Journal of Biotechnology 269 (2018) 16–34

Contents lists available at ScienceDirect

Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Review

Bioreactors in solid state fermentation technology: Design, applications and T

engineering aspects
⁎
Sidharth Arora, Richa Rani, Sanjoy Ghosh
Biochemical Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, India

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

Keywords: In recent years, substantial credibility in employing Solid-State Fermentation (SSF) technique has been witnessed
Solid-state fermentation owing to its numerous advantages over submerged fermentation (SmF). In spite of enormous advantages, true
Bioreactor design potential of SSF technology has not been fully realized at industrial scale. The lack of rational and scalable
Substrate-support bioreactor designs backed by mathematical models and automated control system that could successfully ad-
Mathematical models
dress heterogeneity with respect to heat and mass, and also operate aseptically, remains the prime reason for it.
Heat and mass transfer
As a result, there still exists vast scope in SSF bioreactor research and development to facilitate broad spectrum
Microorganism
of biotechnological applications. The present article reviews state-of-the-art in SSF technology with focus on
bioreactors that have been employed for bioprocess applications, in particular, enzyme production. Based on the
mode of operation, bioreactors are divided into four categories with emphasis on design features, effect of
operating conditions on productivity, applications and limitations. Selected modeling studies developed over the
years, have been revised and presented in problem specific manner in order to address the limitations. Some
interesting designs including few recent ones that have been proposed and/or employed at pilot and industrial
levels are discussed in more detail.

1. Introduction
The recent surge in demand for large quantity of biologically active
secondary metabolites (antibiotics, bacterial toxins, immune drugs, and
alkaloids), single cell proteins, enzymes, industrial chemicals, biofuel,
food, phenolics, feed, and pharmaceutical products (Thomas et al.,
2013; Pandey, 2001) has made SSF technology as an alternate pro-
duction method to submerged fermentation (SmF), the need of the
hour. In addition to the production of bio-active products of commer-
cial interest, there is also a growing popularity of SSF to be used as
waste management technology, applications of which may include
bioremediation, detoxification, bioleaching and biopulping (Thomas
et al., 2013; Singhania et al., 2009; Krishna, 2005). The technique with
its broad application and operational advantage over SmF
(Subramaniyam and Vimala, 2012; Cunha et al., 2012; Sun and Xu,
2009; Holker and Lenz, 2005) (Table 1) has led to significant research
inputs eventually assisting in better reactor design, operation and scale-
up strategies (Mitchell et al., 2006). In spite of advances, major hin-
drance in industrialization of SSF process remains the lack of simple,
efficient and easily scalable bioreactors that could successfully address
heat build-up, heterogeneity (heat and mass), and at the same time
operate with utmost sterility (Papagianni, 2014). This is probably due
to combination of three factors i.e. lack of efficient bioreactor design,
lack of mathematical models describing the transport and kinetic phe-
nomena at micro- and macro-scopic levels and the lack of effective
online process monitoring and control strategies. However, in recent
years there have been reports of few bioreactor systems that have at
least partially overcome these challenges for a specific application,
there still exists a vast scope for improvement to address a broad
spectrum of biotechnological applications.
In this review, based on the mode of operation, SSF bioreactors have
been classified into four categories. Description under each category

Abbreviations: AFR, Air flow rate; APP, air pressure pulsation; APP-SSF, air pressure pulsation solid-state fermentation; ASFB, air-solid fluidized bed bioreactor; aW, Water activity; Bt,
Bacillus thuringiensis; CER, carbon dioxide evolution rate; CFU, colony forming unit; CMC, Carboxy methyl cellulose; D, Axial dispersion coefficient; DDF, dimensionless design factor; DPS,
discrete particle simulation; GDD-SSF, gas double dynamic solid-state fermentation; g-ds, gram-dry-solid/substrate; g-fs, gram-fermented-solid/substrate; HLD, honeycomb loading
device; HC, critical bed height; IM, intermittent mixing; IMC, initial moisture content; IU, international unit; KLa, mass transfer coefficient; NO, nitric oxide; OUR, oxygen uptake rate; Pe,
peclet number; PA, pressure amplitude; PG, pectinase; PBR, packed bed bioreactor; RDB, rotating drum bioreactor; SB, sugarcane bagasse; SmF, submerged fermentation; SL, substrate
loading; SSF, solid-state fermentation; TB, tray bioreactor; TPH, total petroleum hydrocarbons; VAC, volatile aroma compounds; vvm, volume of air under standard conditions per volume
of medium per minute; WB, wheat bran; ∼, Nearly to
⁎
Corresponding author.
E-mail address: ghoshfbs@iitr.ac.in (S. Ghosh).

https://doi.org/10.1016/j.jbiotec.2018.01.010
Received 12 May 2017; Received in revised form 2 January 2018; Accepted 15 January 2018
0168-1656/ © 2018 Elsevier B.V. All rights reserved.
S. Arora et al.
Table 1
Advantages of SSF over SmF.
Parameters Solid State Fermentation (SSF)
Absence of free water Lower reactor volume required.
Negligible chances of contamination.
No foam formation.
Lower cost of treatment of liquid effluents.
Fermentation medium Low cost and natural
Minimal mineral supplement.
Natural Environment Solid nature of the substrate mimics the natural environment of
fungi.
Volumetric productivity Has been reported comparatively higher in many studies
Downstream processing Simpler and easier since the product is highly concentrated.
Environment aspects Use of natural wastes as substrate helps in biomass energy
conservation, waste management and pollution control.
Product quality Heat and pH resistant products reported in few cases.
begins with an introduction of the reactor type and highlights of recent
case studies, with emphasis on enzyme production. The case studies
present a holistic view of reactor configuration, effect of operating
conditions on process productivity, advantages and limitations.
Mathematical models are powerful tools which can aid in bioprocess
optimization, provide scale-up guidelines and facilitate bioreactor
control and automation. For instance, models could be embedded in
control schemes (e.g., Model predictive control, PID control) and the
resulting control algorithms shall control and provide automation,
thereby, significantly increasing bioreactor performance. Selected
modeling studies have been revisited and cited at the end of each ca-
tegory with an objective to address limitations, discuss scale-up stra-
tegies and promote greater interaction between biologists and en-
gineers. However, model assumptions and solution techniques are not
discussed in detail, and can be gleaned from the references cited herein.
Table 2 shows recent examples of SSF bioreactor used for enzyme
production, whereas, Table 3 cites recent examples of SSF bioreactors
employed for the production of spores, antibiotics, pigments, chemicals
etc.
2. Bioreactor classification
The bioreactors have been classified into following four categories,
based on their mode of operation.
I Tray bioreactor
II Packed bed bioreactor
III Air pressure pulsation bioreactor
IV Intermittent or continuously mixed SSF bioreactors
2.1. Tray bioreactor
SSF in trays has traditionally been used for the production of fer-
mented foods such as tempeh, miso, koji, and soy sauce (Zhu and
Tramper, 2013; Nout and Aidoo, 2011) in some Asian countries (Chen
and Zhu, 2013). Trays are generally made of wood, metal or plastic,
with or without perforations, packed with substrate-support and
stacked one above the other in temperature and humidity controlled
rooms (Fig. 1). Scale-up is generally achieved by increasing the surface
area and/or increasing the number of trays. The current section starts
with reports describing the utility of different substrate-supports for
enzyme production in tray bioreactor (TB). Emphasis has been on the
operating bed heights, chemical nature and initial moisture content
(IMC) of substrate-support and their effect on productivity. Operational
difficulties, issues related to poor O2 and moisture level, heat
17
Journal of Biotechnology 269 (2018) 16–34
Submerged Fermentation (SmF)
Relatively large reactor volume is required.
High water activities make the process highly susceptible to bacterial
contamination.
Extensive foam formation and high cost for treatment of liquid effluents.
Highly purified analytical grade chemicals are used which usually cost multiple
times higher than SSF media.
The dissolved nature of substrate does not provide the natural habitat for fungi.
Lower volumetric productivity is often associated with SmF for fungal based
products.
Product concentration and purification costs are higher. Generally defines the
process economics.
Significant environmental sustainability is not possible with the use of synthetic
and processed media constituents of high analytical grade
Compared to SSF, superior product quality has not been observed with SmF.
accumulation in bed and control strategies are discussed herein.
Laccase production by Trametes hirsute was studied using grape
seeds as substrate-support in TB (Rodríguez Couto et al., 2006). Use of
grape seeds as inert support over nylon cube sponge resulted in a
threefold increase in laccase production. Using the same organism, in-
creased laccase activity (12260 U L−1) was achieved when orange peel
was used as substrate-support in a TB of 1 cm bed height (Rosales et al.,
2007). High activity was attributed to high pectin and cellulose content
in orange peels and to the absence of mechanical stress. Application of
laccase production was extended on to the removal and de-colorization
of synthetic dyes in TB (0.5 cm thickness) under semi-solid-state con-
dition using Trametes pubescens (Rodríguez-Couto et al., 2009). Initially,
dyes were adsorbed on to dry sunflower shell seeds which were sub-
sequently used as solid support for fermentation. Remarkably high
laccase activity (40172 U L−1) was obtained when 0.5 mM Cu2+ and
50 μM tannic acid were added as supplements to the growth media on
the 3rd day of cultivation. TB was used for cellulolytic enzyme pro-
duction using a co-fermentation technique involving Trichoderma reesei
and Aspergillus oryzae (Brijwani et al., 2010). Soybean meal and wheat
bran (4:1) were used as substrate (1 cm height) and optimum operating
conditions of temperature (30 °C), moisture (70%) and pH (5.0) were
also obtained. These workers stressed on the importance of an appro-
priate C:N in substrate and concluded that the process not only fa-
cilitated high cellulase titres but also resulted in balanced production of
glucanase (endo- and exo-) and β-glucosidase, which is recommended
in biomass processing for biofuel production. Higher β-glucosidase le-
vels were reported (Dhillon et al., 2011b) during fermentation of apple
pomace using Aspergillus niger and Trichoderma reesei in TB. Still higher
β-glucosidase levels were obtained (91.8 IU g-fs−1) when Aspergillus
niger was cultivated in plastic trays (40 × 25 × 12 cm) (Dhillon et al.,
2011a) and the operating conditions were optimized using response
surface methodology technique. High IMC (> 70% w/w) resulted in
lower enzyme activities possibly due to low O2 levels, decrease in bed
porosity and substrate aggregation. This was an important observation
which asserts IMC, a critical design parameter for TB.
A modification of the traditional TB was proposed for spore pro-
duction by Clonostachys rosea mutant strain CRM-16 (Zhang et al.,
2014). Sporulation area in the bioreactor was two times more than a
traditional TB. Wheat bran and maize meal (3:1, w/w) were covered by
a porous polyethylene membrane both on top and the bottom. With just
two mixing events, sporulation was reported to be ten times greater
than the TB. The porous polyethylene membrane was also claimed to
lower the risk of bacterial contamination. Das et al. (2015) worked on
the production of fumaric acid on plastic trays (35 × 22 × 11 cm) using
Rhizopus oryzae 1526. Apple pomace ultrafilteration sludge and apple
S. Arora et al. Journal of Biotechnology 269 (2018) 16–34

Table 2
Recent examples of SSF bioreactors used for enzyme production.

