Bioresource Technology 323 (2021) 124566

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Recent developments on solid-state fermentation for production of

microbial secondary metabolites: Challenges and solutions
Vinod Kumar a, 1, Vivek Ahluwalia b, 1, Saurabh Saran a, Jitendra Kumar b, Anil Kumar Patel c,
Reeta Rani Singhania c, *
a
Fermentation Technology Division, Indian Institute of Integrative Medicine, Post Bag No. 3, Canal Road, Jammu-180001, India
b
Institute of Pesticide Formulation Technology, Gurugram, Haryana 122 016, India
c
Centre for Energy and Environmental Sustainability, Lucknow 226 029, India

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• SSF emerged as a promising bioprocess

for microbial secondary metabolites
production.
• Certain SMs are produced in SSF with
impressive yield compared to SmF.
• Novel bioreactor designing is major
requisite for large scale application of
SSF.
• Scale-up, heat dissipation and mass
transfer are major challenges of SSF
processing.

A R T I C L E I N F O A B S T R A C T

Keywords: Microbial secondary metabolites (SMs) are the intermediate or the product of metabolism produced during
Solid-state fermentation fermentation process. SMs are produced during stationary phase and play a major role in competition, antago­
Secondary metabolites nism and self defence mechanisms. These metabolites finds application in the pharmaceuticals, food, cosmetics
Antibiotics
etc. These are produced besides primary key metabolites (e.g., amino acids, lipids, carbohydrates etc.). Growth
Bioreactor
Agricultural residues
condition in solid-state fermentation (SSF) resembles microorganism’s own native environment allowing the
microorganisms to adapt best. Recent developments in bioprocessing has identified specific SSF practices that
have a significant impact on SMs production. The practice of SSF, representing new opportunities to design better
bioprocessing with potential genetic development goals for expanding the list of exciting SMs. Current updates
cover advanced techniques on SSF to improve microbial SMs production and their ease of operation and cost-
effective production strategies. Various factors affecting the SSF have been discussed with respect to sustain­
able development of novel SSF strategies for SMs production.

* Corresponding author.
E-mail address: reetasinghania@gmail.com (R.R. Singhania).
1
Equally contributed.

https://doi.org/10.1016/j.biortech.2020.124566
Received 31 October 2020; Received in revised form 13 December 2020; Accepted 14 December 2020
Available online 19 December 2020
0960-8524/© 2020 Elsevier Ltd. All rights reserved.
V. Kumar et al.
1. Introduction
Microbial secondary metabolites (SMs) are complex biological
compounds synthesized by micro-organisms after growth phase. SMs
has no role in the growth and reproductive activities of the microor­
ganisms itself, but they are vital for other secondary needs. These
compounds find applications in pharmaceuticals, food, cosmetics etc.
These are produced besides primary key metabolites (e.g., amino acids,
lipids, carbohydrates etc.). Biosynthetic route of SMs associated with the
pathways of primary metabolite itself and utilize their intermediates and
regulatory machinery for overall regulation such as induction, carbon
catabolite and feedback inhibition etc. Secondary metabolites (SMs) are
produced by bacteria, actinomycetes, fungi, plants and animals. Central
course of growth and development of the organism is not dependent on
these entities, like primary metabolites (like amino acids, nucleotides,
lipids, and carbohydrates) which hold the key for the proper and
balanced growth. These regulations are operated for overall control
which is linked to growth rate (Barrios-Gonzalez et al., 2005). There are
examples of co-existence of same chemical entities both in plants and
animals. For example linamarin is produced both by insect (Zygaena
filipendulae) and plant (Lotus japonicas).. Antibiotics like penicillins,
tetracyclines and gentamicin sulfate have long history and high success
rate as pharmaceutical compounds and are best known example of SMs
(Ahluwalia et al., 2015; Netzker et al., 2018). Reports have suggested
advancement in the application of the SMs for the treatment of human
health diseases (Gertsch, 2016; Singh et al., 2019). Recently, SMs have
also find application in agriculture fields with application as herbicides,
insecticides, plant growth regulators and other developed products like
bio-pigments and surfactants (Ahluwalia et al., 2015; Yan et al., 2018).
In last two decades there is a surge in the discovery and development of
new chemical entities with development of new techniques in different
industrial fields like pharmaceutical, cosmetics, food and agriculture.
The plant-based SMs have the prospective benefit of using plant
extract by well-known chemical methods and identification of bioactive
molecules (Ahluwalia et al., 2014a; Kundu et al., 2013). The microbial
SMs production need fermentation process and then preparation of
extract, SM isolation and identification. Thus, SMs from microbial pro­
cesses has been least exploited by agrochemical industry for pest con­
trol. Abamectin, milbemectin, and spinosad are some important
microbial fermentation products of agrochemical importance (Ahluwa­
lia et al., 2015; Ahluwalia et al., 2014b). In spite of these facts, microbial
factories have several advantages over plant extraction or tissue culture
approach like faster and effortless growth, easy downstream processing,
etc. The microbial production of the secondary metabolites is beneficial
and economical also (Jiang et al., 2020). Hence, fermentation technique
has received considerable attention from pharmaceutical industries for
the production of metabolites useful as drug candidates (Gupta and
Chaturvedi, 2019; Jiang et al., 2020).
SMs production by microorganisms starts during “Idiophase”. It is a
phase of microbial progression in fermentation process when usually
secondary metabolite starts accumulating which is followed by active
growth phase known as “Trophophase”. As production of the secondary
metabolite starts in “Idiophase”, these do not have straight link with
production of cellular material and natural growth (Nigam, 2009). SMs
production starts in stress condition (Isah, 2019). When growth medium
of microorganism is drained of one crucial nutrient like carbon, nitrogen
or phosphate (nutritional shift-down), it starts producing specialized
metabolites. A good example of SMs production in stress condition is
penicillin production by Penicillium chrysogenum. Fungus starts biosyn­
thesis of penicillin when culture medium has very less or no glucose left
for consumption and it starts consuming lactose (Kumar et al., 2010).
The SMs of a particular need can only be synthesized by only particular
microbes because such specified chemicals are not crucial for their
development and reproduction. The production of the secondary me­
tabolites is controlled by growth conditions factors like, composition of
medium, temperature, light and pH of the medium and production can
2
Bioresource Technology 323 (2021) 124566
be enhanced with optimization of growth conditions. New techniques
like genetic engineering and genomics have encouraged the researchers
to find new ways to enhance the production of the secondary metabo­
lites (Nigam, 2009).
Solid-state fermentation (SSF) and submerged fermentation (SmF)
are two major types of bioprocesses employed for microbial metabolites
production. Both types of bioprocesses have been exploited for microbial
SMs. SmF offers advantages of online monitoring and automation which
makes it a better candidate for large scale industrial production of mi­
crobial products, however; SSF imitates natural environment for mi­
croorganisms and any individual performs best in its natural habitat
(Singhania et al., 2009; Thomas et al., 2013). SSF is described as a
fermentation process that utilizes a solid matrix containing adequate
moisture to promote microbial growth without additional free water
(Singhania et al., 2015a, 2015b). The solid material could also be served
as nutrients source or as supporting matter infused with complete
nutrient recipe which are essential for microorganism’s growth (Thomas
et al., 2013; Singhania et al., 2019; Catalán and Sánchez, 2020). SSF is
popular and well stablished bioprocess for enzymes production specially
for biomass degradation (Mekala et al., 2008; Singhania et al., 2015b).
SSF could be an excellent bioprocess for SMs production from agro-
waste and industrial residues. Great quantities of such residues,
involving bagasse, bran, husks, whole pomace, seeds, peels, corn residue
etc. are produced annually as wastes which are under-utilized or thrown
to degenerate on dumping yard. Freshly, significant focus was observed
to use above mentioned materials as adequately present and low-cost
renewable substrates to produce range of valuable compounds. In this
context, few substrates were utilized as solid support in SSF bioprocesses
to produce various microbial SMs (Lima Perez, 2019).
Metabolite production is dependent upon the microbial growth and
SSF helps to achieve it better. The microorganisms have gene regulation
mechanism which depends on the evolutionary heritage while produc­
tion of SMs is dependent on the signals recognized in their locality. In
SmF process some important signals may disappear and this prevents the
full utilization of the microorganism’s production abilities. Though the
literature is scare on the metabolic modifications when culture process is
investigated in SSF or SmF. A comparative account has been made for
cephalosporin C (CPC) production from both SSF and SmF systems.
From this study, despite the Acremonium chrysogenum ability as the only
good CPC producer, it has also addressed the understanding over the
molecular mechanisms for the regulation of antibiotic biosynthesis in A
chrysogenum however it was lacking to deliver deep insights (López-
Calleja et al., 2012). In this context, other studies were also carried out
on the expression of CPC producing genes in A chrysogenum, revealed the
similarities and differences between both SSF and SmF systems under
various process parameters. The study was able to demonstrate exciting
variations in intermediate (Pen N) and expression levels of selective
biosynthetic genes, moreover, able to find out relationships between
physiological characteristics and gene expression (Thomas et al., 2013).
Importantly, expression of various biosynthetic genes was differed due
to the great variations in both the culture environments of SSF and SmF
(Iwashita, 2002). The regulatory mechanisms similarly effect biosyn­
thetic processes in both SSF and SmF modes. Moreover, other parame­
ters in SSF environments which stimulate a diverse physiology. Certain
microbial SMs can only be synthesized through SSF, their production did
not occur in SmF regardless of the impressive microbial growth in SmF.
A potential conoicetin antibiotic could only be produced by SSF which is
quite effective against bacteria and fungi as well as multidrug resistant
pathogen Staphylococcus aureus. Coniochaeta ellipsoidea is good producer
of conoicetin in SSF condition however failed to product it well in SmF
condition despite of attaining a good growth (Segreth et al., 2003).
SSF offers several advantages such as decrease in catabolite repres­
sion and substrate inhibition; superior enzyme harvests and volumetric
outputs, low energy consumption, prolonged product steadiness, no
discharge of organic wastewater and low production expenses which
makes it preferable over SmF (Agarwal et al., 2017; Krishania et al.,
V. Kumar et al. Bioresource Technology 323 (2021) 124566

