch con­
dition [19]. This operation method is analogous to the recycling system
in which the dilution rate (D) can be enhanced up to D = µ without cell
washout, where µ is the specific growth rate. As such, D increases
allowing high productivity of 1,3-PDO. In brief, this cultivation method
can serve as the preliminary test of bacterial immobilization before
upscaling to continuous operation, to determine the operational stability
and biological activity of immobilized cell systems.
The fed-batch process is more efficient than the batch process
because it can control the growth. Fed-batch cultivation strategy allows
a higher cell density due to the extended culture durations resulting
from the supply of nutrients systematically. In other words, fed-batch
cultivation able to overcome the depletion of nutrients in the culture
along with the prevention of high substrate concentration inhibition
[48]. Therefore, a higher 1,3-PDO concentration associated with its high
yield and productivity could be achieved using a fed-batch system than
batch fermentation, as illustrated in Fig. 4 and Fig. 5. To be specific, the
strain performance in batch fermentation acts as preliminary data to get
a prior understanding before applying fed-batch with improved perfor­
mance. Yet, there are some common obstacles to fed-batch cultivation,
such as incomplete substrate fermentation, accumulation of metabolites,
extended duration of the process, low productivity, and difficulties in
optimizing fermentation conditions (feeding quantity, feeding rate,
feeding interval, and feeding time) [102,121].
The common problem in batch and fed-batch is the decline in PDO
production at the end of fermentation which might be due to the accu­
mulated product and coproducts. Another issue is that when dealing
with a large volume of substrate, the space in batch and fed batch re­
actors are limited, turns out the cells have a finite amount of space in
which to grow. One way to combat this issue is through the imple­
mentation of a repeated batch, repeated fed-batch or continuous stra­
tegies where a part of fermentation broth will be removed from time to
time. Repeated batch strategy can be denoted as the batch operation
repeated using a fill-and-draw process. This kind of operation mode is
better than batch and even fed-batch processes. When comparing batch
strategy with repeated batch cultivation, the latter can lead to produc­
tivity enhancement due to the prolonged culture’s life in the batch
bioreactor, at the same time, reduce the time and effort required in the
anaerobic cultivation techniques. Similarly, the merits of repeated batch
over fed-batch are the unlimited working volume of the bioreactor and
the ease of operation [53]. The enhancement of productivity is in
agreement with the results shown in Fig. 7 (repeated batch operation)
where the production rates of Clostridium sp. and Klebsiella sp. are higher
than those in Fig. 4 and Fig. 5 (batch and fed-batch). Therefore, the
repeated batch operation is a result-yielding approach especially suit­
able for anaerobic fermentation, which eliminates the preparation and
sterilization steps for a new medium, eradicates osmotic pressure of
crude glycerol, enhances culture stability towards contamination, and
allows the use of one bioreactor without capacity limitation [53,102,
126].
Apart from that, repeated fed-batch culture is effective in enhancing
the productivity of microbial cultures. When a large portion of culture is
replaced by a fresh medium repeatedly, the products generated are
diluted in each cycle and hence, this can release the inhibition caused by
the metabolites or toxins [120]. Then, a higher productivity could be
sustained throughout the fermentation period. This fermentative system
prevents the seed culture time between two fed-batches to some extent,
and hence enhances the overall product productivity [112].
The sequential fed-batch process is a newly established mode of
operation which provide the advantage of stable fermentation by inoc­
ulating inoculum at an exponential growth period in a new bioreactor.
On top of that, the possibility of fewer cycles in repeated batch and fedbatch fermentation due to the poor seed quality might be resolved by
sequential fed-batch. However, this strategy has certain limitations
where a lot of expenditure is required to reduce the time taken for seed
culture prior to sequential fed-batch cultivation in industrial production
[112].
To study the maximum productivity of a strain, continuous culture
appears to be an effective strategy [94]. Herein, continuous culture
creates a balanced nature of feeding and thence, the achieved steady
state allows a long-term production. The accumulation of toxins and
products which might cause inhibition over time can be avoided using
this fermentative strategy. Continuous production of 1,3-PDO allows the
use of a smaller fermenter and eventually results in a higher product
4.5. Repeated fed-batch operation
Repeated fed-batch cultivation is similar to repeated batch cultiva­
tion where the repeated fill-and-draw process is carried out in fed-batch
mode. A repeated fed-batch glycerol fermentation with immobilized
K. pneumoniae DSM 4799 cells in a fixed bed reactor demonstrated by
Jun et al. [48] had resulted in a productivity improvement when
compared with fed-batch fermentation. Similarly, the 1,3-PDO produc­
tivity of repeated fed-batch culture using K. pneumoniae LX3 was much
higher than the original fed-batch culture (1.5 g/L.h), with more than
3.30 g/L.h for repeated fed-batch cultivation in each cycle [120]. In
every cycle of removal and replacement of fermentation broth, high cell
growth rates that favors 1,3-PDO formation can be restored due to
reduced product concentration. In a recent study conducted by Lan
et al., the adoption of repeated fed-batch for 1,3-PDO production using a
newly identified C. butyricum SCUT343–4 in a fibrous bed bioreactor had
led to the production of 86 g/L 1,3-PDO at the yield and productivity of
0.63 mol/mol and 4.2 g/L.h, respectively [59].
4.6. Sequential fed-batch
A novel sequential fed-batch cultivation mode was established and
examined for its feasibility in 1,3-PDO production using C. butyricum
DL07 by Wang et al. [112]. Before initiating sequential fed-batch
fermentation, the seed culture had tripled up in three bioreactors at
the rate of decreased inoculum size and increased incubation time from
one to another. This strategy is performed by inoculating 2% inoculum
containing C. butyricum DL07 cells which are at an exponential phase in
the prior bioreactor to the next bioreactor with a fresh fermentation
medium. The entire process continues for eight cycles which successfully
maintain the 1,3-PDO concentration. As a consequence, 85 g/L of 1,
3-PDO was formed at 0.63 mol/mol and 6.77 g/L.h of yield and over­
all productivity, respectively. It is worth noting that sequential fed-batch
fermentation could achieve higher productivity than repeated fed-batch
(3.3 g/L.h) in the same study and thus, this cultivation mode possesses a
high potential in feasible 1,3-PDO production [112].