Bioreactor types Substrate used Product Microorganism used Production level Reference

Tray Rice bran, wheat bran, Alkaline protease Aspergillus oryzae 74 U g-ds−1 Fath and Fazaelipoor
soybean meal and wheat (2015)
flour.
Tray Wheat bran Polygalactouronase Aspergillus oryzae 298 U g−1 Demir and Tari (2016)
Tray Sifted pine sawdust, rice Manganese peroxidase Irpex lacteus 950 U L−1 Zhao et al. (2015a,b)
straw and soybean powder.
Tray Palm kernel cake and Protease Aspergillus oryzae 319.3 U g−1 Tsouko et al. (2016)
Palm pressed fiber
Tray Wheat bran, orange peel Polygalatouronase Aspergillus giganteus 180 U g-ds−1 Ortiz et al. (2016)
and lemon peel
Tray Wheat bran, soybean hulls Glucoamylase, protease, Aspergillus awamori Increased enzyme activity in specific Manan and Webb
and rapeseed meal cellulase and xylanase and A. oryzae substrates (2016)
Column-tray Lemon peel pomace Pectinase Aspergillus niger 2181 U L−1 Ruiz et al. (2012)
Tray Wheat bran and linseed Phytase Rhizopus oryzae 148.98 U g-ds−1 Rani and Ghosh (2011)
oilcake
Tray Apple pomace β-glucosidase Aspergillus niger 64.18I U g-ds−1 Dhillon et al. (2011a,b)
Tray Tomato pomace Xylanase Aspergillus awamori 195 IU g-ds−1 Umsza-Guez et al.
(2011)
Tray Grape pomace and orange Xylanase Aspergillus awamori 42.64 IU g-ds−1 Díaz et al. (2013)
peels CMCase 2.16 IU g-ds−1
Packed bed Wheat bran and sugarcane Pectinase Aspergillus niger 22 U g−1 Finkler et al. (2017)
bagasse
Static bed Wheat bran Endoglucanase Aspergillus niger 29.8 IU g-ds−1 Farinas et al. (2011)
Packed bed Soybean bran Cellulolytic enzymes Trichoderma reesei 4.2 FPU g−1, 7.3 U g−1, 1734.8 U g−1 and Gasparotto et al.
NRRL-6156 2.5 U g−1 for filter paper activity, exo- (2015)
cellulase, xylanase and endo-cellulase,
respectively
Static bed with Wheat bran Endoglucanase Aspergillus niger 50.2 IU g-ds−1 Farinas et al. (2011)
forced
aeration
Packed bed Babassu cake Exoamylase Aspergillus awamori 73.4 U g−1 Castro et al. (2015)
Endoamylase 55.7 U g−1
Protease 31.8 U g−1
Xylanases 23.8 U g−1
Cellulases 6.2 U g−1
Packed bed Citrus pulp and sugarcane Pectinase Aspergillus oryzae 37 U g−1 Biz et al. (2016)
bagasse
Packed bed Wheat bran and sugarcane Pectinase Aspergillus niger 20 U g−1 Pitol et al. (2016)
bagasse
−1
Packed bed Pressmud Inulinase Kluyveromyces 300.5 U g-ds Dilipkumar et al.
marxianus (2013)
−1
Packed bed Copra waste Inulinase Penicillium rugulosum 239 U g-ds Dilipkumar et al.
(2014)
Packed bed Wheat bran and sugarcane Endoglucanase Myceliophtorasp I-1D3b 878 U g-ds−1 Zanelato et al. (2012)
bagasse
Packed bed Cane molasses and soybean Inulinase Kluyveromyces 436.7 U g-ds−1 Mazutti et al. (2010)
bran marxianus
−1
Packed bed Waste bread Glucoamylase Aspergillus awamori 130.8 U g Melikoglu et al. (2015)
Protease 80.3 U g−1
Packed bed Wheat bran α-amylase Bacillus sp. KR-8104 473.8 U g-ds−1 Derakhti et al. (2012)
Packed bed Wheat bran Phytase Aspergillus ficuum 87.75 U g-ds−1 Badamchi et al. (2013)
Packed bed Strain immobilised in Endo- and exo- Aspergillus nigerURM 1.18 U ml−1 Maciel et al. (2013)
orange peels Polygalacturonase 5162 4.11 U ml−1
Packed bed Polyurethane foam Tannase Aspergillus niger 7955 U L−1 Rodríguez-Durán et al.
(2011)
−1
Rotating drum Soybean meal Phytase Aspergillus niger 580 U g-ds Saithia and Tongta
(2016)
Rotating drum wheat bran Pectinase Aspergillus niger LB-02- 37% increase in pectinase recovery Poletto et al. (2015)
citric pectin SF
Rotating drum Soybean meal Amylase and Protease Aspergillus oryzae 85000–110000 U g-ds−1 Sukumprasertsri
7800-9000U g-ds−1 (2013)
Rotating drum Defatted rice bran Amyloglucosidase Aspergillus niger 886.2 U g-dm−1 Colla et al. (2016)
Exo-PG 84 U g-dm−1
Rotating drum Empty palm fruit bunch CMCase Penicillium 6.5 U CMCase Kim and Kim (2012)
fiber Avicelase verruculosum 6.8 U Avicelase
Xylanase 8.8 U Xylanase
Rotating drum Empty palm fruit bunch Endoglucanase Aspergillus niger 135 U L−1 Noratiqah et al. (2013)
fiber Exoglucanase 52 U L−1
β-glucosidase 161 U L−1
Rotating drum Palm oil lignocellulosic Cellulase Trichoderma 8.2 FPA g-ds−1 Alam et al. (2009)
biomass harzanium
(continued on next page)

18
S. Arora et al.
Table 2 (continued)
Bioreactor types Substrate used Product
Rotating drum Grape pomace and orange Xylanase,
peels exopolygalacturonase
CMCase
Rotating drum Grape pomace Pectinases, xylanases and
cellulases
Rotating drum Autohydrolyzed algae Fucoidanase
Gas double- – Cellulase
dynamic SSF
Rotating drum – Cellulase
Air pressure Corn cob Xylanase
pulsation Wheat bran
pomace were used as substrate. Fumaric acid yield doubled than what
was observed with SmF. Fath and Fazaelipoor (2015) proposed a novel
trickling TB which consisted of a medium reservoir, a bioreaction
chamber and a product storage tank. The bioreaction chamber housed
perforated trays (17 × 11.5 × 4.2 cm) that acted as the site of fer-
mentation and through which the crude enzymatic solution was made
to trickle down from medium reservoir in batch and semi batch fashion.
Rice bran (primary substrate) was supplemented with wheat bran (to
increase porosity), soybean meal (as nitrogen source) and wheat flour
(inducer for protease). The whole set-up was kept in an insulated wood
cabin at 30 °C and provision for exchange of gases was also provided.
High protease activity (748 U g-ds−1) was achieved on the 3rd day of
cultivation which was however not exceedingly high to that obtained in
flask studies (530 U g-ds−1). Operating bed heights were not mentioned
but high substrate loading (SL) may have been responsible for the
moderate increase. Countercurrent movement of trickling liquid and
fresh air might be an interesting prospect as this is expected to enhance
retention time of fluid in bed and increase interfacial area for heat and
mass transfer. Ruiz et al. (2012) proposed a column TB which consisted
of 8 perforated base trays in a cylindrical column during fermentation
of lemon peel pomace for pectinase (PG) production. Sterile moist air
was circulated through perforations to increase O2 availability and
circumvent bed drying which resulted in high PG activity (2181U L−1).
These workers also emphasized on the importance of substrate particle
size and its effect on bed porosity, heat and mass transfer, bed me-
chanical properties, and PG production. TB (32 × 40 × 5 cm) were
used (Virtanen et al., 2008) for the production of Phlebiopsis gigantia
spores, to be used as biofungicide. Its production using SSF has sig-
nificant commercial potential since yields are higher than SmF (Seiskari
et al., 1992) and the ‘root and butt rott’ caused by Heterobasidiun an-
nosum, a natural antagonist of P. gigantia, results in substantial capital
loss to the forest and wood industry in some European countries. Starch
mash was used as substrate and was supplemented with silica to retain
bed moisture. Spore viability of ∼5.4 × 106 C.F.U g-substrate−1 was
achieved, which was comparable to that obtained in packed bed bior-
eactor (PBR) and disposable plastic bags. These workers asserted that
although TB were simple in design and prevented overheating, their
operation was highly labour intensive since each tray had to be loaded,
monitored, emptied and cleaned individually. Moreover, low SL was
prominent as dead space between trays was necessary for uniform air
circulation. Decrease in lipase activity was reported (Vaseghi et al.,
2013), as substrate (sugarcane bagasse) bed height was increased from
0.5 to 3 cm in a TB consisting of three trays (35 × 25 × 5 cm) mounted
upon each other. Heat build-up within the bed was responsible for
decrease in enzyme production by Rhizopus oryzae. Xie et al. (2013)
studied the effect of SL on conidia production of Beauveria bassiana in a
TB containing rice. Spore yield of 2.70 × 1012 conidia kg-rice−1 was
reported when a 24 × 14 × 2 cm bed was employed. With increase in
bed thickness to 8 cm, the yield reduced to 1.02 × 1012 conidia kg−1
Journal of Biotechnology 269 (2018) 16–34
Microorganism used Production level Reference
−1
Aspergillus awamori 54.4 IU g-ds , Diaz et al. (2009)
8.77 IU g-ds−1,
3.69 IU g-ds−1
Aspergillus awamori 8.77 IU g-ds−1 Diaz et al. (2009)
54.42 IU g-ds−1
3.69 IU g-ds−1
Mucor sp. 3P 9.62 U L−1 Rodríguez-Jasso et al.
(2013)
Penicillium decumbens 18 IU g-ds−1 Chen et al. (2013)
JUA10
Trichoderma viride L3 – Chen et al. (2014a,b)
Thermomyces 8237 IU g-moldy bran−1 Yang et al. (2011)
lanuginosus
which was attributed to inefficient dissipation of metabolic heat.
Maximum bed temperature exceeded 10 °C from optimum (25 °C), most
likely impeding fungal growth. However, when the bed was cut into
100 pieces of size 6 × 4 × 2 cm with 0.5 cm gap between them, conidia
yield increased to 3.94 × 1012 and the maximum bed temperature did
not exceed 31 °C. Similar results were obtained during the production of
Bacillus licheniformis spores using multiple substrates (Zhao et al.,
2008). Spore yield of 1.1 × 1011 CFU g−1 was obtained with a bed
height of 5 cm, however, as the bed height was increased to 10 cm and
15 cm, the spore yield was decreased by 37% and 64% respectively. TB
of different sizes were used for the production of a thermo-tolerant and
acid stable phytase from Rhizopus oryzae (Rani and Ghosh, 2011). Op-
timized growth media consisted of wheat bran and linseed oil cake
(1:1), as main substrate. Different SL (50–1000 g) resulting in different
bed heights (0.2–2 cm) were tried. Increasing the substrate bed height
did not result in significant decrease in maximum phytase yield
(149.2 U g-ds−1). However, a 3.5 cm bed height halved the phytase
yield which was probably due to heat build-up and poor moisture
content (Arora et al., 2017). Similar observations were made during
production of thermo-tolerant phytase by Sporotrichum thermophile
(Singh and Satyanarayana, 2008). The enzyme yield decreased sig-
nificantly as the bed height increased beyond 1.5 cm.
The above studies revealed the drawbacks of employing high SL
rates in TB and necessitates the use of mathematical modeling ap-
proaches, involving system’s transport and kinetic parameters and op-
erating variables, to optimize bed heights and design suitable control
strategy. Rao et al. (1993) investigated the effect of model parameters
such as, bed diffusivity, maximum specific growth rate constant, max-
imum biomass concentration etc., on critical bed height (HC) in a TB.
These workers introduced the concept of HC in TB as shown in Eq. (1):
1
2.De Cg Y ⎞ 2
Hc = 2 ⎛⎜ ⎟
⎝ μmax Xmax ⎠ (1)
Where De is the effective diffusivity of bed, Cg is the atmospheric oxygen
concentration, Y is the yield of biomass, μmax is the maximum specific
growth rate constant and Xm is the maximum biomass concentration.
Using Eq. (1), the effects of model parameters on HC could be explored.
For example, a perforated tray shall double the bed height and which is
why industrial SSF process are generally performed on perforated trays.
Another important factor would be effective diffusivity of fermentation
bed, which in turn would be a function of void fraction, substrate
density and particle size. Similarly, a very fast-growing microorganism
put limits on substrate loading rates as heat dissipation becomes diffi-
cult.
Rajagopalan and Modak (1995) worked on heat and O2 transfer in
TB and took into account the conduction and metabolic heat generated
by microbial activity. Simulation results suggested that for optimum
temperature control, best strategy was to employ thin bed heights
19
 S. Arora et al.