2018; Salgado-Bautista et al., 2020). Thus performance of SSF technique
is considered better for the production of SMs (Dong et al., 2016; Sri­
vastava et al., 2019). Moreover, the implication of agro-industrial
wastes as feedstocks makes this process more cost-effective and eco-
friendly (Pandey et al., 2008, 2000; Singh et al., 2020). Besides above-
mentioned several benefits of SSF, its actual potential was not abso­
lutely apprehended at commercial scale. In this review, SSF bioprocesses
are underlined for microbial SMs production as an alternative bio­
process of SmF. This review provides the recent advancements made in
SSF bioprocessing for industrial exploitation of microbial secondary
metabolites production during the previous decades, especially upon
bioreactor designs, strain improvement and novel process designs to
overcome existing challenges such as microbial biomass estimation, heat
dissipation, mass transfer etc. These major challenges of SSF bioprocess
and their probable solution have been addressed in this review articles.
2. Production of various SMs via SSF
SSF has been referred usually for ‘high volume low value’ products
with low purity index such as amylase, cellulase, protease, etc. However,
in due course of time SSF has been employed successfully for secondary
metabolites (SMs) production such as antibiotics, aroma compounds,
etc. which are high value products having importance in pharmaceutical
industries. Though filamentous fungi have been mostly exploited for
SMs production via SSF; bacteria, actinomycetes and yeast have also
been employed. Table 1 gives an account of various SMs produced by
solid state fermentation.
2.1. Antibiotics
Antibiotics are an important group among various microbial SMs
which are produced by microorganisms for its defense against other
microorganisms. There are several antibiotics available which find ap­
plications in health sector and pharmaceutical industries. Few of them
have been discussed here, leaving the most common ones.
2.1.1. Natamycin
Natamycin is a tetraene macrolide antibiotic with presence of four
conjugated double bonds in lactone ring. It has wide application for
treatment of fungal infection in agriculture field, food industry and
human treatment (Lin et al., 2020; Ojaghian et al., 2020; Patil et al.,
2018). Usually it is used to cure skin diseases, candidiasis found in
mouth and vagina, ophthalmic mycoses, and other general fungal dis­
eases (El-sayed et al., 2019). Streptomyces are great producers of this
antibiotic which includes S. gilvosporeus, S. chattanoogensis, S. lydicus,
S. natalensis are as major producers (Liu et al., 2015). Food and Drug
Administration (FDA) and European Union have specified natamycin as
good antifungal food additive with GRAS status which expand its arena

Table 1
List of Secondary Metabolites produced by SSF along with their yield and potential applications.
Substrate Microorganism Applications Product Yields(mg Remarks References
g¡1)

Corn residue Shiraia sp. SUPER- Photodynamic agents Hypercellins A 4.70 – Cai et al.,
H168 useful for therapeutic and 2010
diagnostic applications
Substrate blend (rapeseed Streptomyces Antifungal Natamycin 9.27 – Zeng et al.,
cake, rice hull, wheat bran gilvosporeus Z28 2019
& crude glycerol)
Sugarcane bagasse A. fumigatus TXD105 Anticancer Paclitaxel 0.146–0.351 – El-Sayed
et al., 2020a
Sesame seed cake Penicillium minioluteum Mycotoxin Monascorubrin 106.29 – Zahan et al.,
ED24 2020
Grapefruit by-products Aspergillus niger GH1 Extraction of Antioxident – – Larios-Cruz
et al., 2019
Vine shoots, pine saw dust Aspergillus niger G131 Anti-oxidant, Anti- Naphtho-gammapyrones 3.40 & 4.20 – Carboué
microbial, Anti-cancer, et al., 2017
Anti-HIV, Anti-
hyperuricuric, Anti-
tubercular,
Macroreticular polymeric Chrysosporium Isoconiolactone, – Ethyl Acetate Le Goff et al.,
adsorbent XAD Amberlite lobatum TM-237-S5 (-)-peniciphenalenin F, (+)-8 extraction from 2019
resin (AMBERLITE hydroxyscleroderodin, and (+)-8- resin and
XAD1600N) hydroxysclerodin mycellium
Malt extract agar A. oryzae KCCM Ketone-citreoisocoumarin, – Ethyl Acetate Son et al.,
12,698 pentahydroxy-anthraquinone, extraction 2018
hexylitaconic acid, oxylipins
Rice, Millet, Corn, Barley Monascus ruber Cholesterol lowering agent Monacolin K 7.12 Ethanolic Zhang et al.,
and Wheat (statin) extraction 2018
Corymbia maculata Leaves Aspergillus terreus Cholesterol lowering agent Lovastatin 4.90 Ethyl Acetate Subhan et al.,
(statin) extraction 2020
Palm kernel cake L. casei Antifungal activity Low molecular weight peptides 43.39** Asri et al.,
2020
Groundnut shell Streptomyces rimosus Oxytetracycline – – Asagbra
et al., 2005
Pearl barley medium Phellinus linteus Antioxidant and Antifungal Hispidin – – Liang et al.,
activity 2020
Fruit peels (apple, Aspergillus niger Antioxidant Gallic acid 13.31 Soxhlet Saeed et al.,
pomegranate, banana & extraction 2020
mango) and seeds (mango,
apple, black plum &
tamarind)
Blend of jatropha cake, vine Trichoderma asperellum Coconut like aroma 6 pentyl alpha pyrone (6 pp) 7.36 Single used Rayhane
shoots, olive pomace & TF1 plastic et al., 2020
olive oil bioreactor