4.7. Comparison between operation modes
Batchwise operation is a relatively simple strategy for 1,3-PDO
production compared to other modes of biotransformation. This oper­
ation mode is typically suitable for early and rapid experimental stages
such as metabolic regulation, optimization of nutrient medium, and
inhibitory effect of substrate [121]. Nonetheless, a low biomass con­
centration is the main obstacle in batch cultivation because of the
shortage of nutrients. Cell growth happens simultaneously with 1,3-PDO
production which is termed as growth-associated 1,3-PDO production;
thus, a lower 1,3-PDO titer, yield, and productivity had observed in
batch cultivation due to its operational limits.
9
K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
yield than batch operation. Moreover, the continuous bioconversion of
glycerol using immobilized cells can further increase the product pro­
ductivity due to the increased cell densities, as shown in several research
studies [31,36,38,39,57,65,77]. The fermentation with the immobili­
zation system able to provide sufficient biomass without the need for
preculture which eliminate the cells’ lag phase, suggesting an acceler­
ated productivity is possible due to reduced reaction time [48]. Other
than that, immobilized scheme is superior over suspended system,
particularly in the prevention of cell washout at high dilution rates (or
low hydraulic retention times), resistance to harsh environment (e.g.
high organic loading rates, extreme pH, presence of acetic acid), cell
reusability for prolonged periods, protection against shear damage,
prolong survival and metabolic activity of cells, and ease of separation
[19,31,38,100]. The immobilization techniques applied in 1,3-PDO
production are for example, adsorption (polyurethane media, pumice
stones, ceramic rings, ceramic balls, polyurethane foam, Vukopor® S10,
glass bead, glass rushing rings, stainless steel wire, keramsite, and etc.)
and entrapment (calcium alginate beads, polyvinyl alcohol hydrogel,
and etc.). In fact, continuous cultivation setup is more complicated and
hard to maintain than other strategies; thence, the future research di­
rection should include the study of continuous operation by creating a
fluidized bed immobilized bioreactor prototype.
Fig. 8 highlights the top-performing strains at respective anaerobic
fermentation mode based on the evaluation of both yield and produc­
tivity [15,19,49,59,100,112]. The strains selected (except sequential
fed-batch) have both equally high yield and productivity when
compared with the other strains at their respective mode of cultivation.
Batch fermentation for K. pneumoniae GLC29 and fed-batch cultivation
using C. pasteurianum K1 are found to be suitable for obtaining high
yield and productivity in respective cases. Interestingly, C. butyricum
strains perform well in continuous, repeated batch, repeated fed-batch,
and sequential fed-batch cultivation. Based on Fig. 8 and Fig. 6,
continuous fermentation obviously stands out among the other culti­
vation methods in terms of yield and productivity; hence, this mode is
promising for future industrial microbiology to produce a high added
value of 1,3-PDO. In short, repeated fed-batch, sequential fed-batch, and
continuous fermentation should be studied intensively in the future to
make further improvements in order to meet the requirement of in­
dustrial application.
5. Aeration condition
Despite the significance of fermentation operation mode, aeration
condition is another key factor influencing the 1,3-PDO producers’
performance. Some microbes can metabolize substrate aerobically
whereas most bacteria can assimilate carbon sources anaerobically. In
other words, oxidative stress induced by the presence of oxygen in the
air would hinder the performance of certain microorganisms in forming
1,3-PDO but some of the microbes will ferment glycerol efficiently under
well-aerated conditions. Since 1,3-PDO is a metabolite of the reductive
pathway, control of the aeration condition is a theoretical technique to
activate the 1,3-PDO biosynthesis. To ensure the feasibility of 1,3-PDO
industrial production, aeration condition ought to be one of the pa­
rameters to be studied and optimized for amelioration in product yield
and productivity.
5.1. Strict anaerobe
The biosynthetic process of 1,3-PDO from glycerol is usually per­
formed anaerobically without any other exogenous reducing equivalent
acceptors. In common, the microorganisms which able to consume
carbon sources and further break down into 1,3-PDO are comprised of
facultative anaerobes, aerotolerant anaerobes and strict anaerobes; they
express a functional anaerobic respiratory chain that supports their
anaerobic tolerance growth under hypoxia [4]. Members of the strain
Clostridium are well known for their strictly anaerobic identity where
oxygen exposure is fatal to them as the GDHt is extremely oxygen sen­
sitive. The anaerobic environment in the fermentation vessel is created
either by sparging gas (N2, CO2, or gas mixture) or through a
self-generated anaerobiosis regime [2,3,9107,112]. The latter is ach­
ieved by the continuous production of CO2 and potentially H2 from the
native strain’s microbial metabolism, particularly the decarboxylation
of pyruvic acid [9].
In a study by Chatzifragkou and the team, the sole utilization of
fermentation effluent gas to create an anaerobic environment had found
to reduce the 1,3-PDO concentration produced by C. butyricum VPI 1718
from 70.8 g/L (use of N2 infusion strategy) to 30.5 g/L. The phenome­
non is possibly ascribed to the insufficient self-generated anaerobiosis
environment which has caused the partial blockage of phosphoroclastic
reaction, eventually leading to the increase in lactate as the main
byproduct instead of 1,3-PDO [9]. Likewise, in the study of Wang et al.
[112], the direct use of fermentation exhaust gas (CO2 and H2) in the
Fig. 8. Best performing strains at different operation mode.