Table 3
Recent examples of SSF bioreactors used for spores, antibiotics, chemical, ethanol and pigment production.

Bioreactor types Substrate Product Microorganism Production level Reference

Tray Rice Conidia Beauveria bassiana 3.92 × 1012 conidia kg−1 rice Xie et al. (2013)
Tray Apple pomace Fumaric acid Rhizopus oryzae 1526 52 g kg-ds−1 after 21 days Das et al. (2015)
Tray Corn forage Lignin degradation Trametes versicolor 71% lignin conversion after 7 days Planinić et al. (2016)
Modified Tray Wheat bran and maize meal (3:1, w/w) Spore Clonostachys rosea 3.36 × 1010 g-dm−1 Zhang et al. (2014)
Static Jar Rice, Corn, whole Sorghum grain, dehulled Pigments Monascus purpureus High Rubropuctamine production with rice as substrate. Srianta et al. (2016)
Sorghum grain and Sorghum bran
Packed bed Citrus processing wastes L-galacturonic acid Aspergillus niger ATCC 1015 and 2.14–2.35 mg g−1 h−1 Kuivanen et al. (2014)
engineered strains
Packed bed Wheat bran and wheat straw Lovastatin Aspergillus terreus 1.86 mg g-ds−1 Kumar et al. (2014)
Packed bed Cane bagasse Citric acid Aspergillus niger 55.90 g 100 g-ds−1 Kumar and Jain
(2008)
Packed bed Sugarcane bagasse and sunflower seed meal Biodiesel Burkholderi acepacia 95% conversion after 46h Salum et al. (2010)
Packed bed Rice straw powder (300 g kg−1) Spores Bacillus licheniformis 0.2 × 1011 CFU g-ds−1 Zhao et al. (2008)
Wheat bran (700 g kg−1)
Glucose (40 g kg−1)
Peptone (20 g kg−1)
Yeast extract (20 g kg−1)
Fixed bed Wheat bran Meroparamycin Streptomycin sp. Strain MAR 01 510 μg ml−1 El-Nagger et al.