Note: Yield unit- *mg ml− 1; ** % of peptide mixture with DH

3
V. Kumar et al.
of application (Kallscheuer, 2018). Commercial natamycin production is
done by submerged fermentation (SmF). Studies have suggested that
solid state fermentation (SSF) has disadvantages in comparison to sub­
merged fermentation like high cost, high energy consumption, high
product concentration and no discharge of organic wastewater. Recently
a study was conducted to evaluate the commercial production of nata­
mycin by S. gilvosporeus using agro-industrial residues by SSF technique.
At 30 L scale-up production, natamycin was produced in good yield
(9.27 mg⋅gds− 1) with benefits like cost reduction, low energy con­
sumption and no waste water discharge (Zeng et al., 2019a, 2019b).
Thus, SSF has advantages in comparison to submerged fermentation like
low cost, low energy consumption, high product concentration and no
discharge of organic wastewater.
2.1.2. Surfactin
Surfactin is a lipopeptide antibiotic with surfactant properties and
have wide applications in cosmetic and pharmaceuticals industries. The
surfactin was produced by Bacillus subtilis in solid state production of
okara and it was found that temperature plays a very important role
(Ohno et al., 1995a, Ohno et al., 1995b). A report showed that okara and
sugarcane bagasse (bulking agent) in 50% proportion can be used as
medium for the production of surfactin. For the production of antibiotic,
Bacillus pumilus UFPEDA 448 was used with reported yield of 3.3 g kg− 1
of dry biomass. Researchers described this as maximum yield from non-
recombinant microorganisms (Slivinski et al., 2012). Plackett–Burman
design was implemented to optimize the key parameters affecting the
surfactin biosynthesis to obtain optimal surfactin yield, and it was found
that medium comprising soybean flour: 5 g, rice straw: 4 g, maltose 2%
(w/w) and glycerol 2.65% (w/w)at pH 7.0 are best conditions. Surfactin
(15.03 mg gds− 1) was obtained in 50L fermenter under optimal condi­
tions (Zhu et al., 2013). Recently, solid and liquid fermentations were
compared for the production of lipopeptides. The type and yield of the
products significantly differ in both cases. Authors reported that under
liquid fermentation surfactin production was 56% as compared to 72%
of total production and rest was production of rutin in solid-state
fermentation medium (Sun et al., 2019).
2.1.3. Sambacide
Sambacide is a novel tetracyclic triterpene sulfate, which was pro­
duced from SSF by using potato as fermentation substrate. Different
fermentation conditions like culture media, temperature and time were
optimized. Fusarium sambucinum B10.2 consumed twenty days fermen­
tation time by SSF technique to produce sambacide in good yield (1.9 ±
0.08 mg gds− 1). The compound also exhibited remarkable antimicrobial
activity against S. aureus and E. coli (Dong et al., 2016).
2.1.4. Neomycin
Aminoglycosides are important class of antibacterials which inhibit
protein synthesis and neomycin is one of the effective aminoglycoside
molecules. Neomycin is efficient against mycobacteria as well as both
gram-negative and gram-positive bacteria. Streptomyces strains like
Streptomyces marinensis and Streptomyces fradiae has a long history for
the production of this drug candidate (Dulmage, 1953; Sambamurthy &
Ellaiah, 1974). The production yield of the neomycin is very low at in­
dustrial scale resulting in its high cost in the market. Commercial pro­
duction of neomycin is completed by Streptomyces fradiae by SmF
process (Mudgetti, 1986). A study was performed to evaluate the in­
dustrial production of neomycin in solid state fermentation from
different sources. The study compared solid residues as substrate and
found that with apple pomace highest yield (4567 µg g− 1 substrate) was
obtained while wheat bran gave lowest yield (2765 µg g− 1 substrate)
(Vastrad & Neelagund, 2011). Recently, Streptomyces fradiae NCIM 2418
was used for neomycin production by solid-state fermentation (SSF) by
utilizing coconut oil cake as solid substrate. The copper sulphate (1% w/
w) and zinc sulphate (1% w/w) enhanced the production of neomycin
(13423 and 12687 g kg− 1 respectively) (Vastrad and Neelagund, 2014).
4
Bioresource Technology 323 (2021) 124566
2.2. Aroma compounds
Flavour and fragrance compounds play a vital role in the commer­
cialization of the food, feed, cosmetic products. These compounds are
also extremely important for pharmaceutical industries. Interest in the
use of SSF in the producing aroma compound is also increasing after
successful utilization of this bioprocess in production of various enzymes
and metabolites (Soccol et al., 2017). Different studies had suggested
that higher yield of the aroma compound can be obtained by using SSF
in comparison to SmF. Filamentous fungi or yeast like Trichoderma vir­
ide, Rhizopus oryzae, Aspergillus niger, Kluyveromyces marxianus and
Ceratocytis fimbriata are best suited for the SSF processes (Mantzouridou
et al., 2015; Try et al., 2018). Aroma compounds synthesized by the
microorganisms by SSF technique are given in Table 2.
2.3. Phenolic compounds
Phenolic compounds are important class of compounds with diverse
biological activities. The diet with phenolic compounds with antioxidant
properties is considered as healthy diet for balanced growth of the
humans. SSF was exploited to enhance the phenolic compounds fraction
in selective food products to improve their antioxidative attributes such
as black beans which are recognized for their great nourishing property
comprising vitamin E, carotenoids, isoflavones, anthocyanins and sa­
ponins (Choung et al., 2001). An enhancement of antioxidant properties
of these beans was observed by employing SSF to make koji, comprising
various filamentous fungi having GRAS status (specifically Aspergillus
sp., Rhizopus sp. etc.), these were deployed to rise contents of phenolics
and anthocyanins (Lee et al., 2008). The SSF was investigated with plum
pomaces and alcohol distillery effluents. Two different fungi, Rhizopus
oligosporus and Aspergillus niger were used. There was increase in the
total flavonoid content. SSF helped in achieving greater lipid recovery
from plum kernels with better oil quality (Dulf et al., 2016). Two
different fungi Aspergillus niger and Rhizopus oligosporus were investi­
gated for phenolic levels and antioxidant activities during SSF of apricot
pomaces. Increase in the total phenolics content with R. oligosporus
(70%) and A. niger (30%) was observed. Total flavonoids content also
increases in SSF process (38 and 12% by R. oligosporus and A. niger
respectively) (Dulf et al., 2017). Recently, due to high consumers de­
mand high-quality processed foods with minimal changes in nutritional
and sensory properties. treatment of pulses was done by Aspergillus
awamori (1 × 10− 6 spores ml− 1) to evaluate the polyphenolics response
release. Under SSF conditions phenolic and flavonoids contents
increased and enhancement in antioxidant potential was also observed
(Saharan et al., 2020). The effect of SSF up on dandelion flavonoids for
its concentration, structure and antioxidant attributes were evaluated by
RSM to optimize fermentation conditions of dandelion. After optimiza­
tions, the highest flavone content was recorded 66.05 ± 1.89 mg g− 1.
The concentration of flavonoid in the fermentative dandelion (FDF)
crude extract was 183.72 ± 2.24 mg g− 1 (Liu et al., 2020). Mulberry is
an important source of quercetin and kaempferol. Edible fungi Monascus
anka was used for the fermentation process of mulberry leaves and it
was found that there was increase in the level of quercetin (5.30 ± 0.35
mg g− 1 dw) and kaempferol (1.87 ± 0.10 mg g− 1) (Guo et al., 2020).
Fungi Aspergillus niger was utilized for the bio-enrichment of Moringa
oleifera (moringa) leaf flour extracts by SSF. Study observed that gallic
acid was major compound with thirteen other bioactive compounds
which were released or produced after fermentation process (Feitosa
et al., 2020). Pearl millet is an important crop comprising several
bioactive molecules such as syringic acid, gallic acid, p-coumaric acid,
ferulic acid, ascorbic acid. Aspergillus oryzae and Rhizopus azygosporus
were used for SSF of pearl millet. Fermented pearl millet is was found
better food supplement as compared to unfermented with high antiox­
idant potential (Purewal et al., 2020).
V. Kumar et al.
Table 2
Production of aroma and flavoring compounds by solid state fermentation.
Microbes Solid state substrate
Trichoderma viride Sugarcane bagasse
Kluyveromycesmarxianus Sugarcane bagasse
(impregnated with L-
phenylalanine and molasses)
C. glutamicum Soyabean enriched with
threonine and lysine
Kluyveromyces marxianus Cassava bagasse (packed bed
reactors)
Saccharomyces cerevisiae, Hanseniaspora Apple pomace
valbyensis and Hanseniaspora uvarum
Trichoderma Sugarcane bagasse
Kluyveromyces marxianus Sugarcane bagasse and sugar
beet molasses
Fomitopsis pinicola Wheat bran
Trichoderma asperellum TF1 mixture of vine shoots,
jatropha cake, olive pomace
and olive oil
3. Effect of factor on the production of secondary metabolites
under solid state fermentation
3.1. Selection of substrate and supplements
SSF uses solid substrate containing adequate moisture to promote
microbial growth. As mentioned earlier that these solid materials can
also be served as nutrients source or just a supporting material infused
with complete nutrient recipe which are essential for microorganisms
growth (Singhania et al., 2019, Thomas et al., 2013). These methods are
different from SmF where free flowing fluids are used (Pandey et al.,
2000). The synthetic materials employed for support are supplemented
with the nutrients while natural materials compositions effectively
provide nutrients (Mulvany, 1969). The growth and physiology are
influenced by the solid support systems in SSF. The overall cost of the
product depends directly on the process and low-cost medium (such as
agricultural residues) will be beneficial for the reduction of the overall
cost. The chemical composition and other physical properties of the
supplements influence the production of secondary metabolites. Various
factors affect the substrates selection for SSF. Important factors includes
cost, availability, and heterogeneity of the substrates. To reduce the
product cost, valorization of the local by-products or agricultural wastes