10
K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
prior bioreactor as aeration gas for the next fed-batch fermentation had
ended up with increased 1,3-PDO productivity yet lower 1,3-PDO yield
and higher concentration of butyric acid. In the same study, the
employment of H2 to create anaerobic environment has resulted in
similar productivity and yield of 1,3-PDO as N2 gassing strategy.
Nonetheless, the use of CO2 itself as aerating gas had led to decrease in
the concentration and yield of 1,3-PDO as well as had shifted the
metabolism to butyric acid when compared to the adoption of N2 gas. In
this regard, most of the anaerobic biotransformation of glycerol by
Clostridium spp. is carried out in the presence of a high flow of N2
throughout the fermentation to ensure strict anaerobiosis. In an exper­
iment carried out by Chatzifragkou et al., N2 continual sparging enabled
an increase of 1,3-PDO yield and biomass formation which was associ­
ated with a decreased production of lactic acid [9]. Under anaerobic
conditions, GDHt is expressed for the 3-HPA formation but it becomes
inactive during oxygen exposure [46]. For that reason, a strict anaerobic
situation should be established for Clostridium spp. to produce fermen­
tative 1,3-PDO from glycerol.
However, there is an interesting phenomenon observed by Katarzyna
and the team. Clostridium bifermentans strain 535 isolated from the forest
soil has high redox potentials, both in anaerobic and microaerobic
conditions (95% CO2 and 5% O2); this strain fermented glycerol and
produced a higher 1,3-PDO concentration in the presence of oxygen
rather than in anaerobic condition. It is noteworthy that this strain
shows the possibility of application of microaerophilic conditions, which
is profitable from an industrial point of view [50].
still promising in fermenting glycerol without any gas supply which adds
merit to the industrial biotechnological application.
Klebsiella spp. can ferment glycerol under different aeration condi­
tions (aerobic, microaerobic, and anaerobic). Yet, the diverse species of
K. pneumoniae give optimum results at different aeration strategies as
shown in Fig. 9. This is in agreement with the statement where the
expression of the enzyme (GDH, GDHt, and PDOR) was reported with
their respective specific activities under different aeration conditions
[114]. Oxygen, as an exogenous electron acceptor helps to improve cell
growth and 1,3-PDO production. Too little or too much oxygen supply
may be detrimental to the glycerol metabolism of the Klebsiella genera to
synthesize 1,3-PDO. In a study by Rossi et al. [89], K. pneumoniae BLh-1
was found to be able to convert glycerol to 1,3-PDO under the aerobic
condition of 1.0 vvm of air. Besides, K. pneumoniae HSL4 was the first
reported wild strain isolated from a marine environment that led to high
PDO concentration (80.08 g/L), yield (0.53 mol/mol), and productivity
(2.22 g/L.h) under aerobic fed-batch fermentation (0.5 vvm air) using
glycerol as the sole carbon source [129].
Several studies apply microaerobic with or without air sparging on
the glycerol fermentation by Klebsiella species. Maintaining micro­
aerobic conditions by supplying 0.04 vvm of air for the degradation of
glycerol to 1,3-PDO using K. pneumoniae CGMCC 2028 had successfully
produced 1,3-PDO at yield and productivity of 0.41 mol/mol and
4.04 g/L.h, respectively [114]. The mentioned phenomenon is
explained by the enhancement of specific activities of enzymes in the
dha operon. The four enzymes located on the dha system are DHAK
(dhaK), GDH (dhaD), GDHt (dhaB), and PDOR (dhaT) where the last
three enzymes’ activities are not suppressed but promoted in the
microaerobic fermentation. In the same study, the microaerobic condi­
tion without air sparging throughout the fermentation was employed for
K. pneumoniae SU6 [91] and K. pneumoniae G31 [84], both resulting in
0.41 mol/mol of 1,3-PDO. In contrast, another study discovered a low
production of 1,3-PDO by K. pneumoniae KKU5 at slightly aerated con­
dition is possibly ascribed to the presence of oxygen [98]. Nonetheless, a
critical level of aeration rate in microaerobic cultivation will ultimately
help to increase the expression of enzymes in dha operon for facultative
anaerobes, according to the literature [114]. Furthermore, Klebsiella
spp. can metabolize glycerol anaerobically, which resulted in a higher 1,
3-PDO yield than the other two conditions as indicated in Fig. 9. The
phenomenon is possibly attributed to the shift of carbon flux to the
oxidative pathway rather than the reductive pathway in the presence of
oxygen supply [114]. Anaerobic conditions established at the initial 2 h
of cultivation by N2 flow enabled a 1,3-PDO yield of 0.73 mol/mol by
K. pneumoniae ATCC 8724 [121]. Also, the glycerol metabolism by
K. pneumoniae LX3 able to reach 0.61 mol/mol of 1,3-PDO yield by
continuous N2 gassing at 0.4 vvm [120]. As displayed in Fig. 9, the yield
of 1,3-PDO under anaerobic conditions, in general, can reach a higher
value than in the other two aeration conditions. For instance, the yield of
K. pneumoniae BLh-1 obtained under aerobic cultivation (0.18 mol/mol)
is much lower than during anaerobic fermentation (0.41 mol/mol),
elucidating anaerobic condition is more suitable for the strain [89]. A
different phenomenon was observed in glycerol fermentation by
K. oxytoca FMCC-197 whereby continual N2 infusion induce a shift of
glycerol metabolism to ethanol as the major product whereas the
absence of N2 sparging led to the production of 1,3-PDO as the principal
metabolite [75]. Thus, the anaerobic condition created by the meta­
bolism gas product (CO2 and H2) coupled with the low-cost of glycerol as
the substrate might become the selling point of the facultative microbes
for the 1,3-PDO industrial production.
Aerobic and microaerobic fermentation are remarkable in elimi­
nating the large volume of pure N2 gas consumption which largely
reduce the operating expenditure in commercial production, provided
the microbes able to perform well under either of these two conditions.