20
(2009)
Glass columns with forced aeration Citric pulp, sugarcane molasses and methanol Citric acid Aspergillus niger (mutants) 278.4, 294.9 and 261.1 g CA kg−1 Rodrigues et al.
(2013)
Rotating drum Different biomaterials (fungal biomass, alkali Biosorbent Aspergillus niger > 60% metal absorption Dhillon et al. (2017)
insoluble material and acid and alkali insoluble
material)
Rotating drum Corncob wastes Red 40 dye degradation Trametes versicolor 96% dye degradation Jaramillo et al. (2017)
Stirred drum Oil palm fond petiole Red pigment Monascus purpureus 5.61 AU g-ds−1 Razali and Said
(2017)
Rotatory drum Apple pomace Citric acid Aspergillus niger 220.6 g kg-ds−1 Dhillon et al. (2013)
Rotatory drum Sugarcane bagasse Ethanol Kluveromyces marxianus 24 g L−1 Lin et al. (2013)
Rotary drum Rice by-product, whey and sugarcane bagasse Ethanol Aspergillus niger 11.7 g L−1 Rocha et al. (2013)
Stirred tank Corn grits Lactic acid Lactobacillus amylovorus 0.91 g-ds−1 Trontel et al. (2011)
Solid-state bioreactor with Wheat bran/rice bran/Soybean cake powder Spore Bacillus cereus DM423 (1.50 ± 0.07) x 1011 CFU g-ds−1 at 40 h Chen and He (2013)
honeycomb loading device
(HLD)
Air pressure pulsation Polyurethane foam Xanthan Xanthomonas campestris 42.62 g L−1 Zhang and Chen
(2010)
Intermittently mixed Winterization oil cake, sugar beet molasses and Sophorolipids S. bombicola ATCC 22214 0.235 g-dm−1 Jiménez-Peñalver
wheat straw et al. (2016)
Intermittently mixed Wheat bran and maize meal Spores Clonostachys rosea – Zhang et al. (2014)
Air solid fluidized bed Sugarcane bagasse Ethanol S, shehatae UFMG-HM 52.2 0.17 g L−1 h−1 Antunes et al. (2017)
Journal of Biotechnology 269 (2018) 16–34
S. Arora et al.
Fig. 1. Schematic diagram of a tray bioreactor and for an individual tray (adapted from
Ali and Zulkali, 2011).
(1–2 cm) and the headspace air temperature to be kept near to the
optimum for growth of microorganism. Increase in biofilm thickness
resulted in low O2 concentration in bed and which may well be un-
avoidable in TB. Importance of optimum operating temperature was
emphasized in a two-phase growth model (Ikasari et al., 1999) where
effect of temperature up-shift was studied on the growth kinetics of
Rhizopus oligosporus. Hyphal tips were exposed to elevated temperatures
regimes at different incubation times of 0–20 h, 20–30 h and 30–60 h.
These workers observed that exposure to elevated temperature (50 °C)
proved detrimental to organism which failed to recover on return to
37 °C. This was corroborated with increase in first order death rate
constant. Modeling approach was used (Rahardjo et al., 2005) to study
the effect of O2 concentration on hyphal elongation, branching fre-
quency and radial elongation rate of Aspergillus oryzae for α-amylase
production. It was observed that biomass and α-amylase production
were severely affected at low O2 concentration below 1%. An improved
model of energy balance (Eq. (2)) was proposed (Smits et al., 1999) by
incorporating removal of heat by evaporation to the model of
Rajagopalan and Modak (1995).
∂H ∂ 2T ∂2c wv
= rH + λ. + ΔHw . Dwv .
∂t ∂x 2 ∂x 2
Where, H is enthalpy of bed, rH is the metabolic heat generation rate, λ
is the effective heat transfer coefficient, the second and third term on
RHS represent conduction within the bed and heat transfer due to
evaporation and diffusion of water vapor, respectively. Simulation re-
sults suggested that the contribution of evaporative heat loss is not
likely to be a major factor if bioreactor is operated in an environment
with saturated air, which may be true with small bed heights in TB.
Figueroa-Montero et al. (2011) studied the effect of internal air
circulation by forced convection on heat and mass transfer in TB. As
shown in Fig. 2, three mechanisms contributed to the heat transfer in
the TB. (a) sensible heat transfer by conduction and convection from
top of the bed (Qsen,t). (b) sensible heat transfer by conduction and
convection from bottom of the bed (Qsen,b). (c) heat transfer due to
evaporation from the bed surface (Qevap). Energy and water balances
were made for the system, and bed temperature, bed moisture, cell
biomass and substrate concentration were measured experimentally.
Journal of Biotechnology 269 (2018) 16–34
Heat and mass transfer coefficients were therefore experimentally ob-
tained using the energy and mass balance equations and the reaction
stoichiometry. These workers emphasized on the effect of Reynolds
number (NRe), a function of airflow in the bioreactor headspace, on heat
removal and mass transfer and maximum possible bed heights. In-
creasing the NRe (2.5–2839) in the headspace increased the heat
transfer coefficient without drying the bed. Findings from their work
could be used to design operating bed height and air flow velocities in
other TB systems. Experimental estimation of process parameters, as
was performed in their work, is of paramount importance for the sa-
tisfactory validation of SSF bioreactor. Parameter values obtained from
related processes may significantly vary from an actual fermentation
process (Khanahmadi et al., 2006). Final water content in substrate bed
was assumed to be function of initial water content, water produced by
microorganism and the water loss due to evaporation. The model
(Figueroa-Montero et al., 2011) however did not incorporate the water
that would be used up in substrate hydrolysis and its uptake by new
biomass, which could be significant as was shown in the model of Nagel
et al. (2001a), where they estimated that water requirement for new
biomass is approximately 45% of that for the evaporative loss.
Although TB constitute a major proportion of commercial SSF pro-
cesses in fermentation industry (nee’Nigam and Pandey, 2009; Binod
et al., 2013), this is not without disadvantages. Heat transfer is mainly
through conduction and because of low thermal conductivity of sub-
strate; heat dissipation is often not efficient thus imposing limitations
on bed height. As a result, control of optimum temperature and
moisture content within the reactor bed is very limited at large bed
height. Moreover, TB requires large operational area and the process is
labour intensive. Most often the substrate requires separate sterilization
and the process is not contained. It is difficult to apply this technology
to sterile processes, except only if large aseptic rooms are built and
procedure and equipment’s are provided for the employees, which may
be prohibitive and highly costly.
2.2. Packed bed bioreactors
Characteristic feature of a PBR is forced aeration through static bed
which aids in replenishment of O2 and moisture, and mitigates accu-
mulation of heat and CO2. PBR are generally employed where mixing is
undesirable or deleterious for microbial growth. They offer better
control and facilitate higher SL than TB. Construction generally consists
of a cylindrical glass or metal tube/drum, which houses the substrate
and the walls of the cylinder or drum may be jacketed. Provision of
cooling plates may also be provided within the bed to facilitate efficient
heat transfer. Fig. 3 represents the traditional design of a PBR. The
section begins by emphasizing the role of forced aeration in PBR, where
some recent reports are reviewed for studying the effect of high air flow
rates (AFR) on bioreactor performance. This is followed by issues re-
lated to pressure drop, bed compaction, air channelling and strategies
(2)
to overcome them. Finally, modeling studies and control strategies
addressing the problems of heat build-up and heterogeneity are re-
viewed herein.
Effect of forced aeration was studied (Melikoglu et al., 2015) on the
production of glucoamylase and protease during fermentation of waste
bread pieces by Aspergillus awamori in a 0.5 and 1 L PBR. AFR of 1.5
vvm (volume of air under standard conditions per volume of medium
per minute) was found to be optimum and a model describing its effect
on enzyme production was developed which successfully predicted the
adverse effect of AFR below and above 1.5 vvm. Similar observations
were made during the production of cellulases from olive oil and wine
processing waste in a glass column. High AFR decreased cellulase
production, which was attributed to low bed water activity (aW) and
high shear stress on Aspergillus uvarum (Salgado et al., 2015). Inulinase
production was negatively affected at high AFR (Dilipkumar et al.,
2014), as the latter caused bed moisture to drop and increased shear
stress on fungus. These workers emphasized on the importance of IMC
21
S. Arora et al.
Fig. 3. Schematic diagram of a traditional packed bed bioreactor.
of copra waste, where a higher than optimum level (60%) resulted in
substrate agglomeration and depleted O2 level, whereas, low levels
were inhibitory for inulinase production. Choice for forced aeration
may also be governed by the type of product in question. Medeiros et al.
(2001) used two different AFR to test the production of volatile aroma
compounds (VAC) in a PBR during fermentation of cassava bagasse by
Kluyveromyces marxianus. They observed that lower specific AFR
(0.06 L h−1 g−1) resulted in higher concentrations of total VAC than
high AFR (0.12 L h−1 g−1), although bed moisture remained near op-
timum for both. Higher production was attributed to lower O2 exposure
to yeast, that resulted in higher concentrations of ethyl acetate, ethanol
and acetaldehyde.
A common issue encountered in a forcefully aerated PBR is high
degree of substrate compaction resulting in air channelling, pressure
drop across bed and subsequent heat and mass heterogeneity. These
problems were observed during the production of Iturin A, an anti-
fungal agent, by a strain of Bacillus subtilis (Piedrahita-Aguirre et al.,
2014). High AFR stimulated the production of poly-γ-glutamic acid
which increased bed viscosity, reduced free passage of air, caused high
pressure drop and consequently decreased oxygen uptake rate (OUR).
The problem was pertinent during pectinase (PG) production, by As-
pergillus niger, in a pilot scale PBR (200 L) (Pitol et al., 2016). 20 kg of
wheat bran (WB) at 30 °C, and with an IMC of 62% (w/w) resulted in
maximum PG productivity (1350 U kg−1 h−1) which was however
lower than that obtained in a lab scale column bioreactor
(1930 U kg−1 h−1). This decrease was attributed to substrate compac-
tion which resulted in shrinkage of bed along the walls of bioreactor
22
Journal of Biotechnology 269 (2018) 16–34
Fig. 2. Schematic representation of heat transfer
mechanisms in TB, adapted from Figueroa-Montero
et al., 2011. (Where, Ta, Ts and Tb are the tempera-
ture of inlet air, bed surface and bed, Ha and Hs are
humidity ratio in inlet air and bed surface, z is the
bed height, mb is the inert support mass and CPb is
the specific heat of bed).
that caused air channelling and bed temperature to rise up to 37 °C. To
address this problem, sugarcane bagasse (SB) was employed to enhance
bed porosity and allow uniform distribution of moist air. 10% of WB
was replaced by SB that avoided air channelling and maintained bed
temperature to near optimum (30 °C). However, this further decreased
productivity (810 U kg−1 h−1) as less WB was available for fermenta-
tion. Increase in SL (30 kg) again led to bed compaction, increase in
maximum bed temperature (43 °C) and moisture gradient. Productivity
increased to 1840 U kg−1 h−1 only when inlet air temperature was
cooled down to 24 °C during peak heat generation. However, issues
pertaining to bed shrinkage, heat build-up and heterogeneity still per-
sisted which suggested that under given operating conditions, high SL
rates were not suitable. Biz et al. (2016) made further inroads for pilot
scale production of PG, using Aspergillus oryzae and enhancing me-
chanical properties of substrate-support that not only resulted in higher
enzyme yields but also circumvented the problems encountered by Pitol
et al. Use of citrus pulp (51.6% w/w) and SB (48.4% w/w) as substrate
(15.5 kg) resulted in pectinase yield of 37 U g−1 which was higher than
obtained by Pitol et al. (20 U g−1). This is a classic example which
underscores the importance of suitable substrate parameters (e.g.,
substrate thermal conductivity, substrate density, substrate specific
heat, substrate particle size, void fraction) and kinetic parameters (e.g.,
specific growth rate constant, metabolic heat yield, maximum cell
biomass concentration) that may significantly affect metabolite yield
and productivity.
Production of hydrolases was investigated during fermentation of
babassu cake by Aspergillus awamori (Castro et al., 2015). Babassu cake
is rich in carbohydrate, known for induction and secretion of enzyme
pool, and exhibits good mechanical properties. PBR was employed to
overcome scale-up limitations encountered with TB, in their previous
study (de Castro et al., 2011). Except for xylanases, similar or higher
enzyme yields were reported for cellulases, proteases, endoamylases
and exoamylases. Interestingly, isoamylase production was only ob-
served with forced aeration. However, heat accumulation and hetero-
geneous bed temperature and aW profiles were prominent across bed
height, as a result these workers called for further improvements. In
PBR, bed temperatures can exceed to more than 20 °C than that of inlet
air (Ghildyal et al., 1994) which could potentially render 20–30%
biomass inactive, particularly in the upper regions of bed (Ashley et al.,
1999). Use of water jacketed system may not be preferred since radial
heat loss is often not significant with a large reactor diameter. Opera-
tion with high superficial air velocity (uS) can be one strategy that may
address heat build-up (Sangsurasak and Mitchell, 1995). Simulation
results on a growth model of Rhizopus oligosporus, including cell death
kinetics, showed that cell death could be minimized if uS of 0.4 m s−1 or
more were employed in 0.3 m bed height. However, this may cause bed
compaction, poor moisture levels and might be energy intensive. Other
alternatives were to cool the inlet air during peak heat generation and
maintain the aspect ratio ≤ 1:1 (Sangsurasak and Mitchell, 1995).
Sangsurasak and Mitchell (1998) worked on an improved model by
including ‘evaporation’ term in the energy balance. Simulation results
showed that convection and evaporation contributed significantly to
S. Arora et al.
Fig. 4. Schematic representation of differential volume element of substrate bed in a
cylindrical PBR and the directions of different modes of heat transfer mechanisms
(adapted from Sangsurasak and Mitchell, 1998).
heat loss. Fig. 4 shows the differential volume element of the substrate
bed in the cylindrical PBR for energy balance across the bed (Eq. (3)).
These workers recommended Eq. (3) as a useful tool for design and
operation for a PBR.
dT dT k dT d 2T d 2T
ρb Cpb + (ρa Cpa + ρa fλ ) VZ = b ⎛ ⎞ + kb ⎛ 2 ⎞ + kb ⎛ 2 ⎞
⎜ ⎟ ⎜ ⎟
dt dz r ⎝ dr ⎠ ⎝ dr ⎠ ⎝ dz ⎠
dX
+ ρs (1 − ϵ) Y
dt (3)
Where, ρb, ρa and ρs are the densities of bed, air and substrate, respec-
tively. Cpa and Cpb are the specific heat of air and bed, respectively, T is
bed temperature, Vz is the superficial velocity of air, kb is the thermal
conductivity of bed, f is the change in water carrying capacity of air
with temperature, λ is the enthalpy of vaporization of water, ε is the
void fraction and Y is metabolic heat yield.
Using pseudo-steady-state approximation, Weber et al. (1999) de-
veloped both enthalpy and water balance to study effect of bed tem-
perature and aw on sporulation of Coniothyrium minitans. Simplification
of enthalpy balance gave a temperature control strategy where AFR was
defined as a function of rate of metabolic heat generation (Eq. (4)) and
further simulations gave real time estimation of bed water content.
−rQ. H
Fair =
ha (Tout ) − ha (Tin )
Eq. (5) shows the simplified water balance equation worked out by
Weber et al. (1999):
d y (Tout ) − yw (Tin )
(1 − ϵ) (x ws . CS ) = (Ywo − x wx Yxo). (−ro) − Fair . w
dt H
Where, H is the height of bed, ha is the enthalpy of moist air, ε is void
fraction, Cs is the concentration of substrate, Yso, and Yxo are yield of
substrate consumption and biomass formation, respectively. yw is the
water content in air and xws is the water content of substrate-support.
Substituting Eq. (4) into Eq. (5) and further numerical integration gave
real time estimation of bed water content. The model assumed that
growth is limited by high temperatures and not by bed aW. However,
significant water loss would be expected where evaporation contributes
greatly (78%) to heat loss. Ashley et al. (1999) worked out a model
Journal of Biotechnology 269 (2018) 16–34
Fig. 5. Schematic diagram of ‘Zymotis’ packed bed bioreactor (adapted from Mitchell
et al., 2006).
describing heat control strategies in PBR. These workers argued that
when bed is aerated from bottom for the entire process, upward tra-
versing air gets hotter with time and loses its ability to cool the bed
after a HC and which is why top regions in a PBR experience over-
heating problems. Using process parameters from the work of Ghildyal
et al. (1994), these workers argued that overheating was inevitable
with unidirectional flow of air. Simulations with air reversal strategy at
different time intervals also proved unsatisfactory with central regions
of bed remaining above critical temperature. However, frequent mixing
was successful as it facilitated distribution of cooler media throughout
the reactor thereby enhancing convective heat transfer. To overcome
overheating in PBR, an advanced version of the packed bed was pro-
