Bioresource Technology xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

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

Review

Waste biorefineries using filamentous ascomycetes fungi: Present status

and future prospects
Jorge A. Ferreira, Amir Mahboubi, Patrik R. Lennartsson, Mohammad J. Taherzadeh ⇑
Swedish Centre for Resource Recovery, University of Borås, SE 50190 Borås, Sweden

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

 Filamentous ascomycetes can be

Ethanol Organic waste materials
cores of lignocellulosic ‘‘waste Gluconic acid Xylanases
biorefineries”.
 Filamentous ascomycetes are sources Metabolites Enzymes
of cellulases and xylanases.
Ascomycetes
 Aspergillus spp. are producers of citric, Cellulases
Itaconic acid Biorefinery
gluconic and itaconic acids. Citric acid
 Fusarium spp. and Neurospora spp. are
potential good ethanol producers. Animal feed Fatty acids
 Protein- and lipid-rich biomass as a Fungal
second product of ‘‘waste biomass
Human
biorefineries”. consumption
Amino acids

a r t i c l e i n f o a b s t r a c t

Article history: Filamentous ascomycetes fungi have had important roles in natural cycles, and are already used indus-
Received 30 December 2015 trially for e.g. supplying of citric, gluconic and itaconic acids as well as many enzymes. Faster human
Received in revised form 26 February 2016 activities result in higher consumption of our resources and producing more wastes. Therefore, these
Accepted 1 March 2016
fungi can be explored to use their capabilities to convert back wastes to resources. The present paper
Available online xxxx
reviews the capabilities of these fungi in growing on various residuals, producing lignocellulose-
degrading enzymes and production of organic acids, ethanol, pigments, etc. Particular attention has been
Keywords:
on Aspergillus, Fusarium, Neurospora and Monascus genera. Since various species are used for production of
Ascomycetes
Biomass
human food, their biomass can be considered for feed applications and so biomass compositional char-
Enzymes acteristics as well as aspects related to culture in bioreactor are also provided. The review has been fur-
Metabolites ther complemented with future research avenues.
Waste biorefinery Ó 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Ascomycetes as core biocatalysts in ‘‘waste biorefineries” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Fermentation with filamentous fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4.1. Cellulases and related enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5. Ascomycetes for ethanol and organic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5.1. Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5.2. Weak organic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

⇑ Corresponding author.
E-mail address: Mohammad.Taherzadeh@hb.se (M.J. Taherzadeh).

http://dx.doi.org/10.1016/j.biortech.2016.03.018
0960-8524/Ó 2016 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Ferreira, J.A., et al. Waste biorefineries using filamentous ascomycetes fungi: Present status and future prospects. Bior-
esour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.03.018
2 J.A. Ferreira et al. / Bioresource Technology xxx (2016) xxx–xxx

5.2.1. Citric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

5.2.2. Gluconic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5.2.3. Itaconic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6. Cell mass. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.1. Ascomycetes biomass for human consumption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.2. Ascomycetes biomass for feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7. Future prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
8. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction can be used for production of superabsorbents (Zamani, 2010), can