As a matter of fact, anaerobic facultative bacterial strains are usually
life-threatening pathogens albeit there is cost-saving advantage in terms
of the use of air or relatively inexpensive gas mixture supply. Even
5.2. Aerotolerant anaerobe
Besides, Lactobacillus spp. is an aerotolerant anaerobe that is
insensitive to oxygen disclosure but without the ability to utilize oxygen.
Therefore, the microbes can grow evenly in the medium because of their
ability to detoxify toxic forms of oxygen. Typically, anaerobic condition
favors the production of 1,3-PDO from glycerol metabolism by Lacto­
bacillus strain. For instance, a fed-batch production of 1,3-PDO from
glycerol using resting cells of L. reuteri was performed anaerobically via
continuous N2 bubbling [18]. Since Lactobacillus spp. manage to survive
in the oxygen-containing environment, the effect of air on the strain
performance can be investigated. In an experiment conducted by Zabed
et al. [124], the performance of L. reuteri FXZ014 was investigated
under three aeration conditions; the microaerobic condition yields the
highest 3-HP concentration whilst the anaerobic environment favors
maximum 1,3-PDO production. In short, the air tolerance ability helps
them to survive which is beneficial in industrial-scale synthesis.
5.3. Facultative anaerobe
In contrast to obligate anaerobes such as Clostridium spp. and
Halanaerobium spp., facultative anaerobes (Citrobacter spp., Entero­
bacter sp., Hafnia sp., Klebsiella spp., Kluyvera sp., Pantoea sp., Shim­
wellia sp.) have the unique abilities to grow with or without oxygen.
These bacteria can adapt well to the changing environment. This means
these microbes are aerotolerant, anaerotolerant and able to consume O2
[4]. Thence, the growth of bacteria distributes evenly in the media but
the majority is at the oxygen-rich surface. The researchers studied the
effect of aeration strategies on the glycerol fermentation by Shimwellia
blattae in which the results show insignificant improvement in the yield
and productivity when supplying high N2 flow, 0.04 vvm (volume per
volume per minute) or 0.08 vvm of air to the bioprocess [87]. Albeit the
additional air supply or gas input have little improvement effect on 1,
3-PDO production, this strain is still promising in terms of the ability
to adapt to various aeration condition. Likewise, the 1,3-PDO produc­
tion capability of newly isolated C. freundii AD119 and H. alvei AD27
were tested under a condition with neither air supply nor N2 sparging
[20]. Although the performance of the aforementioned biocatalysts can
be further improved by optimizing the fermentation condition, they are
11
K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
Fig. 9. Yield of K. pneumoniae strains under different aeration condition.
though K. pneumoniae strains give excellent results in 1,3-PDO produc­
tion, they (except K. pneumoniae BLh-1) are opportunistic pathogens
which would expose biosafety issues, thus restricting the industrial
application. Overall, the anaerobic condition is the best aeration
circumstance as it does not require high-power agitation and long-term
aeration [40].
the research papers published from 2010–2022, as listed in Table 4. The
ability of microorganisms to consume crude glycerol and convert it to
valuable chemicals especially 1,3-PDO effectively is pivotal in the way
to combat the glycerol glut problem nowadays. Section A in Fig. 10
presents a list of microbes which capable of fermenting glycerol to 1,3PDO whilst section B displays the strains with the capability to with­
stand high initial glycerol concentration which is equal to or more than
60 g/L in the fermentation medium. The bacterial strains listed inside
the intersection of the Venn diagram can both tolerate the crude glycerol
as well as the high initial concentration in synthesizing fermentative 1,3PDO, which can be further exploited for the large-scale production
process.
6. Substrate tolerance
In realizing the commercial production of 1,3-PDO via the green
biotechnological method, the constraints encountered such as the uti­
lization of crude glycerol as a carbon source and the inhibition caused by
the high initial substrate concentration ought to be resolved. The Venn
diagram as demonstrated in Fig. 10 summarizes the tolerance of strains
towards crude glycerol and/or initial glycerol concentration based on
Fig. 10. Tolerance of strains towards crude glycerol and/or high initial glycerol concentration. Data are available from Table 4.
12
K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
Table 4
Tolerance of 1,3-PDO-producing strains towards crude glycerol and/or high initial glycerol concentration.
Bacterial strain
Substrate
Operation
mode
C. beijerinckii NRRL B-593
(immobilized)
C. butyricum DSM 5431
(immobilized)
C. butyricum AKR102a
C. butyricum NRRL B-23495
Crude glycerol
Continuous
Glycerol
Crude glycerol
81% w/w crude
glycerol
81% w/w crude
glycerol
85.6% w/w
crude glycerol
0.885 g/g crude
glycerol
Crude glycerol
Raw glycerol
C. butyricum VPI 1718
C. butyricum DSP1
C. butyricum DSM 10702
C. butyricum NCIMB 8082
C. butyricum H304
C. butyricum JKT37
C. butyricum DSM 4278
(immobilized)
C. butyricum DL07
C. butyricum SCUT343–4
C. diolis DSM 15410
C. freundii DSM 15979
(immobilized)