posed by Roussos et al. (1993), called ‘Zymotis’ (Fig. 5). Since this re-
actor had a unique design and could perform satisfactorily among the
existing PBR, its design, mode of operation and modeling studies de-
serve some attention here. The bioreactor was also patented and used
successfully by a German company ‘Prophyta' for the production of bio-
pesticides. As shown in Fig. 5, the bioreactor consisted of a rectangular
box, made up of acrylic sheets. An acrylic-dome shaped cover was
provided on to the top side of the unit to prevent the entry of atmo-
spheric air inside. The outer dome also housed the inlet and outlet of
water circulation circuits, exhaust gases outlet and a handle for lifting
purpose. The reactor consisted of various adjacent sections which were
separated by cooling plates (heat exchanger plates). These sections
contained fermentation media. Entry of moist air, with the provision of
control of flow rate and pressure, was facilitated from the bottom of
each section. The heat transfer mechanism involved conduction from
the substrate bed to the heat exchanger, convection and the evaporative
(4) heat loss. Dynamic heat transfer model were worked out (Mitchell and
Von Meien, 2000; Mitchell et al., 2002), for Zymotis and simulations
suggested reduced temperature gradient across substrate bed than tra-
ditional PBR. Investigation into optimal value for spacing between the
cooling plates revealed that in order to achieve high productivities on
an industrial scale or even at pilot scale, same spacing had to be em-
ployed between the plates as in the laboratory scale. However, insertion
(5)
of cooling plates, especially at higher scale may lead to low SL rates and
could be uneconomical. Roussos et al. (1993), in their evaluation of
Zymotis demonstrated that control of temperature of fermenting solids
by circulating cooling water through the heat exchanger plates proved
to be inefficient during active growth phase, hence, increase in aeration
rate had to be adopted. Furthermore, as the substrate load was in-
creased from 26.68 kg to 40.02 kg, the cellulase activity significantly
decreased. This was however attributed to inefficient heat transfer
during the substrate pre-treatment, a step which makes the substrate
23
S. Arora et al.
more amenable to microbial attack. The sterility of the vessel was
maintained by wiping the box, cover and the heat exchanger plates
which may well be extremely time consuming and unfeasible at a large
scale. Reports of contamination were observed when fermentation time
exceeded 72 h. The autoclaving or steaming of the fermenter was not
possible since it was made of acrylic.
SSF operation in packed beds is a significant improvement over TB
where higher SL rates are possible along with greater control of process
variables. PBR constitutes popular mode of operation where mixing is
undesirable. However, PBRs even at laboratory scale are confronted
with problems of heat build-up, substrate compaction, air channelling,
bed drying and process heterogeneity, thereby imposing limitations on
operating bed heights. Moreover, in situ fermentation operations such
as sterilization, inoculation, product removal and post fermentation
treatment of the bed looks cumbersome. Zymotis arguably has been the
most promising design among existing PBR with its ability to minimize
temperature gradients. However, further improvements in design and
operating strategies would be necessary for PBRs to fully realize the
industrial potential of SSF technology.
2.3. Air pressure pulsation SSF bioreactors
Bioreactors under this category employ periodic pulsation of air
pressure which may be coupled with forced air circulation to enhance
the microbial activity and mitigate process heterogeneity. Systems that
comprise both the pulsation event and forced air circulation are re-
ferred to as gas double-dynamic solid-state fermentation (GDD-SSF).
The high partial pressure of O2 in gas phase during the compression is
intended to increase O2 concentration in the bed while decompression
phase causes air to swell thereby facilitating removal of heat and CO2.
Repeated cycles of the two events simulate mixing action, with an ad-
vantage that forces acting are not shearing but normal. Since this ca-
tegory of bioreactor has received no or very little attention in SSF re-
views (Chen and He, 2012), the section starts with early reports where
it had been employed. Effect of operating variables such as pressure
amplitude (PA), duration, frequency, exhaust time and velocity of air
circulation on bioreactor performance has been reviewed. Heat and
mass transfer studies though only few available have been reviewed
next. The section concludes with a description and performance ana-
lysis of honeycomb loading device (HLD) (Chen and He, 2013), a
bioreactor under this category that works on the principle of air pres-
sure pulsation and forced air circulation.
A SSF bioreactor system was designed (Tao et al., 1996) where
surface area of bed, exposed to surrounding air, was increased by per-
iodic pulsation of air pressure. The pulsating action generated periods
of inhaling and exhaling which these workers referred to as ‘fermenter
breathing’, analogous to lungs action. To ensure O2 was not limiting, air
pressure was varied inside the bioreactor (Tao et al., 1999), which
consisted of perforated trays supported in a stainless-steel cylinder.
Effect of air pressure pulsation (APP) on cellulase production, by Tri-
choderma viride, was studied and threefold increase in cellulase pro-
duction was observed, compared to TB. Effect of GDD-SSF was studied
on cellulase production, by Penicillium decumbens (Fujian et al., 2002),
where 0.20 MPa PA and air circulation at 1.5 m s−1 resulted in high
productivity but further increase in PA resulted in disruption of mycelia
due to gas swell. Electron microscopy revealed that substrate was held
loose and this probably facilitated heat and mass transfer. Use of Air
Pressure Pulsation SSF (APP-SSF) was extended for the production of
alkaline protease using Bacillus pumilus (Aijun et al., 2005). Effect of PA
and its duration were studied on enzyme yield, productivity and SL.
With increase in PA (0.05 and 0.1 MPa), protease activities increased 63
and 95% respectively than at static bed operation. Increase in pulsation
duration caused substrate drying, whereas, a decrease likely resulted in
poor O2 levels. APP-SSF mimicked agitation action as bed was held
loose and incompact. Enzyme yield decreased significantly as bed
height was increased from 1.5 to 6 cm in static bed, whereas only a
24
Journal of Biotechnology 269 (2018) 16–34
marginal decrease was observed with APP-SSF. Feruloyl esterase pro-
duction by Aspergillus niger was studied during fermentation of rice
straw and wheat bran (4:1) (Zeng and Chen, 2009). Static flasks con-
taining substrate were kept in a 25 L cylinder where a PA of 0.2 MPa,
high and low-pressure duration of 20 s and 30 min, respectively, in-
creased enzyme yield and productivity, offered better temperature
control, circumvented steep gas gradients and enhanced respiration
intensity. Hongzhang et al. (2002) argued that frequency of APP should
be employed as a function of organism growth stage and carefully se-
lected, as high frequency may be energy intensive and deleterious to
microbial growth. These workers used GDD-SSF for production of Ba-
cillus thuringiensis (Bt) in a 70 m3 bioreactor, where numerous trays
were stacked in a stainless-steel cylinder. APP with an upper and lower
limit of 1.5 kg cm−2 and 0.05 kg cm−2, respectively, along with internal
air circulation was more effective and yielded maximum Bt activity
(18000 IU). Moreover, temperature gradients were minimized when
trays were kept in horizontal position (0.8 °C cm−1) than in vertical
configuration (4.2 °C cm−1). Yang et al. (2011) emphasized on the
importance of lower and upper limit of PA on production of a thermo-
tolerant xylanase by Thermomyces lanuginosus. Exposure to prolonged
periods of high pressure negatively affected fungal growth and enzyme
yield. Hendges et al. (2011) observed lower pectinase production by
Aspergillus niger under APP. Sudden pressure shock was understood to
be detrimental to fungal mycelia. These reports suggested that opera-
tion with APP should be designed carefully. High xanthan production
was observed by Xanthomonas campestris using polyurethane foam as
inert support (Zhang and Chen, 2010). Higher production, over static
bed, even at high initial glucose concentration (60–80 g L−1) was at-
tributed to better O2 transfer and biomass accumulation. Xanthan
production and OUR were significantly enhanced after 50 h. Similar
observations were made (Chen et al., 2014a) where cellulase produc-
tion was enhanced between 3rd and 5th day of cultivation, which was
the stationary phase of Trichoderma reesei. ATPase activity and cell
permeability were used as a measure of microbial metabolism and their
effect with pressure pulsation was analyzed. Increase in cellulase ac-
tivity corresponded with ATPase activity which suggested that external
stimulation might have enhanced metabolism. He and Chen (2013)
tested the performance of a pilot scale (800 L) GDD-SSF bioreactor for
the production of proteases, pectinases, glucoamylases and cellulases.
The bioreactor consisted of two cylindrical tanks where pre-inoculated
multiple trays could be stacked on a frame and rolled into tanks. In
comparison to static operation, glucoamylase and protease production
were enhanced ∼3 folds and pectinase (at 96 h) and cellulase could be
enhanced ∼2 folds. Moreover, fermentation time was shortened and
axial temperature gradients were reduced in all the four enzyme sys-
tems.
Chen et al. (2013) discussed the importance of exhaust time in APP-
SSF operation for cellulase production. Exhaust time was defined as a
function of pressure pulsation and vent aperture area and its effect on
production efficiency was analyzed. As the PA increased up to 0.2 MPa,
cellulase activity was enhanced. However further increase proved un-
desirable, as this resulted in rapid pressure changes leading to lower
enzyme activity. Vent aperture size was identified as an important de-
sign parameter that had a significant effect on cellulase production.
Using the exhaust time equation (Eq. (6)) for inflation and deflation
system, these authors presented exhaust time (t) as a function of vent
area (S) and bioreactor volume (V) for a pulsation amplitude of
0.20 MPa.
S = 5.45 × V / t (6)
Chen et al. (2005) studied temperature control with GDD-SSF and
found that PA of 0.20 MPa for 5 min duration along with an air circu-
lation rate of 1.5 m s−1 brought down the maximum bed temperature
from 53 °C (in static operation) to 33 °C. At still higher PA (0.30 MPa)
temperature control was satisfactory but large air pressure change
proved detrimental to fungal growth. Axial temperature gradients were
S. Arora et al.
reduced to 0.12 cm−1 which was remarkably improved compared to
trays (3 cm−1) (Rathbun and Shuler, 1983). Chen et al. (2014b) ex-
tended the analysis of GDD-SSF for mass distribution inside the sub-
strate bed. Bed moisture, cell biomass and cellulase activity were de-
termined using multivariate calibration models that involved use of
near infrared spectroscopy and chemometrics. Jian and Yang (2006)
estimated the contribution to heat loss by conduction, evaporation and
convection in static and APP-SSF for production of cellulases by Tri-
choderma koningii. APP-SSF was beneficial since heat loss through
conduction was enhanced (evident from higher wall temperature) and
total water loss doubled, as a result, 657 KJ more energy was dissipated
than in static bed. Zhao et al. (2015b) worked out a model to determine
optimum pressure pulsation frequency and its effect on fermentation
productivity. The model related pulsation frequency, organism meta-
bolism and heat removal through evaporation and convection. A steady
state condition was assumed, where metabolic heat production (rM) was
equal to the heat removal by evaporation (rE) and through exchange
with air (rA) (Eq. (7)), such that:
rM = rE + rA
Information regarding derivation of individual terms in Eq. (7) and
estimation of model parameters, can be found in work by Zhao et al.
(2015b). After substituting various parameter values in above equation,
pulsation frequency (f) and cycle time (t) were related to the CO2
evolution (mCO2) by Eq. (8):
4672.8*(0.2973*mCO2 − 1.7921)
f=
t *(389.0 + 118.4)
Rise in bed temperature, which was related to metabolic heat gen-
eration, was estimated to be a linear function of CO2 evolution. This
however may not necessarily be true for other systems and therefore
such relationship should be verified for each case. These authors ob-
served that when pulsation frequency was high (1/10 min−1), lag phase
was extended to 10 h since microorganism took longer to acclimatize to
high pressures. As a result, a constant pulsation frequency of 1/
20 min−1 was selected and bioreactor performance was analyzed.
Validation with optimum pulsation frequencies (derived from model)
resulted in better cellulase and Bt activity in respective bioprocess as
compared to static pulsation frequency. As corroborated by model (heat
balance) simulations, pulsation frequency could be varied according to
the growth phase of organism wherein a high frequency may be more
useful during log than lag or stationary phase.
Chen and He (2013) designed HLD device (Fig. 6) on the principle of
GDD-SSF. Bioreactor consisted of nine stainless steel tubes being laid on
a metal frame. The metal frame housed two steel discs as end shields
and nine grids were located and welded in between the shields. These
steel tubes had openings with a series of little holes on pipe walls. While
in operation, the inoculated media was transferred into HLD and the
device was pushed into a steam sterilized fermenter and the cover
Fig. 6. Schematic diagram of Honeycomb Loading Device (HLD) (redrawn from Chen and
He, 2013).
Journal of Biotechnology 269 (2018) 16–34
closed. Fig. 7 shows experimental set up for HLD. Performance was
tested by evaluating temperature variance (with GDD-SSF and through
a water jacketed system) and spore viability of Bacillus cereus DM423.
Maximum temperature variation with GDD-SSF was 7.7 °C and that
with circulating water was 19.8 °C. Also, the spore count was better
than what was achieved under static condition. The apparatus gave an
impressing loading coefficient of 66.8% (v/v), which is almost two-fold
higher than conventional bioreactors. Modeling approaches involving
heat and mass transfer studies may be of immense use in designing
optimum operating conditions and predict temperature variations
during scale-up. HLD was applied successfully in the industrialization of
cellulase and biopesticide.
Recent reports suggest that APP/GDD-SSF is a marked improvement
over conventional TB. However, there is a dearth of heat and mass
transfer modeling studies which could answer some important ques-
tions such as: what maximum bed heights are possible with APP for
given transport and kinetic parameters. What would be the effect of
operating variables on SL. Will APP-SSF along with moderate inter-
(7) mittent mixing significantly enhance productivity without affecting
fungal growth. Or will addition of inert support, such sugarcane bag-
gase, perlite, vermiculite, polyurethane, glass fiber (del Campo et al.,
2015) enhance medium porosity and reduce process heterogeneity. All
these questions only underscore the scope and immense research op-
portunities under this category. Fermentation operations such as sub-
strate pre-treatment, inoculation, product extraction etc., are performed
outside the cultivation chamber which may lead to contamination, re-
quire large sterile rooms, procedures and protocols for workers.
(8)
Therefore, development of modular bioreactors with APP/GDD-SSF,
where all the fermentation operation could be carried out in a highly
contained fashion, may be desirable.
2.4. Intermittent or continuously mixed SSF bioreactor
Bioreactors under this category comprise gentle agitation and forced
aeration to enhance heat and mass transfer, and microbial growth.
Mixing enhances convective transport since it increases the surface area
of substrate exposed to moist air and/or cooling fluid. However, the
challenge is to maximize productivity with minimum mixing events
since latter could potentially damage fungal mycelia and also be energy
intensive. Current section begins with reports on the effect of mixing on
biological activity, heat and mass transfer and process productivity in
rotating drum bioreactor (RDB) and other intermittently or con-
tinuously agitated systems. Few recent studies where RDB configuration
has been modified for use in waste treatment have been reviewed. Pilot
and industrial scale bioreactors that have come up over the years, under
this category, have been reviewed next. The section ends with discus-
sions on the utility of modeling studies and control system in designing
appropriate bioreactor configuration and operating conditions.
RDB comprises of a drum-shaped container that may be mounted on
a roller (rotating device) and generally consists of three subsystems i.e.
the wall of the drum, the headspace and the substrate. Air is typically
blown through the headspace above a tumbling bed of substrate par-
ticles and bioreactor may be intermittently or continuously rotated.
Mixing of substrate bed is generally facilitated by rotating action of