be obtained via hydrolysis of chitin from Aspergillus spp. cell walls
The cycles of e.g. carbon and nitrogen in nature have been and these ascomycetes can also be used for production of kerati-
tremendously affected by human activities. In nature, there is no nase hydrolysates (Pandey et al., 2015). Monascus spp. have been
waste, but just the resources for other species. However, the rapid important sources of pigments for the food industry and together
exploration rate of Earth’s resources has been steadily increased, with Aspergillus spp, Fusarium spp. and Neurospora spp. have been
giving rise to depletion concerns and to a rapid generation of waste a source of different human food products (Ferreira, 2015). How-
products. The exploration of fossil fuels is of special relevance to ever, unicellular ascomycetes, that is, yeasts such as Saccharomyces
the carbon cycle, since they have been the main source of energy spp, Pichia spp, and Yarrowia spp. have also been reported to be
and chemicals worldwide and probably the main culprit of the potential sources of organic acids (such as a-ketoglutaric acid, lac-
world rising CO2 emissions (Ferreira, 2015). Similar facts are also tic acid, malic acid and pyruvic acid), polysaccharides such as b-
valid for other elements such as nitrogen, phosphorus, and sulphur glucan, proteins such as collagen, polyunsaturated fatty acids, ster-
due to extensive human exploration of natural resources other ols (e.g. squalene) and lipids (e.g. ceramides) (Pandey et al., 2015).
than fossil fuels. Therefore, there is a need to find alternative The substrates for industrial production of value-added prod-
sources of fuels and chemicals, to improve the processes involving ucts by ascomycetes include mainly refined sugars (Pandey et al.,
the use of resources in order to produce more from less, and to 2015). Therefore, there has been an increasing interest to use other
cope with the increasing amount of waste materials via their con- more cost-effective substrates such as industrial waste materials
version to value-added products in a ‘‘waste biorefinery” concept. mostly based on industrial residuals and lignocelluloses. In this
Research has armed society with diversified tools to give regard, ascomycetes can potentially play a crucial role as core bio-
increasingly higher impetus to the production of fuels and chemi- catalyst in these ‘‘waste biorefineries” given their ability to pro-
cals within a biorefinery context as an alternative to refineries duce enzymes that can break down these recalcitrant structures.
based on fossil fuels. Among the range of strategies developed, By using filamentous ascomycetes, their biomass, normally rich
the biological treatment of a wide range of substrates to produce in proteins and lipids, can represent another value-added product
a panoply of value-added products occupies a relevant position. of the biorefinery. The filamentous growth enables easier biomass
Microorganisms, as metabolically versatile biocatalysts, have been separation from the medium than that if unicellular microorgan-
playing a crucial role in this regard where a variety of products isms are used (Ferreira, 2015).
including enzymes, antibiotics, organic acids, and human food have The present review reports recent developments regarding the
already been successfully produced (Gibbs, 2000). Thus, microor- production of organic acids and ethanol where such type of litera-
ganisms are already directly contributing to the world economy. ture overview is not, to the best of our knowledge, available for the
Nevertheless, as new microorganisms are being isolated world- latter. Especially Aspergillus, Fusarium, Neurospora, and Monascus
wide, the range of substrates that can be used as well as the range are considered. Taking into account the potential of the fungal bio-
of products that can be produced have steadily been increasing. mass for the feasible establishment of ‘‘waste biorefineries”, fer-
Hence, the full potential of the microorganisms is far from reached. mentation strategies and compositional characteristics are also
Filamentous fungi, such as those belonging to the Zygomycetes provided. Moreover, the potential of the ascomycetes for produc-
and Ascomycetes groups, have made great contributions within a tion of cellulases and xylanases has been approached. Future
wide range of industrial sectors. A previous review article from research avenues for the production of the value-added products
our research group has put together some value-added products using ascomycetes have also been included.
produced by zygomycetes using a wide range of substrates
(Ferreira et al., 2013). Comparatively, the ascomycetes are a wider
group of microorganisms where, reasonably, many of them have 2. Ascomycetes as core biocatalysts in ‘‘waste biorefineries
also been of special interest (Ferreira, 2015). The contribution of
ascomycetes to the white biotechnology can be traced back to Filamentous ascomycetes such as Aspergillus spp., Fusarium spp.,
the production of antibiotics by Penicillium chrysogenum, which Monascus spp. and Neurospora spp. are versatile fungi able to grow
together with Aspergillus oryzae, are among the most studied fila- in an expanding array of different substrates. The carbohydrates
mentous fungi at industrial scale (Gibbs, 2000). Aspergillus spp. is range from hexose and pentose monomers to disaccharides such
involved in the production of many value-added products includ- as lactose (Angumeenal and Venkappayya, 2005), cellobiose
ing enzymes such as amylase, protease, lipase, phytase, lactase (Bansal et al., 2014) and sucrose (Bari et al., 2009). In addition to
and catalase. They can also be used for production of cellulase be able to consume the monomers released during the hydrolysis
and xylanase where Trichoderma spp. are also used (Pandey et al., of lignocellulosic materials, the ascomycetes can also grow on car-
2015). Besides, Aspergillus spp. are responsible for a major fraction bohydrate polysaccharides such as xylan, arabinan and glucan
of commercial production of organic acids including citric acid, (Ferreira, 2015). By solid-state fermentation, they can even grow
gluconic acid, itaconic acid and they are also potential sources of on non pretreated substrates such as wheat bran (Bansal et al.,
malic and oxalic acid (Pandey et al., 2015). Moreover, chitosan, that 2014) or wheat straw (Panagiotou et al., 2011).

Please cite this article in press as: Ferreira, J.A., et al. Waste biorefineries using filamentous ascomycetes fungi: Present status and future prospects. Bior-
esour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.03.018
 J.A. Ferreira et al. / Bioresource Technology xxx (2016) xxx–xxx 3

This growth versatility is arguably related to the different fast degradation and fermentation of more recalcitrant structures
enzymes these filamentous ascomycetes can produce depending has been a major research bottleneck.
on the substrates they grow onto. This capacity entails high poten- Filamentous ascomycetes can also play a smaller role in already
tial for these fungi to play core roles within ‘‘waste biorefineries” established industrial processes. For instance, side-streams namely
through valorisation of waste materials from different industrial thin stillage and whole stillage of the ethanol production process
sectors. For instance, filamentous ascomycetes can grow on agri- from sugar-rich or starch-rich agricultural crops can be converted
cultural left-overs such as wheat (Panagiotou et al., 2011) and corn to value-added products by filamentous ascomycetes (Ferreira,
(de Almeida et al., 2014) straw, wheat bran (Bansal et al., 2014), 2015). The inclusion of a new biological conversion step in an
rice hulls (El-Metwally et al., 2015), sugarcane bagasse (Jabasingh established facility is arguably faster to be realised than an entire
and Nachiyar, 2011) or corn cobs (Panagiotou et al., 2011). The new facility; some of the equipment is at place rendering lower
use of these materials would allow the full crop to be used for investment costs (Lennartsson et al., 2014). For instance, if ethanol
biotechnological applications as well as lower the pressure on side-streams are converted to ethanol and biomass for feed by fil-
the use of sugar-rich crops or starch-containing grains that can amentous ascomycetes, the distillation column for the former, and
alternatively be used for human consumption (Ferreira, 2015). Fil- the dryer for the latter, are already available.
amentous ascomycetes could also be used for valorisation of Due to their metabolic versatility, filamentous ascomycetes can
wastes generated after handling of fruits and vegetables including produce various value-added products from waste materials such
banana, orange, pineapple, carrots, onions and potato peels (Bansal enzymes, organic acids, ethanol and biomass for feed applications.
et al., 2014), empty fruit bunches from palm oil industry, or apple The use of waste materials is of special importance either for
pomace (Dhillon et al., 2011b). Other examples of waste materials reduction of the production cost of already available industrial
that filamentous ascomycetes can grow on include tea waste products or for the establishment of new processes producing
(Sharma et al., 2008), waste office paper (Ikeda et al., 2006), other value-added products. Clearly, filamentous ascomycetes
lactose-rich wastes from dairy industries such as cheese whey detain high potential to be core biocatalyst in such ‘‘waste biore-
(Angumeenal and Venkappayya, 2005), cream or crème fraiche fineries”. The range of possible substrates and produced value-
(unpublished results of the authors), and even spent grains from added products in filamentous ascomycetes-based ‘‘waste biore-
the brewery industry (Xiros and Christakopoulos, 2009). However, fineries” is presented in Fig. 1.
the economical feasibility of using ascomycetes for production of
value-added products from lignocellulosic materials will arguably 3. Fermentation with filamentous fungi
be limited by the level of expressed enzymes that will dictate the
required time for hydrolysis and fermentation. Actually, finding The cultivation of filamentous fungi is generally carried out via
microorganisms with natural powerful enzymatic machinery for submerged fermentation (SmF) or via solid-state fermentation