C. freundii ATCC 8090
C. freundii FMCC-8
C. freundii FMCC-B 294 VK-19
C. pasteurianum DSMZ 525
C. perfringens GYL
H. saccharolyticum DSM 6643
K. oxytoca FMCC-197
K. oxytoca MIG 01
K. pneumoniae DSM 4799
K. pneumoniae SU6
K. pneumoniae HE-2 (immobilized)
K. pneumoniae HE1
K. pneumoniae GenBank no.
27FHM063413 (immobilized)
Klebsiella sp. Ana-WS5
K. pneumoniae BLh-1 (immobilized)
K. pneumoniae GLC29
K. pneumoniae ATCC 8724
K. pneumoniae L17
K. cryocrescens NBRC 102467
P. agglomerans DSM 30077
(immobilized)
S. blattae ATCC 33430
75.40% w/w
crude glycerol
Crude glycerol
78% crude
glycerol
95% glycerol
Crude glycerol
54.35% w/v
crude glycerol
80% crude
glycerol
79.4% w/w
Crude glycerol
79.4% w/w
Crude glycerol
Pure glycerol
Crude glycerol
56% m/v crude
glycerol
Crude glycerol
Glycerol
80% w/w crude
glycerol
27% crude
glycerol
Crude glycerol
Glycerol
Crude glycerol
Glycerol
81.8% w/w
crude glycerol
Biodiesel
glycerol
Crude glycerol
Crude glycerol
79.3% w/v crude
glycerol
54.35% w/w
crude glycerol
65.8% w/w
crude glycerol
1,3-PDO
titer (g/L)
Yield
(mol/
mol)
Productivity (g/
L.h)
Reference
45.00
18.00
0.66
6.40
Gonen et al.,[32]
Continuous
60.00
33.80
0.70
16.9
Suratago, Nootong[100]
Fed-batch
Batch
20.00
55.00
61.50
32.30
0.64
0.71
5.00
N/A
Wilkens et al.,[115]
Metsoviti et al.,[74]
Continuous
80.00
19.3
0.54
N/A
Chatzifragkou et al.,[8]
Repeated
batch
Fed-batch
100.00
62.00
0.53
1.68
70.00
36.10
0.59
0.72
SzymanowskaPowalowska[102]
Loureiro-Pinto et al.,[66]
Batch
Repeated
batch
Batch
60.00
86.34
32.18
42.39
0.63
0.59
2.38
2.14
Martins et al.,[72]
Zhang et al.,[126]
20.80
10.65
0.62
1.33
Tan et al.,[106]
Repeated
batch
Fed-batch
40
12.60
0.42
3.50
Dolejš et al.,[19]
40
94.20
0.63
3.04
Wang et al.,[112]
Batch
Batch
Continuous
80
54.15
92
42.80
26.00
26.1
0.65
0.61
0.56
1.78
0.72
2.47
Lan et al.,[59]
Kaur et al.,[52]
[36]
Batch
16
4.35
0.28
N/A
Fed batch
20
37.70
0.67
0.69
(Ferreira, T.F. et al.,
2012)
Maina et al.,[70]
Batch
71.8
31.30
0.54
1.09
Maina et al.,[70]
53.70
39.30
2.40
N/A
0.58
0.50
0.90
2.67
N/A
Groeger et al.,[33]
Guo et al.,[40]
Kivistö et al.,[55]
Continuous
Batch
Batch
Initial substrate
concentration (g/L)
80
100
5
Fed-batch
Batch
Fed-batch
40
75.6
40
50.10
15.85
71.10
0.40
0.49
0.67
0.90
N/A
1.51
Metsoviti et al.,[75]
Rodriguez et al.,[88]
Jun et al.,[48]
Fed-batch
60
45.35
0.36
1.94
Batch
Batch
Continuous
50
70
40
10.50
N/A
2.50
N/A
0.37
N/A
N/A
0.53
4.80
Sattayasamitsathit et al.,
[91]
Wong et al.,[117]
Wu et al.,[119]
Gungormusler et al.,[39]
Batch
Repeated
70
65
18.40
24.99
N/A
0.58
0.85
6.29
Yen et al.,[123]
De Souza et al.,[17]
Batch
49
27.60
0.43
2.30
Neto et al.,[80]
Fed-batch
Batch
Batch
40
9.21
20
62.72
2.48
5.28
0.73
N/A
N/A
1.74
N/A
0.28
Yang et al.,[121]
Kong et al.,[56]
Loh, Abel[64]
Continuous
40
N/A
N/A
3.6
Casali et al.,[6]
Batch
30
13.84
0.53
1.15
Rodriguez et al.,[87]
6.1. Crude glycerol
the cost incurred for feedstock. The source of substrate will have a
substantial influence on the 1,3-PDO production, corresponding to how
well the applied strain can fully consume the substrate without any in­
hibition effects. As mentioned in the earlier section, pure glycerol and
crude glycerol differs from each other in terms of glycerol content and
impurities. A few studies investigate the relationship between crude
glycerol as the feedstock and the 1,3-PDO-producing bacteria. Crude
glycerol originating from the biodiesel industry will contain a high di­
versity of impurities, such as residual methanol, fatty acid methyl/ethyl
ester, fatty acids (e.g., stearic acid and oleic acid), heavy metal ions or
A paradigm shifts in fuels and chemicals toward renewable energy
and material has drastically elevated biodiesel production and subse­
quently, this has caused a huge accumulation of raw glycerol. A great
deal of research effort has been devoted to the utilization of low-quality
glycerol as a substrate in microbial conversion since the last decade. The
main cost driver of the microbial fermentation to produce 1,3-PDO is the
raw material, which accounts for more than 50% of the total expenses
[115]; thence, 1,3-PDO yield becomes the parameter to aid in evaluating
13
K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
salts, soaps, and matter organic non-glycerol (MONG) [60,78,118].
Theoretically speaking, the impurities content in crude glycerol will act
as the inhibitory agent that affect the morphology and metabolism of the
bacteria; thence, certain pretreatment is necessary prior to fermentation.
Impurities present in crude glycerol had shown a noteworthy impact on
the yield and productivity of 1,3-PDO; however, there was no substantial
effect on the cell growth of K. pneumoniae DSMZ 2026, particularly the
viability and biomass [60]. Gram-positive bacteria such as Clostridium
spp. are generally more susceptible to crude glycerol than gram-negative
microbe like Klebsiella spp. [10,78]. In this regard, the raw glycerol was
pretreated with acid to get rid of soap and free fatty acids for
C. butyricum strains consumption which ended up in an improved 1,
3-PDO concentration [78]. Also, pretreatment of biodiesel-derived
crude glycerol using ion exchange resins was carried out to remove
impurities; the pretreated glycerol fermentation by C. butyricum DSM
10702 had resulted in the improvement of 1,3-PDO productivity from
0.52 g/L.h to 0.99 g/L.h [66]. The use of pretreated glycerol by
C. butyricum AKR102a [115] and C. butyricum JKT 37 [106] had led to
notable performance increases compared to crude glycerol.