like oil crop waste, wood waste can be used as feedstock (Carboué et al.,
2018; Hamrouni et al., 2019). The key drawbacks of utilising solid
substrates as supporting material is, it undergo physical and symmetri­
cal changes in the structure along the course of microbial growth which
ultimately cause decrease in the heat and mass transfer. It can be
overcome by replacing an inert substrate to fulfil both requirements
while maintaining its physical structure during entire period. It will also
provide an appropriate control upon heat and mass transfer.
Liu et al., 2018a, 2018b reported, Okara (substrate) and buckwheat
husk (inert support) were evaluated for the production of erythritol
production by two-stage solid state fermentation (SSF). Authors re­
ported that okara-BH mixture (5:2, w/w) and sodium chloride (0.01 g
gds− 1) were optimal condition for the production of erythritol (143.3 mg
gds− 1). Production of undesired mannitol and citric acid was also
inhibited (Liu et al., 2018a, 2018b). Another study showed erythritol
yield of 185.4 mg gds− 1 in single stage SSF by oil crop wastes (Liu et al.,
2019). In 2009, Aspergillus terreus was used in solid-state fermentation
for the production of lovastatin (Baños et al., 2009). Medium optimi­
zation for the production of lovastatin was done and it was found that
barley (8 g) and yeast extract (1% w/w) are best composition for
Bioresource Technology 323 (2021) 124566
Flavor and odor References
6-Pentyl-α-pyrone, δ-Octalactoneγ-Nonalactone, Fadel et al., 2015;
γ-Undecalactoneγ-Dodecalactone, δ-Dodecalactone Hamrouni et al.,
2019b
2-Phenylethanol2-Phenethyl acetate Rose like Martínez et al.,
2017
Pyrazines Nutty Fadel et al., 2018
chocolate-like
flavor
Volatile compounds (acetaldehyde, ethyl acetate, ethanol, fruity aroma Medeiros et al.,
propyl acetate, butyl acetate, ethyl propionate, ethyl 2001
isobutyrate, isoamyl alcohol and acetate)
Saccharomyces strains accumulated higher levels of fatty Madrera et al.,
acids and their corresponding ethyl 2015
estersHanseniaspora genus yeasts produced acetic acid esters
6-pentyl-α-pyrone (6-PP), an unsaturated D-lactone coconut-like de Aráujo et al.,
aroma 2002
Volatile ester fruity-odor Martínez et al.,
2017
4-ethyl-2-methoxy-phenol Sala et al., 2020
6-pentyl-α-pyrone coconut-like Rayhane et al.,
aroma 2020
optimum yield (34.97 mg g− 1) (Atlı et al., 2019). By using solid matrix,
insoluble in the medium, the high yield of the secondary metabolites can
be obtained. Care must be taken that the medium should be easily
available during pilot scale production. The main advantage of SSF is its
ease of operation and resemblance to the natural habitat of several mi­
croorganisms. The efficiency of SSF for producing high product titer
depends on several factors including a suitable solid substrate, micro­
organism’s performance, and a specialized bioreactor growth condition.
3.2. Particle size
Particle size greatly affects the ratio of surface area-to-volume of the
particle which is primarily accessible to the microbes and the packing
density within the surface mass. It is a crucial factor to facilitate an
effective gaseous exchange which helps in efficient heat and mass
transfer at the interface of particles. The size of the substrate determines
the void space, which is occupied by air. Because the rate of oxygen
transfer into the void space affects growth, the substrate should contain
particles of suitable size to enhance mass transfer (Singhania et al.,
2009). Smaller the particle size, larger the surface area would be for
microbial action but particles which are too small may result in substrate
agglomeration interfering with microbial respiration/aeration and thus
result in poor growth. However, larger particle size provide better
respiration/aeration efficiency but provide limited surface for microbial
action (Mitchell et al., 1992). Smaller particle size offers advantages for
heat transfer and exchange of oxygen and CO2 between the air and the
solid surface.
3.3. Inoculum size/type
Microbial growth in the fermentation process is dependent upon the
inoculum size, however low concentration of inoculum may not be
adequate to start growth of microbe and high inoculum leads to the is­
sues such as limitation in mass transfer. Inoculum concentration is an
important parameter in the SSF (Pandey, 2003; Singhania et al., 2009;
Thomas et al., 2013). There are reports which shows that higher inoc­
ulum concentration results in reduced metabolism (Kashyap et al.,
2002) which could be related to mass transfer limitations. There are
many reports described the effect of inoculum size on the microbial
secondary metabolite’s biosynthesis. The effect of inoculum size on the
caffeine degradation by Trichosporon asahii and Fusarium solani has been
discussed (Lakshmi and Das, 2010; Nanjundaiah et al., 2016).
5
V. Kumar et al.
Fermentation using SSF is generally initiated using a mycelial or spore
inoculum. There are several advantages of using spore inoculum,
including homogeneity, prolonged shelf life and ease of manipulation.
However, in wheat straw fermentation by Chaetomium cellulolyticum, the
use of mycelial inoculum is more effective in yielding high level of
proteins, presumable due to instant availability of hydrolytic (cellulase
and hemi-cellulase) enzymes. Another important factor is the density of
the inoculum, as a higher density minimizes contamination with unde­
sired organisms (Singh et al., 2017).
Hence optimization of inoculum size is critical for SMs production
via SSF process.
3.4. Hydrogen ion concentration (pH)
In solid state fermentation system, pH is very important and most
affecting growth parameter for metabolism and growth of microorgan­
isms. The substrate used in SSF itself having a buffering effect due to its
complex chemical composition, however, it is difficult to monitor the
changes due to heterogenous characteristic of the substrate and no free
water. Bacterial contamination can be prevented in fungi and yeasts by
cultivating at pH other than suitable for bacteria as fungi and yeast have
a broad range of pH for growth that can be exploited (López-Calleja
et al., 2012). Ammonium salts have been used in combination with urea
or nitrate salts in SSF to neutralize the effect of acidification and alka­
lization (Singhania et al., 2019). Thus, characteristics of solid substrate,
absence of free water or low water content, heterogeneity in the biore­
actor conditions, and lacking an appropriate on-line pH monitoring
device are major challenges for pH optimization. Several microorgan­
isms react differently against the varying pH range under the fermen­
tation atmosphere. Bacteria are known to prefer pH near to neutrality for
growth whereas fungus and yeast preferably need slightly acidic pH for
their growth, and actinomycetes prefer above the neutral pH value.
The pH of the medium must not go beyond the desirable range for
efficient production of SMs. A recent study carried out at varying pH
condition for the production of cordycepin, and it was observed that 5.5
was the optimal pH range to synthesize maximum cordycepin yield
(Adnan et al., 2017). Study showed that pH is a crucial and active
parameter to regulate the production of metabolite cephalosporin C in
SSF, as well as in SmF. A study on SmF showed that cephalosporin C
production is actively regulated by pH, which worked relatively in a
narrow pH range. When the production of cephalosporin C carried out in
SSF using sugarcane bagasse as solid substrate, its biosynthesis occurred
in a strictly defined pH range (Cuadra et al., 2008). Moreover, a
comparative account between SmF and SSF showed that cephalosporin
C biosynthesis took place at the same pH range of 6.4–7.8 in both the
culture systems (López-Calleja et al., 2012).
3.5. Moisture content and water activity
Substrate’s moisture and water content perform a critical role in the
SSF process. An optimal moisture level is needed for microbial growth.
Lower moisture content causes reduced solubility of substrate’s nutri­
ents whereas higher moisture levels hamper the enzyme yield as the
porosity of the solid matrix is reduced (inter-particle spaces), thus
interfering with oxygen transfer. Microbial growth along with evapo­
ration, reduce the moisture of the substrate during fermentation leading
to dryness.
Water activity (Aw) is the available or accessible water for the mi­
croorganisms growth. It gives the amount of unbounded water available
in the immediate surroundings of the microorganism. It is closely
related, but not equal to, the water content. The definition of the water
activity is:
Aw = Ps /Po (1)
where Ps is the equilibrium vapor pressure of water within the solid
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Bioresource Technology 323 (2021) 124566
substrate and Po is the vapor pressure of pure water.
In general, fungal and yeast cultures have been found to be more
effective than bacteria in SSF processes. This has been essentially based
on the theoretical concept of water activity, as bacterial cultures have
higher water activity requirement (around 0.8–09 Aw) whereas, fungi
and yeast have lower water activity requirements (0.5–06 Aw). Thus
bacterial culture are less suitable for SSF processes. However, it must be
remembered that the choice of microorganism for use in the SSF is
dictated by the nature of the substrate to be fermented.
3.6. Effect of temperature
Temperature is another important factors affecting the SSF process.
Growth of microorganisms, production of enzymes, secondary metabo­
lite synthesis, etc., depend on temperature. Fungi have a wide range of
temperatures for growth; however, the optimal temperature for product
formation and growth may differ greatly (Carboué et al., 2020; Kumar
et al., 2020). Temperature inside the fermenter increases with the
growth rate and metabolic activity. Thus, the mass transfer difficulty
and metabolic heat driven temperature rise are the major challenges
occuring in SSF system. If there isn’t an efficient system to remove the
heat generated, this will adversely affect the product formation and
growth. Heat generated due to metabolic processes of microbes harm the
growth most which needs to remove from bioreactor to avoid the loss of
growth and product yield due to overheating. Several bioreactor designs
have been tested in SSF system to remove the rising heat efficiently