Citric acid
Gluconic acid
Itaconic acid
Filamentous Kojic acid
Metabolites
ascomycetes Oxalic acid
enzymes Malic acid
Pigments
Ethanol
Filamentous
Raw materials Hydrolysis ascomycetes Biomass Feed
Wheat straw cultivation Chitin
Wheat bran Hexoses
Corn straw Rice hulls Pentoses Amino acids
Cellulose Glucan Variable-length Lipids
Xylan Arabinan saccharides Fatty acids
Cellulose Sugarcane bagasse Sterols
Orange peel Banana peel
Enzymes Amylases
Pinneapple peel Onion peel
Cellulases
Potato peel Corn cobs Xylanases
Keratin Carrot peel Proteases
Apple pomace Empty fruit bunches Lipases
Tea waste Waste office paper
Phytases
Cheese whey Cream
Crème fraiche Laccase
Brewer spent grain
Thin stillage Catalase
Whole stillage
Sago starch Thatch grass Keratinase
Coir pith Lactose
Sucrose
Fig. 1. Potential substrates and produced value-added products in filamentous ascomycetes-based ‘‘waste biorefineries”.

Please cite this article in press as: Ferreira, J.A., et al. Waste biorefineries using filamentous ascomycetes fungi: Present status and future prospects. Bior-
esour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.03.018
4 J.A. Ferreira et al. / Bioresource Technology xxx (2016) xxx–xxx