Due to discrepancies in the tolerance of microbial strains to crude
glycerol impurities, the findings of several works of literatures are
controversial. The researchers found out that K. pneumoniae DSM 4799
utilizes raw glycerol more effectively than pure glycerol, through the
evidences of higher 1,3-PDO concentration and productivity obtained by
raw glycerol [48]. Besides, C. butyricum VPI 1718 possesses the adapt­
ability to high concentrations of NaCl, presence of stearic acid and
methanol, indicating a wide spectrum of raw glycerol is possible to be
used for this specific strain [10]. A similar finding was discovered by
researchers where C. butyricum B-23495 is promising in consuming
55 g/L of 81% w/w crude glycerol under batch anaerobiosis and even­
tually manage to produce 32.3 g/L of PDO with a yield of 0.71 mol/mol
[74]. This interesting finding indicates that the impurities present in raw
glycerol have insignificant adverse effects upon bacterial glycerol
fermentation. Rodriguez and coworkers discovered that the substrate
uptake rate, biomass growth, and final 1,3-PDO concentration of raw
glycerol fermentation by Shimwellia blattae ATCC 33430 are higher than
that obtained in pure glycerol degradation; this scenario is possibly
ascribed to the phosphates and salts present in crude glycerol which
create a pH buffer for fermentation medium in the early stages, and
subsequently improve the biocatalyst’s fermentation performance [87].
Similarly, the presence of few impurities in crude glycerol has an
insignificant inhibitory effect on the fermentation by K. oxytoca
FMCC-197 [75], C. perfringens GYL [40], and C. butyricum DL07 [112] as
well. Fig. 10 displays some examples of strains which can utilize crude
glycerol directly for 1,3-PDO synthesis. In brief, since the oil types,
catalysts, alcohols, and specialized biodiesel technologies for respective
manufacturers vary, the effects of crude glycerol’s impurities on 1,
3-PDO production performance cannot be ruled out and should be
emphasized for feasible industrial biotechnological application.
concentration of glycerol was applied [35]. The negative effect of high
initial glycerol concentration or high concentration feeding is often
associated with the significant residual glycerol concentration at the end
of fermentation. Consequently, the cell metabolism and substrate
diffusion in the fermentation medium would be greatly affected [121].
Also, the high viscosity of the system resulting from the high concen­
tration of residual glycerol will act as a physical barrier to mass transfer.
No amelioration of 1,3-PDO production at high initial glycerol concen­
tration indicates the channel of carbon flow toward the formation of
organic acids instead of biomass and 1,3-PDO [59].
Based on the literature, the typical initial substrate concentration is
in between 20 and 60 g/L [76,87,100,106,118]. Several studies on the
effect of initial glycerol concentration reveal that the maximum initial
glycerol concentration is recommended at 60 g/L to maintain microbial
activity and prevent impediment of 1,3-PDO production [100,123].
Very high glycerol concentration will lead to incomplete substrate
consumption by microbes, for example, K. pneumoniae CGMCC 2028
[114] and C. freundii FMCC-B 294 (VK-19) [76], at the same time, excess
substrate is inhibitive to cell growth or might enhance lactic acid pro­
duction. This means the shift of the pathway from acetic acid (formation
of ATP) to ethanol and lactic acid will increase the competition for
NADH2 with 1,3-PDO, resulting in a low titer and conversion yield of the
targeted product [114]. In general, a higher substrate concentration will
lead to a greater amount of byproducts ultimately [104]. Loureiro-Pinto
et al. observed that the maximum 1,3-PDO titer synthesized by
C. butyricum DSM 10702 corresponded to 70 g/ L of crude glycerol
where more than 100 g/L of glycerol concentration gave a marked
inhibitory effect on the fermentation [66]. As such, a high initial sub­
strate concentration does not favor efficient 1,3-PDO production due to
the suicidal inactivation of glycerol dehydratase (GDHt) of Clostridium
spp. in the reductive metabolic pathway [41].
Still, the tolerance toward initial glycerol concentration differs
among the strains. There are quite a few microorganisms manage to
endure high initial glycerol concentration of above 60 g/L, as listed in
Fig. 10. For instance, 140 g/L of glycerol was converted to 67 g/L of 1,3PDO in batch fermentation by C. butyricum DSP1, which indicates
C. butyricum DSP1 has a high tolerance of osmotic pressure at high
glycerol concentration [104]. C. perfringens GYL had been tested on the
tolerance of different glycerol concentrations; it is a powerful strain
which able to grow at initial pure glycerol concentrations of 200 g/L and
120 g/L of crude glycerol [40]. Similarly, 115 g/L of glycerol was used
by C. butyricum SCUT343–4 from an initial glycerol concentration of
140 g/L for glycerol metabolism [59]. Besides, for the case of Klebsiella
spp., a high glycerol concentration can encourage the 1,3-PDO forma­
tion by limiting the channel of carbon flux into glycolysis which
generate pyruvate-derived byproducts such as ethanol [89]. Yet,
140 g/L is the critical concentration that causes weak 1,3-PDO pro­
duction, reduced number of bacterial cells, and weaken cells’ metabolic
activity [104]. The biodiesel manufacturing process and its raw mate­
rials will cause a different composition of crude glycerol, thus, the extent
of tolerance of initial substrate concentration might differ among 1,
3-PDO-producing strains. Nonetheless, higher substrate concentrations
are essential to improve production efficiency. To enable the effective
use of high glycerol concentration by microbes, other stress factors like
toxic metabolites in fermentation should be taken into consideration.