(Soccol et al., 2017). This challenge becomes severe during large scale
process where rising heat leads to substantial moisture loss which
greatly affects the microbial growth. Such heating problem ultimately
lead to condensation which results considerable amount of water in the
fermented solid matrix, accumulated water cause heterogeneity and
causes difficulty to maintain homogenous ideal temperature range in
solid substrate and negatively affect the growth and fermentation. To
overcome this challenge, significant air needs to blow into the SSF sys­
tem to remove the heat through gaseous vent. Temperature could be
controlled to an extent by aeration and agitation. Humid air could
control the temperature by reducing the water activity of the substrate
Carboué et al., 2020.
An incubation temperature for the proper growth of the microor­
ganism is very important (Adnan et al., 2017; Kumar et al., 2020). Study
is required for the proper identification of these environmental param­
eters for individual microorganism for the optimal growth and optimum
yield of the SMs (Carboué et al., 2020).
Cordycepin is an important therapeutic compound produced by
Cordyceps militaris. Effect of the temperature (10 ◦ C, 15 ◦ C and 20 ◦ C) on
the yield of cordyceplin was analyzed. It was found in the study that at
low temperature the yield was low and maximum yield was obtained at
25 ◦ C (Adnan et al., 2017). The temperature effect was investigated for
the production of SMs by Aspergillus tubingensis G131 at fixed moisture
(66%) at four incubation temperatures (25–40 ◦ C with 5 ◦ C interval).
The study observed that particular temperature is required for the
maximum production of SMs. For example, ergostrol production was
highest at 35 ◦ C, optimum temperature for the fonsecin B production
was 30 ◦ C, for dianhydroauras perone C 35 ◦ C and for asperpyrone E it
was 30 ◦ C (Carboué et al., 2020).
3.7. Effect of sterilization
Microorganisms are capable of causing infection and are constantly
present in the external environment. These are the major source and
responsible for contamination. The aim of sterilization is the remove or
destroys them from the material or surface. In order to maintain the pure
culture and to make the industrial fermentation process successful,
sterilization is an essential operational requirement for every biotech
industry (Deindoerfer, 1957). Sterilization can be achieved by different
methods either by the combination of heat, chemicals, irradiation, high
V. Kumar et al.
pressure and filtration or dry heat, ultraviolet radiation, gas vapor
sterilants, chlorine dioxide gas etc (Liu et al., 2013b). Effective sterili­
zation techniques are essential for working in a lab or in industry. The
sterilization batch time on a large/ industrial scale is 3–6 h as against
that of 15 min at 121 ◦ C at laboratory scale in the batch sterilization
mode. One of the major blockages in solid-state fermentation industry is
the solid medium sterilization. Autoclaving is the only technique which
is currently used for solid medium sterilization; however, it has many
disadvantages high discharge, energy waste, which results in high cap­
ital cost and low process efficiency (Zhang et al., 2007) it also includes
longer sterilization time which leads to nutrients destruction. Thus,
there is an urgent need to have a very efficient, less time consuming solid
medium sterilization technique which causes less nutrient depletion and
gives complete sterilization by killing all the microbes (Zhao et al.,
2015). High-temperature and very short-time sterilization process can
be a useful recommendation for developing the new sterilization
technique.
4. Challenges for the production of SMs via SSF
SSF bioprocess faces a wide range of challenges, some of which are
associated with its operation, while others are associated with fermen­
tation monitoring and product harvesting. The substrates used in SSF
differ greatly in composition, chemical nature, mechanical properties,
particle size (including inter- and intra-particle spaces), water retention
capacity, surface area, etc. These factors affect the overall process design
and product development (Srivastava et al., 2019. While several of these
are of generic in nature, they still hold a significant impact and need to
be considered in a holistic manner. These include the selection of
microorganism and substrate, optimum physical–chemical and biolog­
ical process parameters along with purification of the desired products
Dong et al., 2016. These are all ongoing challenges for SSF. The opti­
mization of downstream processing is considered to be the most
important challenge in SSF. Separation of product from the solids is a
technically and economically challenging process, and, although there is
much advent in biochemical engineering, SSF is generally being used
only for the production of metabolites where low levels of purity are
required (Agarwal et al., 2017; Krishania et al., 2018).
4.1. Scale-up
One of the challenges in solid state fermentation for metabolite
production is scale-up as is mainly limited to flask scale. High inoculum
density is used in large scale of SSF so as to reduce the risk of contam­
ination and to produce the desired product faster. Inoculum generation
becomes a separate unit operation in large-scale production (Pandey,
2003). In case, the inoculum has to be generated in liquid medium,
large-scale fermenters should be used. Because of the ease of uniform
mixing of spores with a moist solid substrate, a spore inoculum is
preferred over an inoculum generated in liquid medium. Generating
spore inocula, takes more time, especially in fungal cultures, and the
chance of mutation is quite high. To reduce the potential chance of
mutation, a minimal amount of sub culturing must be done during
inoculum development (Thomas et al., 2013).
Scale-up practices are process specific. Only scarce information on
bioreactors design and conditions on biomass production by microor­
ganisms is available and little is known on the particularities of a certain
process at the onset of the fermentation development. Though the scale-
up in the SSF has been a major concern since years; it is expected that
with bioreactor designing, one will be able to address most of the
shortcomings that are still associated with the actual SSF. Biochemical
engineering has stated to overcome the challenges of scale-up with the
designing of the bioreactors. Bioreactor design plays an important part
in obtaining maximum yield of SMs (Ashok et al., 2017). Researchers
around the globe are still working on scale-up processes along with its
downstream processing. Scale-up may pave a way for efficient
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Bioresource Technology 323 (2021) 124566
validation of the system effectiveness and economic evaluation the SSF
processes.
4.2. Heat dissipation and mass transfer
One of the major challenges to overcome in scale-up of SSF processes
includes the heat accumulation due to heterogeneous nature of the
substrate and metabolic activities, comprising a three-phase
gas–liquid–solid multiphase system during bioprocess. In order to
circumvent these, it is required to estimate and understand the heat and
mass transfer parameters, which would help in developing the mathe­
matical models, considered as the key for scale-up data (Thomas et al.,
2013). The bioreactors provide unique environment for the cultural
growth of microorganisms. With the progress in the fermentation pro­
cess, due to the metabolism and breakdown of carbon compounds en­
ergy is generated that increases the heat inside the reactor and removal
of this heat is very crucial for proper fermentation process. Heat pro­
duced in the bioreactor can be reduced by cooling it from outside (Ashok
et al., 2017). One another method of heat removal is water evaporation,
but this process ends with water depletion inside the reactor.
Another challenge faced by SSF is mass transfer. Diffusion of the
oxygen in the solid matrix is quite challenging as after growth of mi­
croorganisms; the solid substrate along with culture biomass causes
reduction in permeability of oxygen through the matrix. Oxygen in
appropriate quantity is required for the proper growth of the microor­
ganisms and aeration is a major problem while utilizing solid matrix. For
the better yield of product of interest during SSF, solid supports should
be homogeneous and it also helps for oxygen transfer (Mitchell et al.,
1992; Singhania et al., 2009). The heterogeneity results in temperature
and concentration gradients which affect the product yield adversely.
The development of new bioproduct discovery system for compounds
delivery remains dependent on culture conditions, media preparation,
bioreactor design, product recovery (López-Gómez et al., 2020). Recent
advancement in the bioreactor design for SSF has enabled on-line
monitoring of parameters like heat and mass transfer.
4.3. Biomass estimation
As microbial biomass is embedded with the substrate in SSF it is
almost impossible to separate the microbial biomass completely and do
its estimation. However, there are number of indirect methods for its
estimation which is discussed elsewhere (Singhania et al., 2019; Ashok
et al., 2017). Nucleic acid determination, glucosamine content analysis,
ergosterol determination, protein content determination are the
methods which are applied for biomass determination in SSF and can be
referred from Singhania et al. (2019). Botella et al., 2019 proposed dry
weight model based on capacitance measurements and metabolic data
for estimation of biomass of Aspergillus awamori during SSF. Air inlet and
outlet CO2 measurement could be a method for biomass estimation in
aerobic fermentation process. The growth kinetic parameters can also be
indirectly estimated by determining the amount of oxygen intake,
assuming logistic growth kinetics and that O2 is consumed for growth of
the microorganisms and its maintenance (Assi et al., 2009).
Biomass growth estimation and its monitoring is necessary for ki­
netic studies of fermentation. However, separation of biomass from solid
substrate is not possible in SSF, and accurate measurement of biomass is
impractical. Almost every method for biomass estimation available, has
its own drawback.
4.4. Downstream processing
A suitable downstream process is crucial for obtained product when
SSF bioprocesses are performed. Recovery of product in SSF is a chal­