drum around its central axis (Fig. 8 a), however, stirred drum bior-
eactors may also be used where paddle mounted on a central shaft carry
out mixing with drum remaining static (Fig. 8 b). The drum may come
with internally screwed baffles or lifters (Fig. 8 c) of different sizes and
shapes to facilitate mixing.
A two-fold increase in cellulase production was reported in a 50 L
RDB, compared to flask studies, using 4 kg of empty fruit bunch as
substrate (Alam et al., 2009). The increase was attributed to better
aeration and mixing in RDB. Simultaneous saccharification and fer-
mentation of alkali pretreated sugarcane bagasse was performed in a
100 L RDB with internal baffles (Lin et al., 2013). To achieve high
ethanol productivity, a thermo-tolerant yeast, Kluveromyces marxianus
25
S. Arora et al.
Fig. 7. Schematic diagram for experimental set up for HLD (redrawn from Chen and He, 2013).
was employed which could grow optimally at temperatures required
(40–50 °C) for prolific saccharification. Gentle rotation at low intensity
and frequency resulted in high ethanol yield (79%). Effect of agitation
on the production of hydrolases was studied (Diaz et al., 2009) on a lab
scale RDB (0.25 L) where Aspergillus awamori was grown on grape po-
mace and orange peels (1:1). Maximum activities for xylanases, exo-
polygalactouronase and CMC were obtained at high AFR (120 and
200 mL min−1) and very low agitation rate (1 min day−1). Constant
agitation without aeration caused substrate agglomeration, resulting in
poor growth. Intermittent rotation was found favourable for citric acid
production during fermentation of apple pomace by Aspergillus niger
(Dhillon et al., 2013), however, continuous mixing resulted in 34%
reduction in citric acid production. Mixing resulted in significant re-
duction in endoglucanase, glucosidase and xylanase activity during
fermentation of wheat bran by Aspergillus niger (Cassaro et al., 2015).
26
Journal of Biotechnology 269 (2018) 16–34
Cellulase activity decreased by 17% with increase in mixing frequency
in a novel bioreactor using Aspergillus niger (Lee et al., 2011). Even if the
organism is unable to tolerate mixing, it may still be necessary to
aseptically mix the bed during inoculation (Moilanen et al., 2015) and
sampling (Lee et al., 2011) to ensure uniform distribution.
On the contrary, several reports have corroborated improvement in
heat and mass transfer and overall productivity as a result of mixing
action. Nagel et al. (2001b) observed that continuous mixing facilitated
heat and mass transfer and there was negligible damage to microbe as
the fungus mainly grew inside the wheat grain. Also, respiratory pro-
files of continuously mixed cultures were similar to those of the un-
mixed one. Effect of intermittent mixing (IM) was evaluated on so-
phorolipids production by Starmerella bombicola in 0.5 L flasks, using a
mixture of winterization oil cake and sugar beet molasses (4:1) as
substrate-support (Jiménez-Peñalver et al., 2016). Mixing action
Fig. 8. a. Schematic diagram of rotating drum bior-
eactor, b. Stirred drum bioreactor, c. Cross section of
RDB showing arrangement of baffles (c) (adapted
from Mitchell et al., 2006).
S. Arora et al.
enhanced sophorolipids yield and OUR by 31% and 15%, respectively,
in comparison to static operation. Effect of IM was studied on cellulase
production (Flodman and Noureddini, 2013) by Trichoderma reesei
using wet corn distilled grains in Erlenmeyer flasks and glass columns.
Mixing had an overall positive effect on microbial activity, however,
marginal (5–10%) decrease in cellulase activity was observed. Com-
pared to static bed, a 23% increase in lovastatin production, by Asper-
gillus flavipes, was observed when IM was coupled with forced aeration
(Valera et al., 2005). Various other reports corroborate that IM did not
negatively affect product yield (Lonsane et al., 1992; Mitchell et al.,
1988; Silman, 1980). No substantial decrease was observed in methy-
lesterase production, CER, OUR of Aspergillus tamari during IM of sub-
strate bed (Nava et al., 2011). Schutyser et al. (2003) argued that it may
be impractical to derive a general mixing strategy for a broad spectrum
of bioprocess and that the choice for mixing shall be a function of fungal
morphology, chemical and physical nature of substrate, bioreactor
configuration and mixing regime employed.
Few interesting configurations of RDBs have been reported for
biological treatment of pollutants. Nitric oxide (NO) removal efficiency
was evaluated in a rotary drum biofilter (Wang et al., 2006). Bioreactor
comprised of an aluminium chamber which housed a spongy material
with perforated plates at both ends. Inoculum consisted of a con-
centrated sludge derived from a waste water treatment plant and was
supported on the sponge material. The inside of the drum contained
nutrient medium which was distributed to growing cultures through
drum’s rotatory action. However, this also resulted in the formation of
liquid biofilm across the sponge which offered resistance to the in-
coming NO stream. High rotation speed increased thickness of liquid
film that resulted in low NO bioconversion. Rodriguez-Meza et al.
(2010) used a bench-scale RDB to study bioremediation of soil con-
taminated with total petroleum hydrocarbons (TPH). Removal effi-
ciency of TPH was analyzed as a function of length to diameter ratio (L/
D), rotational speed (N) and aeration in reactor. Slurry, filled to 30%
reactor capacity, consisted of mineral medium and polluted soil. 59.6%
of TPH removal was observed at L/D of 1.5 and with helical fillers this
was enhanced to 62%. Treatment efficiency strongly depended upon N
and aeration rate.
An industrial scale IM SSF bioreactor (15 ton) was successfully
employed for koji process by Nagata Brewing Industry Co. Ltd, Japan
(Sato and Sudo, 1999), however, detailed working operation was not
made available. Another bioreactor (1 ton) in this category was de-
veloped for single cell protein production (Durand and Chereau, 1988).
Mixing action was provided with screws that lifted the substrate with
their rotating action, and was coupled with intermittent water addition.
It was successfully extended for production of enzymes and bio pesti-
cides (Durand, 2003). Similar bioreactor configuration (10 ton) was
employed (Xue et al., 1992) for the production of microbial protein
using Aspergillus tamari. Pérez-Correa and Agosin (1999) used a 200 kg
capacity bioreactor for gibberlic acid production by Gibberella fujikori.
Unlike the one developed by Durand and Chereau (1988), agitator as-
sembly was fixed, whereas solid material was rotated. However, only a
40–60 cm bed height could be employed. Fig. 9 a. represents schematic
diagram for intermittently agitated and forcefully aerated bioreactors,
and most of the systems mentioned in this paragraph have similar de-
signs. Above mentioned bioreactors are however likely to be limited in
their capacity to provide sterile operation, simple product extraction
and residue treatment. Several reports are available, (Durand et al.,
1993; Mirón et al., 2010; Takashi et al., 2009; Agosin and Aguilera,
1998; Bandelier et al., 1997) where bioreactor was operated under
sterile conditions, however, working capacities were 50 kg or less and
hence there appears to be a trade-off between sterility and scalability.
Modular bioreactor configurations are promising prospects for next
generation SSF bioreactor system as they help circumvent process
heterogeneity (Thomas et al., 2013) and operate aseptically. Böhmer
et al. (2011) worked on a mixed modular bioreactor for laccase pro-
duction by T. hirsutae using pine wood chips and orange peels.
27
Journal of Biotechnology 269 (2018) 16–34
PLAFRACTOR, a modular design patented by Biocon Ltd.
(Suryanarayan and Mazumdar, 2001) claimed for a self-contained SSF
device that combined all fermentation operations i.e., sterilization, in-
oculation, cultivation, extraction and post extraction treatment, in a
single unit. It was validated for sterile production of proteases, cy-
closporine, amylases and lovastatin. Multiple modules were stacked
vertically (Fig. 10a), where each module consisted of a base plate and a
frame which formed the sides of a structure that served as a container
for holding media (Fig. 10c). The base plate consisted of two separate
set of channels i.e., communicating (carrying nutritive, extractive,
sterile fluids) and non-communicating channels (carrying heating and
cooling fluids) (Fig. 10b). Mixing in each module was achieved with the
help of two concentric shafts, at the centre of mixing arrangement. A
working bed height of 4–8 cm was assumed in each module and the
temperature control was claimed by conduction, which however is the
least contributor in heat removal (Gutierrez-Rojas et al., 1996). Use of
mathematical models could be of great utility as they would help op-
timize SL. Random and un-optimized bed heights could result in det-
rimental temperature especially at top of the bed.
Novozymes Bio A/G patented (Andersen et al., 2013) a novel
bioreactor design with an objective to maximize automation in opera-
tion. As shown in Fig. 11, the bioreactor comprised of an upper and
lower compartment separated by a perforated plate. The upper com-
partment housed the substrate-support and was the site of fermentation,
whereas lower chamber facilitated transfer of sterile air to the upper
chamber. Each compartment housed one or more apertures, of which at
least one was used for air supply, and could be controlled by an electric
or pneumatic valve allowing change of direction of air flow across the
perforated plate. Access ports were provided to enable withdrawing of
solid samples via utensils or vacuum. The upper compartment also
housed one or more nozzles for the supply of water, inoculum, ex-
tracting fluid etc. Provision for agitators was made to reduce or elim-
inate differential environment across the bed. Bioreactor units were
connected to fermentation stations for supply/exhaust of liquid/gases,
extraction and washing. The transport and handling of bioreactor units
was assigned to automated guide vehicles and industrial robots. Mul-
tiple such units could be employed (10–2000) depending upon the scale
of operation. As with PLAFRACTOR, all the fermentation operations
could possibly be carried out, aseptically, in a single unit. The hor-
izontal expansion of the bioreactor (100–400 cm or 150–300 cm) was
greater than the vertical height (1 m, 2 or 2.5 m) and was estimated to
replace 50–100, 2 kg plastic SSF bags. Bed heights of 10–50 cm were
claimed, which may well be possible since similar bed heights have
been employed successfully in PBR (Biz et al., 2016). However, op-
timum substrate bed height shall also be governed by system’s transport
and kinetic parameters and the operating conditions. As modeling
studies and subsequent control strategies have shown (von Meien and
Mitchell, 2002; von Meien et al., 2004), it is prudent to optimize SL
based on system parameters and operating variables. Here, inlet AFR,
temperature and humidity, mixing regime, direction of air flow and the
system’s substrate and kinetic parameters can be used to design oper-
ating bed heights, which may well be greater than claimed by the in-
ventors.
In RDB, useful space for fermentation is usually 30% of total drum
volume (Chen and He 2013). As corroborated by modeling studies
(Stuart and Mitchell 2003), high SL may lead to process failure unless
reactor design and operation are backed by sound knowledge of heat
and mass characteristics, mixing behaviour and control strategies. Jin
et al. (2010) investigated the effect of air spargers and lifters on mass
transfer coefficient (KLa) during bioleaching process in RDB. Zhang
et al. (2012) extended mass transfer studies in 18 L bioreactor (Jin
et al., 2010) and developed a correlation between sherwood number,
reactor configuration and operating parameters for scale-up. They ob-
served that KLa was significantly enhanced by the number of lifters only
at higher aeration rate that increased internal convection and prevented
bubble to coalesce. The effect of increased lifter width on mass transfer
S. Arora et al.
Fig. 10. a. Schematic representation of PLAFRACTOR bioreactor showing multiple modules stacked vertically. b. Plate schematic. c. Arrangement of plate and frames to form vertical
stack. (redrawn from Suryanarayan and Mazumdar, 2001).
was more pronounced at higher rotational speeds. Wang et al. (2013)
studied the effect of operating conditions and reactor configuration on
power consumption in a RDB. These workers proposed a scale-up
strategy where power number was correlated with reactor design for
liquid and solid/liquid operations. Liu et al. (2013) did extensive work
on power consumption in a 703 L RDB (Jin et al., 2010) and concluded
28
Journal of Biotechnology 269 (2018) 16–34
Fig. 9. (a) Schematic diagram of a forcefully aerated
and intermittently mixed bioreactor (b) schematic
representation of gas and solid phases in the inter-
mittently mixed bioreactor (adapted from Mitchell
et al., 2006).
that specific power requirement was significantly less than stirred tank
reactor of similar size. AFR, drum rotational speed and SL were ac-
counted in Peclet number (Pe) and its behaviour was used for esti-
mating KLa (Hardin et al., 2002). Using this approach, KLa for different
RDBs could be calculated at different Pe values, thus simplifying com-
parative studies and scale-up. However, these workers recommended
S. Arora et al.
experimental determination of parameters before applying it for dif-
ferent RDB configurations. Hardin et al. (2000) improvised on the
modeling work of Saucedo-Castaneda et al. (1992) and developed a
design tool which they called as dimensionless design factor (DDF).
DDF was defined as the ratio of kinetic (rate of metabolic heat gen-
eration) and transport (rate of heat removal) processes, in reactor, and
was used to select operating variables such as AFR, inlet air tempera-
ture and humidity to control bed temperature and guide scale-up.
Nikakhtari et al. (2014) studied the effect of SL, rotational speed and
bead loading on KLa in bead mill and baffle RDB. These workers pro-
vided scale-up strategies by correlating KLa with Froude, Reynolds and
Schmidt number. Marsh et al. (2000) studied mixing behaviour in RDB
and determined axial dispersion coefficient (D). Correct estimates of D
could predict mixing speed and time required for complete distribution
of inoculum, water or nutrient within substrate bed, as well as the
degree of substrate agglomeration. However, estimates were made in
the absence of microorganism and mixing characteristics may con-
siderably differ in an actual fermentation. Discrete particle simulation
(DPS) was employed (Schutyser et al., 2002, 2003) to characterize
mixing in two and three dimensions in the presence of Aspergillus niger.
Tensile strength of aerial mycelia of A. niger at different fermentation
times was obtained and used in DPS in order to optimize mixing events.
Based on the physical characteristics of particles the model could pre-
dict interaction, corresponding movements and also the size and
quantity of aggregates obtained after the first mixing event. Schutyser
et al. (2002) extended DPM to three dimensions with inclusion of axial
mixing and mixing was characterized for different reactor
Fig. 12. Schematic diagram of different mechanisms contributing to water balance during SSF of substrate particle in RDB (adapted from Nagel et al., 2001a).
29
Journal of Biotechnology 269 (2018) 16–34
Fig. 11. Schematic representation of modular bior-
eactor adapted for automation (Novozymes Bio A/G)
(adapted from Andersen et al., 2013).
configurations. Results implied that mixing was more homogenous and
fast with curved baffles in RDB and discrete particle simulation tech-
nique can be a useful guide in order to scale up RDBs and other mixed
SSF bioreactors.
Stuart and Mitchell (2003) did a comprehensive study on the
modeling of RDBs. They characterized models into three subsystems i.e.
substrate bed, headspace and bioreactor wall and developed energy
balances for them. The experimental results co-related well with model
predicted values. Simulated results showed that temperatures greater
than 45 °C were reached which were detrimental to fungal growth. Half
and one third of the fungal biomass was predicted to be dead at 10 and
50 rpm, respectively. Control strategies such as lowering inlet air tem-
perature, employing higher flow rates and reducing the initial loading
to half did not work. However, a combination of these three along with
lower ambient temperature did bring the maximum temperature down
to 36 °C. This shows the utility of modeling in SSF systems, but also
highlights the difficulty in controlling bed temperature in RDBs at high
substrate loading rates. The model could further be modified con-
sidering the heat transfer in axial direction, the effect of shear forces on
fungal growth, volume losses of substrate to CO2 as these factors are
likely to affect the transfer coefficient. Mathematical modeling in RDB
was extended for control of temperature (Nagel et al., 2001b) and
moisture (Nagel et al., 2001a). These workers emphasized on the im-
portance to distinguish between extracellular and intracellular water
content. Fig. 12 shows the schematic overview of different contribu-
tions in water balance on SSF of moist substrate in RDB. Water required
for substrate hydrolysis (1), metabolic water generation (2), water
S. Arora et al. Journal of Biotechnology 269 (2018) 16–34