(SSF). The SmF has a wider biotechnological application probably the production of enzymes in order to degrade more complex sub-
due to better heat and mass transfer and culture homogeneity ren- strates entails higher energy expenditure by the cells. Thus, good
dering it a more reliable, reproducible, flexible and easier to mon- access to ATP generating processes is required, stressing further
itor character (Fazenda et al., 2008). However, constraints exist the importance of the supplied air (Lennartsson et al., 2014).
especially in larger scale. These constraints are mostly related to SSF has attracted increasing interest over the years as an alterna-
the morphology of the filamentous fungi: they can grow as uni- tive to SmF processes for production of value-added products. SSF
form and long filaments evenly distributed through the medium involves the growth of microorganisms on moist solid substrates
or the filaments can get entangled into clumps or pellets (Gibbs, similarly to their natural habitat, in the absence of free flowing
2000). The rheological properties of the medium (particularly the water (Barrington et al., 2009). The degradation of the substrate will
viscosity) are influenced by the fungal growth form. When fungi be catalysed by free extracellular enzymes or enzymes organised in
give rise to dense mycelial suspensions, the medium is more vis- cellosomes (Section 4), produced by the growing microorganism.
cous and so oxygen and other mass transfer resistances can Thus, SSF is influenced by the strain and substrate in addition to
become limiting factors. The problem is exacerbated when fungi several process parameters including carbon and nutrient composi-
are grown in stirred-tank reactors. The filaments can wrap around tion, moisture content, particle sizes, incubation temperature, pH
the internal parts of the bioreactor including impellers, baffles, pH, and inoculum density (Bari et al., 2009; Pandey et al., 2001). Advan-
O2, CO2, or temperature probes leading to low-performance tages of SSF include lower energy requirements and so lower oper-
biotechnological processes (Gibbs, 2000). When fungi grow as pel- ation costs, less wastewater produced (Pandey et al., 2001), and
lets, the media are generally less viscous due to the lower impact of higher productivities (Sreedharan et al., 2016). Nonetheless, low
the pellets in the bulk medium (Gibbs, 2000). The growth of fungi surface aeration (the air provided and made available to growing
as pellets is generally preferred for biotechnological application. ascomycetes on moisturised substrate surfaces is crucial for high
However, pellets can suffer shear stress and, according to their size process performances), temperature gradients, moisture variations
or fluffiness, hollow oxygen centre can arise for larger pellets and and restricted gas exchange can hamper high SSF performances as
substrate and oxygen transfer rates limitations can occur when reported for citric acid production by A. niger in a column bioreactor
fungi grow as compact pellets (Gibbs, 2000). Generally, small fluffy (Barrington et al., 2009). SSF has been used for food fermentation,
pellets are the most suitable form for high-performance fermenta- enzyme production, mushroom production, mould-ripening of
tions (Ferreira et al., 2013). For instance, the pellet form is viewed cheese and partial composting of agricultural residues (Barrington
as the most advantageous fungal growth morphology for enhanced et al., 2009). Additionally, SSF has widely been used for production
production of citric acid by Aspergillus niger (Dhillon et al., 2013b). of enzymes for lignocellulose degradation that will be used in a sec-
Medium supplementation of lower alcohols, such as methanol has ond step where the monomers generated by the enzymes will be
been reported to induce pellet morphology (Dhillon et al., 2011a). converted to value-added products such as ethanol using yeast.
When grown in bioreactors, the supplied air plays a crucial role The production of a homogeneous and high volume of spores for
for optimal cultivation performances. For instance, the production conversion of glucose to gluconic acid by intracellular glucose oxi-
of organic acids as being dependent on the tricarboxylic acid cycle dase can also be carried out by SSF (Ramachandran et al., 2006).
intermediates or on enzymes that use oxygen as substrate, is a Another example of application of spores produced by SSF includes
strict aerobic process. Therefore, obtaining high oxygen transfer those of Trichoderma spp. that function as biological control agents
rates is crucial for good process performances. At bioreactor scale, in agricultural and forest pest management. Trichoderma spp.
higher oxygen transfer rates demand increased power input; for shares around 50% of the market of fungal biological control agents,
instance by faster stirring if a stirred-tank reactor is used (Okabe mostly as soil/growth enhancers (Verma et al., 2007). Fungal-based
et al., 2009). Although stirred-tank reactors have widely been used biological control agents detain broader spectrum regarding dis-
in biotechnological processes, bubble columns have alternatively ease control and production yield compared to that of bacteria
been constructed due to their simpler design, that is, absence of (Whipps and Lumsden, 2001).
internal mechanical parts and so the constraints when cultivating
filamentous fungi are considered to be lowered (Yoshida, 1988). 4. Enzymes
Airlift bioreactors have also been developed which have an internal
or external loop giving rise to a different mixing pattern in compar- Filamentous ascomycetes have gathered intense research inter-
ison to that in a bubble column. This, in turn, has been shown to est due to the cocktail of enzymes they can produce when ade-
lead to better oxygen and other mass transfer rates (Merchuk quately induced. Their relevance is further stressed considering
and Siegel, 1988). Using airlift bioreactors can represent an impor- that the cheaper substrates sought to be used for production of
tant step towards cheaper industrial processes since its energy value-added products are at a great extent lignocellulose- or
demand is about one third of that needed for running a stirred- starch-based substrates. Taking into account the available research
tank reactor (Träger et al., 1989); the medium mixing is promoted during the last decade, the present review could be only based on
by the supplied air and further medium density differences. The production of some lignocellulolytic enzymes namely cellulases or
lower power requirement with concomitant better performances xylanases. An overview was tentatively made upon different sub-
by an airlift bioreactor in comparison to a stirred-tank reactor strates and cultivation parameters towards improved production
has been shown by research towards gluconic acid production with of these lignocellulolytic enzymes since they dominate the recent
Aspergillus terreus (Okabe et al., 2009). Nonetheless, the better sub- research by the four genera. The use of SSF for enzyme production
strate and oxygen transfer rates when using an airlift bioreactor seems to play a more prominent role when compared to the avail-
instead of a bubble column might depend on the experiment set- able research works using alternatively SmF. Here, filamentous
up. Ferreira et al. (2015) have reported similar performances when fungi have advantage over unicellular microorganisms such as bac-
growing Neurospora intermedia in thin stillage for production of teria and yeasts due to their extracellular enzymatic system cou-
ethanol and biomass for feed using an airlift bioreactor and also pled with hyphal penetration (Sharma and Arora, 2015).
when the bioreactor was used as a bubble column. Moreover, the
supplied air is crucial for consumption of xylose, a very prominent 4.1. Cellulases and related enzymes
pentose in lignocellulosic materials and that filamentous fungi can
convert to ethanol, which is not possible under anaerobic Filamentous ascomycetes are capable of growing both directly
conditions due to redox imbalance (Lennartsson, 2012). Besides, on lignocellulosic materials or after those had been through a

Please cite this article in press as: Ferreira, J.A., et al. Waste biorefineries using filamentous ascomycetes fungi: Present status and future prospects. Bior-
esour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.03.018
 J.A. Ferreira et al. / Bioresource Technology xxx (2016) xxx–xxx 5

Table 1
Enzyme activities for different filamentous ascomycetes when grown in different substrates under both solid-state fermentation (SSF) and submerged fermentation (SmF).

Ascomycete Substrate Mode of operation Endoglucanase Exoglucanase b-Glucosidase Xylanase References

A. nidulans NaOH-pretreated sugarcane bagasse SF, SSF 28.8 U/g – – – Jabasingh and Nachiyar (2011)
MTCC344
A. niger FGSCA733 Jatropha seed cake + 10% SF, SSF 3974.0 U/ga 6087.0 U/g Ncube et al. (2012)
H2SO4-pretreated thatch grass
A. nidulans NaOH-pretreated coir pith SF, SSF 28.6 U/g 10.2 U/g 4.3 U/g Jabasingh (2011)
A. niger NS-2 Wheat bran SF, SSF 463.9 U/g 101.1 U/g 99.0 U/g Bansal et al. (2014)
A. niger MA1 Rice hulls SF, SSF 15.0 U/g 3.4 U/g 49.1 U/g El-Metwally et al. (2015)
F. verticillioides Corn straw SF, SmF 8.0 U/mL 0.6 U/mL 0.3 U/mL 114.0 U/mL de Almeida et al. (2014)
F. oxysporum F3 Wet exploded pre-treated wheat SF, SmF 9.1 U/mL – 0.3 U/mL 34.3 U/mL Panagiotou et al. (2011)
straw + corn cobs (1:2)
F. chlamydosporum Sugarcane bagasse + wheat bran SF, SSF 281.8 U/g 95.2 U/g 135.2 U/g 4720.0 U/g Qin et al. (2010)
N. crassa DSM Wheat straw + wheat bran SF, SSF 492.8 U/g 1.1 U/g 26.7 U/g 297.8 U/g Dogaris et al. (2009)
1129