lenge and is extracted from the solid fermented matter by extraction
with solvents (aqueous or other solvents mixtures). There are important
variables that influence in the product extraction such as the type of
V. Kumar et al.
solvent and its concentration, ratio of solvent to the solid and also pH of
the solvent. In SSF as the metabolites get diffused within the solid ma­
trix, product recovery becomes tedious when compared to SmF. The cost
of purification depends rely on degree of purity obtained for the product.
For SMs extraction degree of purity must be high. SMs having high
purity are used in bulk in the pharmaceutical and health industry and its
purity is governed by stringent regulations which necessitates to go
through specific purification strategy (Nigam, 2009). After the fermen­
tation course, fermented matrix is subjected to the extraction process in
which appropriate solvent selection is highly important for effective
product extraction from the fermented matrix. Solid handling and waste
management are also drawbacks in this case. If the final product is not
the fermented solid itself, expenses of waste-treatment add heavily to
the cost of product. Various management strategies could be beneficial
such as recycling of the spent solid as animal feed or biogas generation
(Rodríguez-Fernández et al., 2012; Díazetal., 2007; Castilhoetal., 2000).
Separation of ellagic acid have been achieved from pomegranate
husk by utilizing size-exclusion chromatography (SEC). Effective
extraction of secondary metabolites was carried out from fermented
matrix through manual pressing while adding ethanol appropriately.
Subsequently culture soup was poured on a Sephadex LH-20 separation
column to obtain purified ellagic acid (Sepúlveda et al., 2018). Likewise,
purification of seven bioactive compounds synthesized from Strepto­
myces cavourensis TN638 have been achieved through a complex puri­
fication steps employing combination of silica gel column (eluted with
ethyl acetate), followed by medium pressure liquid chromatography
(MPLC) with repeated stepwise gradient of eluting solvents (ethyl ace­
tate in heptane and ethyl acetate in cyclohexane) to get four compounds
at this stage, then sephadex LH20 column chromatography comprising
an eluting solvent MeOH–CH2Cl2 (4:1) to obtain three purified com­
pounds at this last step (Kaaniche et al., 2020). Another study addressed
the purification of polysaccharides produced from Fusarium solani DO7
via solid-state fermentation. Purification of Fusarium polysaccharides
was achieved using DEAE-52 column chromatography while employing
the 0.4 M NaCl eluting solvent, followed by Sephadex G-200 columns to
obtain purified polysaccharide fraction (Zeng et al., 2019a; Zeng et al.,
2019b).
5. Aspects of design of bioreactor for the production of SMs in
SSF and overcoming limitations
Different bioreactors structure has been used in SSF to obtain
desirable products. Standard design of bioreactor is required to provide
the unique environment for the growth of the micro-organisms. Meta­
bolic heat is generated inside the bioreactor during bioprocess is the
primary concern, and it is harder to remove the waste heat generated
from a bed of fermented matter in which the inter-particle phase is
occupied by air. Several basic bioreactors for SSF operation have been
discussed in Singhania et al. (2019).
5.1. Tray fermenters for SSF
Tray fermenters are most common and economical when referred to
solid state fermentation. These are metallic trays with a cover in which
solid substrate along with inoculum are placed and are loosely filled so
as to get space for mycelia or other microorganisms to penetrate and
grow evenly. Though the tray culture fermenters are operated in an
aseptic condition, it is not a “contained” system as the workers are
usually exposed with the product during the handling of the tray
fermenter and harvesting processes. Hence, the tray cultures are not
suitable for handling sporulating cultures for example the culture of
Trichoderma viride with a high spore content is prepared to be used as
biological control agent. High spore count is must in this case for the
product to be effective hence, a huge spore dust is formed during harvest
which may cause allergy and breathing issue. This is the reason that tray
fermenters are employed for the products that are regarded as ‘safe’,
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Bioresource Technology 323 (2021) 124566
such as, fermented food products and enzymes (Mahmoodi et al., 2019).
These are not cytotoxic and few antibiotics and immunosuppressants
also fall in this category. The systems using mixed beds are also not fully
‘contained’. For example, the sterilized substrate is inoculated in one
place and then transferred in another place for incubation to the mixed
bed. Subsequently, the fermented matter is transported to a harvesting
vessel for downstream processing. This is a batch fermentation process
where the fermented matter is removed after fermentation in one go.
Rolling bed bioreactor provides few advantages over tray fermenters.
It is needed to remove metabolic heat from the fermenting bed.
Among the major challenges the water activity and the heterogenous
characteristics of the solid substrate are significant ones. These attri­
butes are due to thermal conductivity and heat capacity of a continuous
aqueous phase, are superior to those of a bed of moist solids with inter-
particle air. to An effective approach to remove any kind of metabolic
heat generated inside the bioreactor is cooling from the outer surface. It
would lead to protect the contents of the bioreactor because of the
rotation of the fibre column rapid cooling could be achieved. The rota­
tion helps to properly expose the fermented matter providing a better
surface area for the reaction and also facilitate the heat dissipation for
higher productivity (Ashok et al., 2017). However, in large scale colling
from outside reactor is difficult, as the water requirement is too high. For
certain product biosynthesis or maximizing the production of SMs in
SSF, specialized bioreactor design is highly important.
As discussed above, for scale-up and designing of any bioreactors in
SSF, heat dissipation, mass transfer and mixing are the important pa­
rameters to be taken care of (Benz, 2011). Several strategies have been
tried to circumvent the above limitations with great performance, but
very few of them have been able to achieve success. Single used plastic
bioreactors have been employed for 6 pp production along with group of
hydrolytic enzymes by T asperellum TF1 which is a new and emerging
issue. In this work authors have compared production of 6 pp via SSF in
shake flask, Raimbault column and single use plastic bioreactor (Ray­
hane et al., 2019). The system was able to process 5–7 kg solid DM (solid
medium), while the ease of operation and design simplicity were
considered attractive features, which could lead to immediate technol­
ogy transfer and adoption by the agricultural and industrial sector. Use
of single used bioreactor with the application of forced air on the SSF
system had a positive effect on the group of enzymes, 6 pp and conidia
produced at high titers by T. asperellum TF1. Production of these me­
tabolites could be related to the necessity to maintain adequate and
homogeneous levels of temperature and moisture into the system,
allowing transfer of nutrients, metabolites and the oxygen
solubilization.
5.2. Rolling bed fermenters for SSF
Novel design of bioreactor for the SSF comprising multiple rotating
drums were arranged, in which each of them were having individual
feed inlets as shown in Fig. 1 by Ashok et al. (2017). Moisture level was
maintained inside the bioreactor and reaction were carried out in each
bioreactor for reaction time after which the final products were collected
through the output. According to bioreactor design, the substrate must
be added along one end of the bioreactor and aeration was maintained
depending on conditions. Heat transfer effect was controlled by
continuously cooling the bioreactor using a sprinkler overhead. To
prepare the transparent bioreactor acrylic polymer tubes were used
which were flexible, and non-corrosive and could perform at varied
range of temperature. Fig. 1 represent the design of bioreactor which
was derived by studying the facts of bioreactor designing from several
studies (Benz, 2011; Suryanarayan, 2002; Zhang et al., 2015). It is
important to optimize the speed for the rotation when the process is not
static because mycelia are shear sensitive and at higher speed it might
get disrupted. However, low rates of agitation may limit the mass and
energy transfer at large scale production. In case the cultivated micro­
organisms can tolerate agitation; agitated bioreactors could be used to
V. Kumar et al.
Fig. 1. Bioreactor with multiple rotating drum.
resolve the scale-up challenges in SSF. Agitated bioreactors could be
employed for continuous SSF to overcome productivity drawbacks
associated to convention batch process. (Fig. 1)
5.3. Continuous stirred tank reactor (CSTR)
In continuous bioprocess substrate is constantly fed to the reaction
vessel, discharging the same amount of fermented material to maintain
the volume constant. Continuous stirred tank reactor (CSTR) is used in
continuous SSF (Liguori et al., 2016; Varzakas et al., 2008). The CSTR is
an ideal model specifying perfect mixing to have homogenous compo­
sition of the bed but having heterogenous residence time (Fig. 2).
An up-flow column bioreactor was employed for the production of
anti-cancer prodrug ‘camptothecine (CPT)’ via SSF by an endophytic
fungus Fusarium oxysporum (NCIM 1383). A bench-scale-up-flow biore­
actor (Fig. 3) was developed by optimizing SSF conditions, to verify its
use for continuous CPT production which could yield up to 128 mg/gds
CPT after 48 h of incubation (Bhalkar et al., 2016).
Important process parameters which are effectively maintained or
regulated by bioreactor design in SSF include temperature, moisture
Fig. 2. Representation of Up-flow column bioreactor.
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Bioresource Technology 323 (2021) 124566
Fig. 3. Representation of Plug flow bioreactor.
level of the solid substrate, aeration or oxygen transfer, agitation or
mixing, and heat transfer. To achieve an efficient oxygen transfer and