uptake by microorganism (3) and evaporative water loss (4) was ac- forcefully aerated PBR. Simulations showed that varying inlet air
counted in the overall water balance (Eq. (9) and Fig. 12): temperature and keeping it saturated along with intermittent water
dWwh supply was a good strategy but irrespective of the control strategy
= Fair . (CWin − Cwout ) + Xw, x . Y x O . rO2. Mwx − Y W . rO2. MWw employed, temperature gradients could not be avoided when a 2 m bed
dt 2 O2
height was employed. This only underscored the utility of such tech-
+ Yhyd. Y S . rO2
O2 (9) niques while designing bioreactor operation.
Intermittently mixed SSF bioreactors offer better control than TB
Where, Wwh is the extracellular water content, Fair is the volumetric
and PBR, and also augur high SL rates. Limitations with some of the
air flow, Cw is the water concentration in air, Xw,x is the water content
earlier reported designs are non-sterile operation, cumbersome product
of biomass, Yx/O2 is the yield coefficient for biomass on oxygen, rO2 is the
extraction and post fermentation residue treatment. Modular design,
oxygen consumption rate, Mwx is the molecular weight of water, YW /O2 is
such as those patented by Biocon Ltd. (Suryanarayan and Mazumdar,
the yield coefficient for water on oxygen, Yhyd is the water required to
2001) and Novozymes Bio A/G (Andersen et al., 2013) are promising in
hydrolyze starch and YW /O2 is the yield coefficient for substrate on
their ability to scale-up and aseptically perform all the fermentation
oxygen. These workers suggested the use of water and elemental bal-
operations in a single module. Automation ensures that process runs are
ance for online control of water activity. Wang et al. (2010) worked on
homogenous, less labour intensive and less injury prone to workers.
the modeling of RDB for ethanol production under anaerobic condi-
However, such system should be validated for various bioprocesses and
tions. The experimental results agreed well with the predicted values.
supported by suitable models and control strategies. At present, very
However, the biomass declined after 20 h could not be explained
few performance reports are available for automated modular system
probably since the model did not consider cell death kinetics. Decline in
and vast scope for reactor development and validation exist under this
growth rate may be attributed to the heat accumulation or poor mass
category.
transfer of solutes/gases.
Air-solid fluidized bed bioreactor (ASFB) is another category in SSF
Although IM makes it possible to replenish and homogenously dis-
bioreactor where substrate and microorganism are constantly kept in a
tribute water within the bed, it is not an easy task to aseptically de-
fluidized state by action of upward flow of air. The fluidized state ef-
termine online bed aW during actual fermentation. Khanahmadi et al.
fectively increases the surface area of substrate available for microbial
(2006) estimated moisture content of bed by determining inlet and
growth (dos Reis and Silva, 2011) and efficient heat transfer is also
outlet gas temperature. A model for water balance was used to derive
achieved due to a higher degree of turbulence arising from high su-
relationship between the rate of change of water and dry matter with
perficial air velocities. Fig. 13 shows schematic diagram of ASFB.
rate of metabolic heat generation.
However, its application is limited mainly for the production of volatile
A model for water balance was used to derive relationship between
compounds and the process is highly energy intensive. There is almost
the rate of change of water (Eq. (10)) and dry matter in the substrate
no recent report available for SSF bioreactors under this category; as a
bed with rate of metabolic heat generation (Eq. (11)), respectively.
result, it is not dealt in detail here. However, its performance com-
dW parison with other SSF bioreactor is shown in Table 4.
= −3.19 × 10−7. Qm
dt (10)
3. Conclusion
ΔMdm = −7.3 × 10−8. Qm (11)
Where W is the total water content of substrate, ΔMdm is the total loss of Over the years, SSF bioreactors have been in the process of rapid
dry matter and Qm is the total heat removal from bed. Qm (t) was de- modifications and advancement so as to maximize the productivity and
termined using two thermocouples at inlet and outlet at different time increase the commercial utilization of SSF processes. It includes the
intervals. The moisture content of the bed could then be determined as development and exploration of new processes employing different
a function of time using Eq. (12): engineering tools to accomplish the desired outputs. With increasing
W improvement and application of rational methods in engineering, SSF
x w (t ) = can reach higher levels in industrial standardization. The above-
W + Mdm (12)
Although these generalizations were arrived after numerous as-
sumptions and simplifications, they can still serve as useful tool in order
to monitor and control aw during fermentation. However, it was as-
sumed that air is saturated and remains in thermal equilibrium with
solid which may not hold true where dry air is used to cool the inter-
mittently mixed bed. As shown in Fig. 9b, (von Meien and Mitchell,
2002) air and solid were considered two different phases, not at equi-
librium, and energy and water balances were developed for them, se-
parately. The model predicted heat, water and biomass profiles in axial
direction, although the model could also be modified to include radial
gradients. O2 was not considered to be limiting which may be a valid
assumption with forced aeration operations. Model assumptions,
equations and numerical solution techniques can be accessed in their
work (von Meien and Mitchell, 2002). Control strategy based on model
simulations suggested satisfactory control of moisture in the bed with
fewer mixing events. However, at low mixing frequency, the bed tem-
perature quickly returned to a high value and a better control strategy
was necessary.
Control strategy holds paramount importance especially with op-
eration in large scale bioreactors where significant radial and axial
gradients are expected. von Meien et al. (2004) tested proportional
integral derivative (PID) and dynamic matrix control (DMC) strategy to Fig. 13. Schematic diagram of air-solid fluidized bed bioreactor (adapted from Mitchell
et al., 2006).
the model (von Meien and Mitchell, 2002) for intermittently mixed