SF—Shake-flasks.
a
Enzymatic activity for the whole cellulase system.

pretreatment step. In both cases, a second biocatalyst is often used At industrial scale, SmF is the preferred strategy due to afore-
to convert the released simpler sugars to value-added products. mentioned advantages, despite the long fermentation times with
This is a very prominent case where Aspergillus spp. or Fusarium low production in comparison to that when SSF is used. The latter
spp. hydrolyse lignocellulose-based substrates and yeast convert has more recently been dominating research towards production
the released sugars to ethanol. The ascomycetes can also be used of cellulases and xylases due to lower costs and higher enzyme
within the concept of consolidated bioprocessing (CBP) where production (Sreedharan et al., 2016). A panoply of different ligno-
the fungus is responsible for the hydrolysis of the substrate and cellulosic substrates has been studied for enzyme production using
conversion of the simple sugars to value-added products (Jouzani both strategies. Research towards production of those enzymes by
and Taherzadeh, 2015). The ascomycetes can also be used for iso- ascomycetes in focus in this review, has at all times been domi-
lation of cellulases and related enzymes retaining high enzymatic nated by studies using Aspergillus spp. most likely due to the fact
activities that can serve different purposes in a variety of industrial of being a well-known industrial microorganism. Over the past
sectors. Cellulases can be used in textile, paper and pulp, food and decade the cellulase activities obtained when using Aspergillus
animal feed, fuel, chemical, waste management, and pharmaceuti- spp. ranged from 3 to 321 U/mL where the highest values (83–
cal industries (Jabasingh and Nachiyar, 2011). The industrial pro- 321 U/mL) were obtained during SmF using wheat or maize straw
duction of cellulases is presently dominated by Trichoderma spp. as substrates whereas coir waste, banana peel and grass gave rise
and Aspergillus spp. (Pandey et al., 2015). to the lowest activity values (3–12 U/mL) (Sreedharan et al.,
The complete hydrolysis of lignocellulosic substrates to mono- 2016). It is worth mentioning that among those substrates just coir
meric sugars to be converted to value-added products is dependent waste suffered a pretreatment step before enzyme production dur-
on the availability of a consortium of different enzymes by the ing Aspergillus spp. growth. As shown in Table 1, the cellulase sys-
growing microorganism which directly imposes the first constraint tem produced by some Fusarium spp. when grown on wheat straw
for feasible industrial realisation. Such enzymatic consortium or pretreated wheat straw gave rise to around 10 U/mL. Over the
includes delignification enzymes (lignases), cellulose-hydrolysing last decade, cellulase activities of Aspergillus spp. when grown in
enzymes (cellulases) and hemicellulose-hydrolysing enzymes SSF have been within the range 3–581 U/g of dry substrate, where
(hemicellulases which includes variable enzyme classes where the highest values were obtained when using corn stover or wheat
xylanases are of utmost importance due to xylan predominance) straw as substrates and the lowest values achieved when grass,
(Sreedharan et al., 2016). Ascomycetes are not generally good can- wheat bran, or combinations of wheat bran with rice straw or
didates for growth on lignocellulosic materials with high lignin wheat straw (Sreedharan et al., 2016). However, as it can be
contents when compared with white-rot fungi basidiomycetes observed in Table 1, higher cellulase activities were obtained with
(Kersten and Cullen, 2007). However, the comparatively slower wheat bran or its combination with wheat straw when other Asper-
growth in culture of the latter discourages their bioprospecting gillus spp. as well as Fusarium spp. and Neurospora crassa were
(Banerjee et al., 2010). used. Xylanase activities are comparatively higher than cellulase
The produced cellulases in fungi are available extracellularly activities and also varies among substrate and strain used
and have three components namely endoglucanase, exoglucanase, (Table 1).
and b-glucosidase (Sreedharan et al., 2016). In a production as Clearly, the production of enzymes is dependent on various fac-
single-product context, aerobic fungi are preferred than anaerobic tors including substrate, strain, cultivation strategy, medium sup-
bacteria and fungi since their cellulase are extracellular, adaptive plementation, and cultivation parameters. Moreover, the
in nature and usually secreted in large quantities during growth. existence or not of a pretreatment step, in order to remove the lig-
In contrast, cellulases in anaerobic bacteria and fungi are organised nin and to make the cellulosic component more accessible to the
into tight multi-enzyme complex, often membrane bound as cel- fungus, can also influence the final production of enzymes. Such
losomes, being difficult to recover individual active enzyme species improvement has been reported for sugarcane bagasse (Jabasingh
(Gincy et al., 2008). Some recent examples of activity for those cel- and Nachiyar, 2011) and coir pith (Jabasingh, 2011) although for
lulase components plus xylanase, from different substrates, have the latter, 11 days were needed to achieve the highest enzyme
been gathered in Table 1. The potential of the ascomycetes is there- activity. Several studies over the years have been focused on the
fore huge since the addition of enzymes mainly for hydrolysis of optimisation of cellulase and xylanase production by the
lignocellulose is always a hurdle for feasible scale up. ascomycetes genera here in focus under SSF and SmF considering