6.2. Initial glycerol concentration
Glycerol concentration is regarded as an imperative limiting factor in
fermentation. The concentration of glycerol will directly affect the
metabolic flux distribution and induce osmotic pressure inside the cell
that to a certain extent, can cause damage to the microorganisms in the
event of the excessive residual substrate [103]. According to the liter­
ature, both lower and higher glycerol concentration will eventually
decrease 1,3-PDO productivity. At low or high glycerol concentrations,
glycerol passes the membrane by glycerol uptake facilitator protein
(glpF) or via passive diffusion, respectively [80]. In the case of low
substrate concentration, the transport of itself across the membrane is
apparent until little glycerol remains for bioconversion [15]. In a pro­
teomic study of C. butyricum strain, the expression levels of enzymes in
both oxidative and reductive were found reduced in addition to lower
cell growth rate and substrate consumption rate when an elevated
7. Formation of 1,3-propanediol and other metabolites
A wide range of metabolites is produced by indigenous bacteria with
the ability to metabolize carbon sources. Table 5 summarizes the
possible metabolites generated via microbial glycerol fermentation
which was investigated in several research papers and the bold com­
pounds are normally found in huge amounts. Even though a high 1,3PDO concentration is desired at the end of fermentation, concentrated
1,3-PDO accumulated in the broth will indeed inhibit cell growth and
1,3-PDO production as well [120]. 1,3-PDO is more inhibitory than
14
K.Y. Tey et al.
Journal of Environmental Chemical Engineering 11 (2023) 110998
Subsequently, a large amount of energy is used to remove hydrogen ions
from the cell via proton pumps [102]. Other than acetic acid and butyric
acid, production of lactic acid by Clostridium spp. is possibly due to some
blockage of the usual fermentation pathways, lack of iron for further
break-down of pyruvate, carbon monoxide gassing, the substrate in
excess or insufficient anaerobiosis condition [9,66]. Consequently, the
abundant organic acids contained in fermentation broth will decrease
culture pH rapidly and thus causing inhibition of cell growth and 1,
3-PDO metabolic pathways in those organic acids-producing strains.
To be clear, the premature cessation of those organic acid-producing
strains’ growth, as well as incomplete substrate consumption will
occur because of the culture acidification. Thus, switching pH control
appears as an effective strategy to obtain the target products [121].
Typically, the terrestrial strains were reported to have decreased 1,
3-PDO production owing to the toxic effects caused by the high quan­
tity of byproducts such as acetic acid and lactic acid in the culture broth.
Conversely, K. pneumoniae HSL4 isolated from mangrove sediment is a
salt-tolerant strain that also partly resistant to the salt of acetic acid and
lactic acid (acetate and lactate) up to 6.0 g/L and 9.0 g/L, respectively
[129].
The other byproducts formed in fermentation broth will bring sig­
nificant impacts to the 1,3-PDO production. The mixture of organic acids
and alcohol in fermentation broth will exhibit an extremely strong in­
hibition to cell growth. The flux from glycerol to 1,3-PDO for
K. pneumoniae CGMCC 2028 will be reduced when a greater amount of
2,3-BDO and lactic acid is formed, as these metabolites consume NADH2
[114]. Furthermore, a lower ethanol formation indicates more reducing
power of NADH available for 1,3-PDO production [120]. As such, the 1,
3-PDO production will compete with the formation of all byproducts
that possibly produced during fermentation, which leads to a reduced 1,
3-PDO yield. These evidences demonstrate that the formation of
byproducts should be minimized to ensure a higher yield of 1,3-PDO.
Thence, Clostridium spp. is a promising cell factory in terms of lesser
byproducts formed at the end of fermentation with appreciable yield and
productivity. The coproduction of byproducts with 1,3-PDO will
compromise the yield of 1,3-PDO and increase the complexity of product
purification. The tolerance to 1,3-PDO, organic acid, and salt will assist
the development of large-scale fermentation.
Table 5
Metabolites produced by species of Citrobacter, Clostridium, and Klebsiella.
Species
Citrobacter spp.
Clostridium spp.
Klebsiella spp.
Byproducts
1,3-PDO
Acetic acid
Ethanol
Formic acid
Lactic acid
Succinic acid
1,3-PDO
2,3-BDO
Acetic acid
Citric acid
Ethanol
Formic acid
Lactic acid
Succinic acid
H2
References
[2,20]
1,3-PDO
2,3-BDO (only C. beijerinckii)
Acetic acid
Butyric acid
Ethanol
Formic acid
Lactic acid
n-butanol
Succinic acid
H2
CO2
[32,35,49,50]
[60,114,119]
glycerol as the former will affect PDOR negatively and eventually cause
an accumulation of 3-HPA, an anti-microbial agent that is strongly toxic
to cells [41,59]. Nonetheless, C. butyricum VPI 1718 was found to have
excellent resistance to a high 1,3-PDO concentration of more than
70 g/L, indicating the strain is superior in adapting to the culture
environment [8]. From Guo et al. [40] investigation, C. perfringens GYL
can tolerate 1,3-PDO production up to roughly 70 g/L. Yet, the high
osmotic pressure of concentrated 1,3-PDO which lead to increased
cellular fluidity will damage the bacterial cell and even worst will cause
cell death. In this regard, the removal of fermentation broth by interval
or continuously might help to lessen the effects of 1,3-PDO concentra­
tion towards strain performance as the concentration are being diluted.