30

Table 4
Performance comparison of SSF bioreactors.
Performance Bioreactors Types
parameter
Tray
Heat transfer Primarily through
capability conduction, low thermal
conductivity of substrate
often leads to inefficient
heat dissipation.
Scalability Mostly done by increasing
the surface area and the
number of trays
Sterilization/ Not suited for sterile
Containment applications and the
31
issues process is not contained.
Loading Capacity Low substrate bed loading
due to limitations in
operational bed heights.
Modularity A single tray my serve as a
module so the system can
be modular in nature.
In-situ product May not be feasible
recovery
In-situ residue May not be feasible.
treatment
Maintenance Generally, maintenance is
requirements not intensive.
Damage to fungal No damage to the fungal
mycelia mycelia due to shear.
Packed Bed
Conductive, convective and
evaporative heat transfer at play, but
at large scale the process is often
confronted with bed compaction & air
channeling. Therefore, maintenance
of optimum bed temperature and
moisture is challenging.
Due to issues of bed compaction, air
channeling, heterogeneity, traditional
PBB are not suited for scale-up.
In-situ sterilization may be achieved
by injecting high pressure steam,
however this may not be feasible with
high bed loading.
High substrate loading coefficients are
generally not possible due to problems
of bed compaction and air channeling.
Zymotis among PBR is partly modular
in nature.
May not be feasible.
May not be feasible.
Due to its simple design, maintenance
is generally not intensive.
No damage to the fungal mycelia due
to shear.
Air Pressure Pulsation & Forced Air
Circulation
Forced air circulation and periodic
pulsation of air pressure employed
to achieve high degree of
convection. Limited reports are
available on heat transfer at
industrial scale.
Scalability is generally obtained by
increasing the number and surface
area of trays since increasing air
pressure beyond a certain limit is
detrimental.
Since most fermentation operations
are carried outside the cultivation
chamber, current APP-SSF reactors
do not provide contained
environment.
Impressive loading coefficient of
66.87% has been reported for
Honeycomb loading device (HLD).
A single tray my serve as a module
so the system can be modular in
nature.
May not be feasible
May not be feasible
Regular monitoring and
maintenance may be necessary as
the system involves the use of high
air pressure.
Fungal mycelia could be disrupted
owing to high air pressure pulsation
and circulation velocity and time.
Rotating Drum Bioreactors
Heat transfer is achieved
through conduction, convection
and evaporative cooling. At
high substrate bed loading
temperature control is off limits.
The working volume is usually
30% of the reactor volume.
RDBs are generally not suited
for scale-up.
In situ sterilization may be
achieved by injecting high
pressure steam. The process can
be operated in a contained
environment.
Low substrate bed loading.
Usually useful space for
fermentation comprises only
around 30% of total volume.
RDBs are not modular in nature.
Has been possible in some
reactor types.
Has been possible in some
reactor types.
Generally, not intensive but
may be required in large scale
RDB with mixers.
Shear forces come into play.
Trade-off between the rotational
speed and fermentation.
Intermittently Mixed Forcefully
Aerated
Mode of heat transfer includes
conduction, convection and
evaporative heat loss. Mixing action
increases the surface area of substrate
exposed to incoming moist air. This
expedites heat transfer.
Potentially scalable if the design and
operational strategies are guided by
design equations.
In-situ sterilization has been reported
with modular bioreactor designs.
However, in most of the designs, trade-
off between scalability and sterility is
observed.
Substrate bed loading generally higher
than TB, PBR and RDBs.
Except PLAFRACTOR most other
designs are not modular.
Has been reported in some reactor
types (e.g., PLAFRACTOR).
Has been possible in some types. (e.g.,
PLAFRACTOR).
For bioreactors with large ancillary
equipments, extensive maintenance
may be required by skilled workers.
The mycelia may be subjected to
disruption during mixing.
Optimization of mixing events
becomes critical.
S. Arora et al.
Air-Solid Fluidized Bed
Efficient heat transfer achieved
through forced convection as
the bed is kept in a fluidized
state by the action of upward
flow of fluid.
The present designs are not
amenable to scale-up because
of high shear stress and very
high energy requirements.
In situ sterilization may be
possible by sending in high
pressure steam.
Low bed loading, since large
reactor volume is needed for
fluidization of substrate.
The existing bioreactors under
this category are not modular
in nature.
Possible in case of volatile
products.
May be possible.
Continuous monitoring and
maintenance may be required
by skilled workers.
Intensive shear forces into
play, bioreactor may not be
suited for aseptate fungi.
Journal of Biotechnology 269 (2018) 16–34
S. Arora et al.
mentioned bioreactors, categorized on the basis of their operating
mechanisms, represent a holistic picture on the existing technology and
what can indeed be done to truly realize its potential at the industrial
scale. The relevant details clearly suggest that most current available
design models neglect many of the important observable facts which
are important for scale-up. The integration of maximum possible fea-
tures in a single bioreactor system along with mathematical models
aiding in automation and control of the fermentation parameters and
thereby facilitating its potential to be scaled-up to the production level,
is desired to cash in on the immense socio-economic opportunities that
SSF technology holds. However, a lot of investigations are still needed
to be carried out to identify sustainable processes and developments to
maintain productivity and quality of the products for intensive bio-
technological applications.
Conflict of interest
All authors declare no competing interest.
Acknowledgments
Authors gratefully acknowledge the constant support provided by
Indian Institute of Technology Roorkee (IITR) and Ministry of Human
Resource and Development (MHRD).
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