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esour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.03.018
6 J.A. Ferreira et al. / Bioresource Technology xxx (2016) xxx–xxx

temperature, pH and medium supplementation. Since constraints within a biorefinery context where value-added products are pro-
such as poor aeration and decreased dissipation of heat are verified duced from the enzymatically released simpler sugars. However,
in SSF, the weight or bed height of the substrate has also been there seems to be a lack of research studies regarding the hydrol-
included in the optimisation studies. Some interesting outputs ysis of lignocellulosic materials using SSF or SmF at larger scales.
include the increase in xylanase production by Aspergillus nidulans Moreover, higher enzyme activities were reported for agricultural
when Jatropha curcas seed cake was supplemented with 10% of wastes such as wheat straw, wheat bran or corn stover than those
acid-pretreated thatch grass or ammonium chloride, whereas no reported for other lignocellulosic substrates. Therefore, optimisa-
positive influence on cellulase concentration was observed tion studies are also needed in order to show a potential wider
(Ncube et al., 2012). Bansal et al. (2014) have carried out an exten- application of ascomycetes cellulases and xylanases. Nonetheless,
sive study on the influence of supplementation of wheat bran for taking into account the importance of a direct use for the
production of cellulase by a strain of A. niger. The influence of a lignocellulose-based materials increasingly generated, extensive
wide range of carbon, nitrogen, phosphate, sulphate, and surfac- research is expected to be performed in order to hopefully fill in
tant sources as supplementation to wheat bran on cellulase pro- those gaps.
duction has been investigated. Supplementation with cellobiose
led to the highest activity of endoglucanase, whilst cellulose led
5. Ascomycetes for ethanol and organic acids
to the highest activity of exoglucanase and salicin to the highest
b-glucosidase activity. Peptone as nitrogen sources lead to the
Due to their enzymatic capabilities, filamentous ascomycetes
highest activity of endoglucanase, whereas tryptone led to the
have widely been investigated for production of metabolites such
highest amount of exoglucanase and b-glucosidase. Among the
as ethanol and organic acids using mostly lignocellulose-based
phosphate sources examined, K2HPO4 had a stimulatory effect on
materials. Nowadays, the entire commercial production of organic
the production of cellulase. When testing different sulphate
acids including citric, gluconic and itaconic acids is ensured by
sources, MnSO4 led to the highest activity of endoglucanase, whilst
Aspergillus spp. using, at great extent, refined sugars (Pandey
CuSO4 led to the highest amount of exoglucanase and (NH4)2SO4 to
et al., 2015). Therefore, the possibility to produce these organic
the highest activity of b-glucosidase. Surfactants are thought to
acids within, for instance, lignocellulose-based ‘‘waste biorefiner-
alter the permeability and fasten the secretion of enzymes
ies” would lower the overall production costs with concomitant
(Deswal et al., 2011); triton-X enhanced the production of
increase in the range of possible applications. The same holds for
endoglucanase and b-glucosidase, whilst SDS enhanced the activity
the production of ethanol industrially dominated by the unicellular
of exoglucanase. Activities reported in Table 1 relate to the opti-
ascomycete Saccharomyces cerevisiae using sugar-rich or starch-
mised medium via supplementation of wheat bran. When Bansal
rich agricultural crops (Ferreira, 2015). Recent research towards
et al. (2014) increased the amount of wheat bran from 5 to
production of ethanol has been carried out using Fusarium spp.
1000 g either in shake-flasks or shallow trays decreased enzyme
and Neurospora spp. (Table 2), whilst Aspergillus spp. expectedly
activities were observed. The crude enzyme were used for saccha-
still dominates the recent research towards production of organic
rification of various steam-pretreated lignocellulosic residues
acids (Table 3). Bacteria can also be used for production of ethanol
including dry potato peels, carrot peels, composite waste mixture,
or organic acids from lignocellulose-based substrates but the
orange peels, onion peels, banana peels, pineapple peels where cel-
capacity of producing the necessary concentrations of enzymes
lulose conversion efficiencies of 92–98% were achieved.
for complete saccharification of pretreated substrates is still lim-
de Almeida et al. (2014) have optimised conditions for produc-
ited to fungi (Amore and Faraco, 2012).
tion of the cellulase system in Fusarium verticillioides using corn
straw as substrate, where sodium nitrate was found to have a pos-
itive effect at a corn straw concentration of around 5% and the cul- 5.1. Ethanol
tivation time for optimum enzyme activity was within 140–150 h
in SmF. In contrast, sodium nitrate had no significant effect on Ethanol, or ethyl alcohol, is a volatile, flammable, and colourless
xylanase activity. An aspect found by the authors and worth liquid. Its production at commercial scale is dominated by USA and
reporting was that lactose was found to have a negative effect on Brazil that produce annually 50 billion and 23 billion litres of etha-
endoglucanase and xylanase activities, whereas it is a known cellu- nol, respectively (ePURE, 2014). The dominant feedstock in the USA
lase inducer in Trichoderma species (Fekete et al., 2008). is corn, whereas sugarcane dominates in Brazil. The potential as an
Panagiotou et al. (2011) have tested different ratios of wet- additive or a replacement of gasoline in the transport sector has
exploded pretreated wheat straw and corn cobs where the highest been the main driving force behind the production of ethanol from
cellulase and xylanase activities were found at a ratio of 1:2 using renewable feedstocks. Nonetheless, ethanol finds applications in a
Fusarium oxysporum. Qin et al. (2010) have also reported cellulase variety of industries including chemical, cosmetic, pharmaceutical
and xylanase production by Fusarium chlamydosporum when and medical, as well as the automotive and beverage sectors
grown in sugarcane bagasse together with wheat bran. Dogaris (ePURE, 2014).
et al. (2009) have obtained the highest activities of cellulases and Research has been performed on ethanol production using
hemicellulases with N. crassa when using a mixture of wheat straw Fusarium and Neurospora species (Table 2). Aspergillus species
and wheat bran (25/5, w/w), ammonium sulphate as nitrogen was also used in ethanol production. However, they are mainly
source, initial pH of 5.0 and initial moisture of 70.5% (w/w). Cellu- used for production of enzymes for further conversion to ethanol
lolytic and hemicellulolytic abilities have also been found in N. using yeast. Fusarium and Neurospora are used within a context
intermedia which was able to degrade sugar polymers such as glu- of consolidated bioprocessing (CBP) where the biocatalyst is
can, xylan and arabinan present in the fibres of thin stillage, a by- responsible for the production of enzymes, saccharification of sub-
product from the ethanol industry (Ferreira et al., 2015). Cellulases strate carbon sources to monomeric sugars and further fermenta-
and xylanases produced by ascomycetes genera in focus seems to tion to ethanol (Jouzani and Taherzadeh, 2015). The advantage of
be stable at a wide range of temperature and pH where maxima using filamentous fungi such as ascomycetes becomes clear due
activities have been reported within the ranges of 25–80 °C and to their ability to assimilate and convert xylose released during
pH 3.0–9.0, respectively. hydrolysis of lignocellulosic materials to ethanol (Ferreira, 2015).
Altogether, ascomycetes are undoubtedly a potential source of In contrast, baker’s yeast, the most widely used biocatalyst for
enzymes either for enzyme cocktail preparation or for application industrial production of ethanol, it unable to consume pentose sug-