heat removal, a moisture with water and solid matrix mixing, mainly
two approaches are often used. The first technique is the move of air
around the solid matrixs and the second one is, air is going across the
unmixed, intermittently blended and continuously blended matrix beds.
(Durand, 2003). In a report of a bioreactor with control on inter­
mediate times to exposure for air (tEA), Sarkar et al. (2009), an ultralow-
speed rotating disk bioreactor was designed keeping 50% of the disk
immersed in liquid medium. Production of the SMs actinomycin-D was
studied by an estuarine isolate viz Streptomyces sp. MS 310. Employing
this 25 L bioreactor, higher actinomycin-D productivity was obtained
when compared to a shake flask culture or a stirred-tank bioreactor, by
operating at a disk rotational speed of 1 revolution/day. In this context
SSF would be more efficient process compare he SmF processes in
pharmaceutical industries.
5.4. Plug flow reactor (PFR)
The PFB ideal model indicates perfect mixing in the radial dimension
and no axial dispersion so that the solid particles move inside the
bioreactor with uniform kinetic profiles thus exhibiting similar resi­
dence time (Van’t Riet and Tramper, 1991). Thus, the product concen­
tration in PFB is constant at any single point. However, approximating
the time studying growth kinetics is challenging as several parameters
needs to be measured.
Carboué et al. (2019) by using reactor PFB which was prototype
bioreactor (made by Vinovalie company (FranceIt consists of a feeder
screw made up of stainless-steel. The feeder screw contains 14 threads,
each confining a separate compartment that can be filled with solid
material (Fig. 3). These compartments serve to define the various
V. Kumar et al.
positions along the reactor, they communicate with each other during
the rotation of the screw as some solid particles are exchanged between
two of them placed nearby. Inside the bioreactor this alimentation
compensated the fermented material that exit the PFR to attain a mass
balance. However, it was complicated to follow a single particle during
its residence inside the bioreactor also to study the culture behaviour.
Fungal compound was successfully produced in a semicontinuous mode
by cultivating Aspergillus niger with a PFR prototype.
6. Strain improvement for SMs production using SSF
Stain improvement is another important approach for maximum
production of secondary metabolites. For strain improvement, popular
physical methods (e.g., chemical, radiation); and genetic methods have
been greatly exploited (Parekh et al., 2000). These methods are popular
even today for improving the microbes. All these approaches make
changes in strain characteristics via mutation or genetic recombination
which can be selected based on altered properties through random se­
lection (mutagenesis) or rational selection (genetic recombination). For
commercial production of a product it is necessary that yield must be
significantly high to make the process commercially viable. Wild type
strain usually screened to give the best yield at optimized parameters but
it is great possibility that the production yield may increase even 10
times by strain improvement before the process is taken to commer­
cialization (Ismaiel et al., 2014; El-Sayed et al., 2020b). Even today,
classical mutation is most practical and popular technique to enhance
the product yield. In case of fungi, pelleted morphology is the most
desirable attribute of the microbes in SmF, whereas in SSF fast growing
filamentous characteristics are preferred in order to colonized the fungi
effectively over solid matrix. With acclimatization in optimum growth
condition, these strains become more specialized producers and exhibit
altered growth response for example less or vigorous growth than their
wild counterparts. They sporulate relatively less than the wild type
strain, therefore their capability to cause opportunistic infection is
significantly decreased.
With these desirable improvements of microbial strains, they are able
to decrease the economic constrain of the processes by yield improve­
ment of the metabolites (Parekh et al., 2000). Such Improvements in
producer strains can be achieved by mutagenesis for which chemical and
physical mutagens are quite practical. Gamma radiation found highly
energetic ionizing radiation between numerous physical mutagens and
recommended as most preferred mutagen (Chopra, 2005). Gamma ra­
diation effectively induces mutations in the cells via structural changes
which includes deletions in the double or single strand genomic DNA.
The DNA break commonly oxidize the bases and cross links of DNA–­
protein complex (Cadet et al., 1999).
There are several reports recommending both UV and gamma radi­
ations for product enhancement of numerous fungal secondary metab­
olites (Ismaiel et al., 2014, 2015; Chopra, 2005; El-Sayed et al., 2020a,
2020b, 2020c). The effect of both UV or gamma radiation examined on
the fungal growth, a substantial reduction in the concentrations of
glucosamine was detected with respect to their respective controls.
Similarly, two strains of P. roqueforti exhibited great reduction in their
fungal biomass yields upon irradiation of UV or gamma (El-Sayed et al.,
2019b). Moreover, the ochratoxin producing A. ochraceus fungus also
exhibited mycelial growth reduction upon 1⋅5 KGy dosing of gamma
radiation (Paster et al., 1985). Importantly, the gamma radiation has
been a classified ionizing radiation to exhibit strong effects on cellular
mutagenesis. It induces effective genes modifications via DNA repairing
mechanisms in the cells (Thacker, 1999).
7. Plants derived SMs compounds enhanced by SSF employing
microorganisms
Various plant species are also able to produce a wide variety of SMs e.
g., flavonoids, phenolic acids, lignans, sterols, stanols, salicylates,
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Bioresource Technology 323 (2021) 124566
glucosinolates etc. (Hooper and Cassidy, 2006; Ventura et al., 2008).
Plant based SMs compounds enhanced by SSF have been summarized in
Table 3. SSF have been investigated to enhance the structure, property
as well as extraction of naturally occurring active substances embedded
in plant materials via microorganisms. These includes antioxidants, anti-
cancer, antidiabetic, anticoagulating agents, etc (Hooper and Cassidy,
2006; Ventura et al., 2008). The waste residue of diverse fruits and
vegetables peels are commonly considered as zero cost waste as they has
been often utilized by researches to obtain several valuable compounds
with active ingredients thus finds application as pharmaceuticals (Par­
ashar et al., 2014). Rashad et al. (2015) produced antioxidant com­
pounds and anticancer agent from pineapple waste using SSF which also
enhanced the content of protein, fiber, phenolics in spite of antioxidant
activity. Duda-Chodak and Tarko, (2007) have examined the antioxi­
dant characteristics, whole polyphenols, and tannin content of peels and
seeds of selected fruits. Due to these compounds, extract have significant
scavenging activity. The polyphenol contents were recorded more in the
peels as compared to the seeds. Similarly, the highest antioxidant ac­
tivity determined in extract obtained from pomegranate peel as
compared to orange and lemon peel (Singh and Genitha, 2014). Field
plant residues such as stem, leaves, and stalks have also been examined
for antimicrobial and antioxidant activities in their extracts. Parasar
(2017a, b) investigated the effect of SSF on extraction of phenolic
compounds with antioxidant activity from plants such as Lablab pur­
pureus (seim), Oryza sativa (rice), moreover from filamentous fungi (with
GRAS status) e.g., Aspergillus oryzae and also detected a significant rise in
the level antioxidant.
Treviño-Cueto et al. (2007) demonstrated the Larrea tridentata plant
waste as promising source for tannase and gallic acid production which
was modulated by A. niger Aa-20 during SSF. Higher ellagic and gallic
acids contents have also been obtained by A. niger PSH deployed SSF of
tannin repleted Larrea tridentata aqueous extracts infused in poly­
urethane foam (Ventura et al., 2008). Fungus Aspergillus niger GH1
deployed SSF was reported for ellagic acid production using Larrea tri­
dentata as substrate and the process showed remarkable ability to hy­
drolyze ellagitannins into ellagic acid (Aguilera-Carbo et al., 2009).
8. Future prospects of SSF for SMs production
SSF is no more associated with low value bulk products but is very
well employed for production of pharmaceutically important high value
products. SMs production via SSF has taken up SSF to a higher level
where high value products are being produced by choice. Even today
SSF is associated with few drawbacks limiting its industrial application
but with the advent of new bioreactor designs for SSF coming continu­
ously, soon it will be a preferred bioprocess for industrial products as
SMs. Heat dissipation, mass transfer, biomass estimation is being
addressed by researchers worldwide working on SSF. These are the
challenges limiting scale-up of SSF and there are solutions to overcome
these. Novel bioreactor designs fabricated specially for SSF processes
will surely resolve these shortcomings. Even continuous solid-state
fermentation is possible using PFB or CSTB type bioreactor in SSF.
Strain improvement might improve productivity and quality of the
product by several times and with new technologies such as CRISPR-
Cas9 multiple genes could be targeted for modification resulting in
desired genetic combination. Robust industrial strains are needed to
perform at industrial level bioprocess for large scale metabolites pro­
duction. Specific process conditions are required by particular microbial
strain for specific products which implies long rigorous research before
commercializing a product. Even SSF process vary for each microbial
product by specific microorganisms. Thorough study is needed for
optimization of the process. Microorganisms may preferably produce
antibiotics in SSF compared to SmF even though having optimal growth
in SmF. In future, with more engineering research, newer fermenter
design will be developed for large scale SSF based fermentation pro­
cesses. These new reactors will surely be sophisticated, compact, less
V. Kumar et al. Bioresource Technology 323 (2021) 124566