Apart from that, the increased concentration of the other metabolite
will induce toxic stress that has noteworthy influences on 1,3-PDO
production [103]. Microbial glycerol metabolism will yield a number
of cometabolites such as 2,3-BDO, acetic acid, butyric acid, citric acid,
ethanol, formic acid, lactic acid, n-butanol, succinic acid, H2, CO2 which
varies accordingly based on the bacterial species. Almost all of the 1,
3-PDO producers produce acetic acid, ethanol, formic acid, lactic acid,
and succinic acid, according to Table 5. Only Enterobacteriaceae (and
Clostridium beijerinckii) will produce 2,3-BDO whereas butyric acid and
n-butanol are merely produced by Clostridium [40]. Kluyvera cry­
ocrescens was found to produce n-butanol and acetone alongside 1,
3-PDO [64]. Typically, acetic and butyric acids are the usual
end-fermentation byproducts produced by almost all members of Clos­
tridium as they possess the B12-independent GDHt. C. freundii and H. alvei
mainly produce organic acids (e.g., acetic acid, lactic acid) with 1,
3-PDO; K. pneumoniae produce mostly alcohols (e.g., 2,3-BDO,
ethanol) as coproducts [20]. Besides, fermentation effluent gases like
CO2 and H2 are detectable in the fermentation of some species of
Clostridium.
The yield of the desired principal product greatly relies on the
combination and stoichiometry of oxidative and reductive pathways
[50]. The 1,3-PDO production greatly relies on the reducing power
generated from pyruvate oxidation in the metabolic pathway. A greater
yield of 1,3-PDO can be achieved when it is produced along with acetic
acid as the sole metabolite in the oxidative pathway. As a matter of fact,
1,3-PDO and acetic acid formation are cell growth-associated [120]. In
the case of Klebsiella spp. and Clostridium spp., as each acetic acid
molecule is formed from the pyruvate pathway, an additional ATP is
formed at the same time, available for biomass synthesis. On the other
hand, more energy generation (ATP) and biomass are produced from
butyric acid formation than acetic acid for the fermentation using
Clostridium spp. as the 1,3-PDO producing strain [66,125]. Nonetheless,
it should be noted that in the case of Clostridium spp., the production of
other metabolites in glycerol oxidative pathways (except acetic acid)
will compete with 1,3-PDO synthesis because all of them are
NADH2-needing.
On the contrary, the weak acids present in the culture will undeni­
ably increase the concentration of hydrogen ions around the cells and
inside the cells after extracellular and intracellular dissociation.
8. Conclusion
Biodiesel-derived crude glycerol has emerged as the abundant and
essentially low-prized feedstock for the environmentally benign micro­
bial conversion to value-added products; the present scenario coupled
with the existence of numerous microorganisms born with the capability
of fermenting glycerol into 1,3-PDO making this bioproduction an
attractive option for further research and development. This biotech­
nology is anticipated to resolve waste management problems and at the
same time, fully replace the conventional energy intensive chemical
synthesis process of 1,3-PDO. A variety of native bacterial strains were
investigated on their glycerol fermentabilities to produce 1,3-PDO under
different modes of cultivation; continuous fermentation possesses
certain robustness over the other operational processes in terms of yield
and productivity, while fed-batch enables high titer. Among the
different oxygen gaseous requirement, glycerol fermentation under
anaerobic condition is desirable to ensure the best performance of strict
anaerobes which able to give high product yield and productivity. Be­
sides, the very osmotolerant microorganisms with insignificant response
towards high initial substrate and impurities in the crude glycerol are
desirable for future industrial 1,3-PDO production. The formation of
byproducts (alcohols, organic acids, and effluent gases) is inevitable
during the fermentation process but should be minimized to ensure the
maximum yield of 1,3-PDO. In summary, industrially useful microbes
should be identified to study the fermentation feasibility in order to
pursue the vision of a sustainable circular bioeconomy, opening new
doors for new business opportunities, especially in Europe and Southeast
15
Journal of Environmental Chemical Engineering 11 (2023) 110998
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9. Future prospective
Despite the significant results obtained in 1,3-PDO synthesis from
glycerol fermentation, substantial research efforts are still required to
effectively commercialize the microbial fermentation process of this
high value chemical derivative. Since there are numerous bacterial
strains which have been tested with their glycerol-fermenting ability on
a lab-scale fermentation, the future study should move forward to the
scaling-up of microbial 1,3-production using those high-performing
microorganisms. There are limited studies on the production of micro­
bial 1,3-PDO from glycerol fermentation in larger bioreactors or fer­
menters under continuous operation. By doing so, a more realistic
performance of certain microorganisms for fermenting glycerol can be
assessed, and then further move on to industrial scale of 1,3-PDO pro­
duction. Besides, bacterial immobilization should be explored further to
break through the bottleneck of productivity by increasing the cell
concentration and its tolerance towards the fluctuating environment
conditions. Techno-economic analysis should be derived to provide in­
sights on the feasibility of such biotechnologies in multiple aspects.
Besides, future studies should focus on cost-cutting solutions in the in­
dustrial context. For instance, the high price of N2 sparging gas and the
difficulty in maintaining large-scale strict anaerobic conditions have
always been the bottlenecks for commercialization. The microaerobic
study (oxidoreductive potential/redox potential) on strict anaerobic
strain had been conducted; thus, it is suggested to carry out a related
study to either explore new strains with such capability or discover the
potential of currently available microbes. The direct utilization of crude
glycerol is a huge advantage in the aspect of industrial capital costs;
however, the impurities content might influence the performance of
strain as well as the downstream processing. As such, a simple yet
effective pre-treatment process of crude glycerol should be developed to
overcome the aforementioned limitation.
Declaration of Competing Interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
Tan Jian Ping reports financial support was provided by Ministry of
Science, Technology and Innovation Malaysia (MOSTI). Tey Ker Yee
reports financial support was provided by Malaysia Toray Science
Foundation (MTSF). Tan Jian Ping reports financial support was pro­
vided by Xiamen University - Malaysia.
Data Availability
No data was used for the research described in the article.
Acknowledgement
This material is based upon work supported by the Ministry of Sci­
ence, Technology and Innovation Malaysia (MOSTI) under Technology
Development Fund 1 (TeD 1), [project number TDF07211418];
Malaysia Toray Science Foundation (MTSF) [grant number
220527STRG0160]; Xiamen University Malaysia Research Fund
(XMUMRF) [project number XMUMRF/2022-C10/IENG/0047].
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