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esour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.03.018
 J.A. Ferreira et al. / Bioresource Technology xxx (2016) xxx–xxx 7

Table 2
Examples of performance of ascomycetes towards the production of ethanol under submerged fermentation.

Microorganism Medium Mode of Production Yield (g/g) Productivity References

operation (g/L) (g/L/h)
F. oxysporum 7.5% (w/v) alkali-pretreated brewer’s 2 L STR 8.00 0.11 0.07 Xiros and
F3 spent grain Christakopoulos
(2009)
F. verticillioides 20 g/L glucose SF 9.44 0.47 0.05 de Almeida et al.
(2013)
20 g/L xylose 5.68 0.46 0.03
10 g/L glucose + 10 g/L xylose 5.60 0.50 0.05
45 g/L pre-treated sugarcane bagasse 4.6 0.31 0.03
F. oxysporum Sequentially dilute acid and dilute alkali SF 8.64 0.27 0.05 Magesh et al.
MTCC 284 pretreated tapioca stem (32 g/L) (2011)
F. oxysporum 20 g/L cellulose 6 L STR anaerobic 7.00 0.35 0.04 Panagiotou et al.
F3 (2005)
F. oxysporum Wet exploded pre-treated wheat straw SF, anaerobic 4.9 0.04 0.03 Panagiotou et al.
F3 (110 g/L) (2011)
N. crassa DSM 8% w/v dilute-sulphuric acid pretreated SF, anaerobic 8.1 24.8% of theoretical yield based on 0.04 Dogaris et al.
1129 sweet sorghum bagasse cellulose and hemicellulose (2012)
N. intermedia 7.5% dilute-phosphoric acid pretreated SF 10.1 0.135 g/g of dry biomass loading (95% 0.21 Nair et al. (2015)
CBS 131.92 wheat bran theoretical yield based on glucan)
N. crassa DSM 75 g/L NaOH-pretreated brewer spent 2 L STR 74.0 0.17 g/g (36% of the theoretical yield 0.77 Xiros et al. (2008)
1129 grain based on glucose and xylose)
N. intermedia 15.6% solids whole stillage + 1 FPU/g SS SF 8.7 0.06 0.18 Bátori et al. (2015)
CBS 131.92 cellulase
N. intermedia 9% solids thin stillage SF 5.0 0.16 g/g of reduced solids 0.14 Ferreira et al.
CBS 131.92 (2014)
N. intermedia 9% solids thin stillage 26 L ALB, 5.0 0.303 g/g reduced solids 0.57 Ferreira et al.
CBS 131.92 continuous (2015)
(0.1 h 1)

Table 3
Performance of ascomycetes towards production of different organic acids using different experiment set-ups.