Table 3
Plant based secondary metabolites produced by solid state fermentation.
Plants Microbes Metabolites References

Dandelion Lactobacillus plantarum (CGMCC No. 1.12934) and Under optimized conditions,maximum flavone content was 66.05 ± 1.89 mg Liu et al., 2020
(Taraxacum Saccharomyces cerevisiae(CGMCC No. 2.1190) g− 1 and flavonoid content in crude extract of fermented dandelion was
officinale) 183.72 ± 2.24 mg g− 1.
Mulberry leaves Monascus anka Quercetin and kaempferol Guo et al., 2020
Teff (Eragrostis tef Mushroom-mediated solid-state fermentation. Flavonoids to o-quinones Gebru and
(Zucc.) Trotter) Ganoderma lucidum Sbhatu., 2020
Plum pomaces Aspergillus niger and Rhizopus oligosporus Quercetin-3-glucoside major phenolic compounds Dulf et al., 2016
(Prunus domestica
L.)
Apricot (Prunus Aspergillus niger and Rhizopus oligosporus Chlorogenic acid, neochlorogenic acid, rutin, and quercetin 3-acetyl- Dulf et al., 2017
armeniaca L glucoside
Rice bran A. brasiliensis, A. awamori, and A. sojae Phenolics, Flavonoids, Kojic acid, and Phenolic acids Ritthibut et al.,
2021
Rice husk Beauveria bassiana and Trichoderma harzianum Fungal biopesticide Sala et al., 2020

energy consuming, economical, efficiently using low cost substrate,
dependable and very promising. Hence, the fact cannot be denied that
these newly design SSF rectors will provides natural environment to
microorganisms which allows best adaptation causing them to perform
efficiently.
9. Conclusions
SSF offers several benefits over SmF for production of metabolites
like SMs such as high volumetric productivity, lower energy re­
quirements, less effluent generation and simple to work on. Novel bio­
reactors in SSF are designed to overcome some of the existing challenges
such as heat accumulation, mass transfer, homogenous mixing, etc.
Moreover, with new approaches it can be strategically utilized for
effective regulation of SSF competing the existing SmF system. Thus, it
can be anticipated that SMs production via SSF will be the technology of
the future and is constantly evolving as a safer natural bioprocess.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
Authors VK and SS are thankful to Director, Indian Institute of
Integrative Medicine, Jammu and author VA is thankful to Director,
Institute of Pesticide Formulation Technology, Gurugram for continuous
support and encouragement.
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