Microorganism Medium Mode of Titre Yield Productivity References

operation (g/L) (g/g) (g/L/h)
Citric acid
A. niger NRRL 567 100 g apple pomace, SSF SF, SSF 127.9 g/kg 0.13 1.33 g/kg/h Dhillon et al. (2011b)
Apple pomace sludge (56.2 g/L carbohydrates) SF, SmF 18.2 0.32 0.13
A. niger NRRL 322 HCl-pretreated jack fruit carpel fibre SF, SmF 73.9 0.53 1.03 Angumeenal and
supplemented with glucose (140 g/L) Venkappayya (2005)
A. niger NRRL 567 3 kg apple pomace + rice husk (1% w/w) 12 L rotating drum type 294.2 g/kg 0.29 2.45 g/kg/h Dhillon et al. (2013a)
solid-state bioreactor
A. niger NRRL 567 270 g wet peat moss (80% moisture) + 40 mL 0.89 L column reactor 123.9 g/kg 17.8% 0.43 g/kg/h Barrington et al. (2009)
glucose (250 g/L)
A. niger NRRL 567 Apple pomace ultrafiltration sludge 7.5 L STR 40.3 0.61 0.31 Dhillon et al. (2013b)
(66.0 g/L carbohydrates)
Gluconic acid
A. niger NCIM 548 Deproteinised whey (9.5% lactose) + 0.5% SF, SmF 69.0 0.69 1.44 Mukhopadhyay et al.
glucose (2005)
A. niger IAM 2094 Enzymatically hydrolysed waste office paper SF, SmF 46.0 0.92 0.64 Ikeda et al. (2006)
(glucose adjusted to 50 g/L)
Enzymatically hydrolysed waste office paper SF, repeated batch 110.0 0.92 1.53
(glucose adjusted to 120 g/L)
Enzymatically hydrolysed waste office paper 0.8 L turbine blade reactor, 72.0 (average) 0.60 0.99
(glucose adjusted to 120 g/L) repeated batch (average) (average)
A. niger ARNU-4 Tea waste + 20% molasses SF, SSF 82.2 0.88 0.28 Sharma et al. (2008)
(mutant strain)
A. niger ORS-4.410 Rectified grape must (120 g/L glucose) Sf, SmF 73.2 0.81 0.51 Singh et al. (2005)
(mutant strain)
Rectified banana must (120 g/L glucose) Sf, SmF 69.3 0.72 0.48
Hexacyanoferrate-treated sugarcane molasses SF, SmF 60.3 0.61 0.41
(120 g/L glucose)
Itaconic acid
A. terreus TN 484-M1 140 g/L sago starch hydrolysed with HNO3 to 3 L STR 48.2 0.34 0.40 Dwiarti et al. (2007)
pH 2.0
A. terreus LU02b Glucose (180 g/L) 15 L STR 86.2 0.86 0.51 Kuenz et al. (2012)

Please cite this article in press as: Ferreira, J.A., et al. Waste biorefineries using filamentous ascomycetes fungi: Present status and future prospects. Bior-
esour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.03.018
8 J.A. Ferreira et al. / Bioresource Technology xxx (2016) xxx–xxx

ars. CBP is normally a two-stage process including a first aerobic high pace. From the data gathered in Table 2, there seems to be a
saccharification followed by ethanol production under oxygen- need of a potent microorganism that will lead to higher ethanol
limited conditions. Reasonably, the success of CBP will be depen- yields and productivities. Genetic tools might give an important
dent on the strain used (Ferreira, 2015). Among the genus Fusar- input in this regard towards the isolation of mutants with better
ium, F. oxysporum is the species most widely used for production performances. However, this may also hamper the use of the bio-
of ethanol but F. verticillioides, F. equiseti and F. acuminatum have mass for feed applications (Section 6).
also been investigated. Substrates for ethanol production included
brewer spent grain where 60% of the theoretical yield based on
total glucose and xylose content was achieved (Xiros and 5.2. Weak organic acids
Christakopoulos, 2009). A high-affinity transporter for hexose
(Hxt) has been characterised in F. oxysporum where high expres- 5.2.1. Citric acid
sion levels were observed at low glucose concentrations, a com- Citric acid, an intermediate of the tricarboxylic acid cycle with
mon situation under CBP. The transporter was further reported annual growth demand, is mostly used in the food industry
to transport both C5 and C6 sugars (Ali et al., 2013). F. oxysporum (>70%) but it has also applications in phytoremediation of heavy
has also been used for production of ethanol from wheat straw metals and manufacturing of biodegradable polymers for medical
as pure culture or co-cultured with S. cerevisiae. Whilst the produc- applications (Dhillon et al., 2011a). A. niger is nowadays used for
tion of ethanol was similar between F. oxysporum and the mixed large scale production of citric acid under submerged fermentation
culture, the productivity was three times higher when the latter using beet or cane molasses, sucrose or glucose syrup as substrates
was used (Panagiotou et al., 2011). The source of nitrogen has also (Soccol et al., 2006). Citric acid production has also been studied
been reported to influence the production of ethanol and alcohol using yeast; however, comparatively lower yields are obtained
dehydrogenase activity where higher values were registered when due to the production of isocitric acid. Citric acid produced by syn-
urea was used instead of yeast extract with F. equiseti and F. acumi- thetic route cannot compete with that produced by A. oryzae due to
natum (Anasontzis et al., 2011). the higher price of the starting materials in comparison to that of
N. crassa is the most widely used among the Neurospora genus citric acid (Papagianni, 2007). The production of citric acid is the
for ethanol production. The fungus has been used for production second largest fermentation product after industrial ethanol fer-
of ethanol from sweet sorghum bagasse, the lignocellulosic solid mentation (Dhillon et al., 2013a). However, as an effort to lower
residue obtained after extraction of sugars from sorghum stalks, citric acid production costs, other more cost-effective substrates
where 24.8% of the theoretical yield based on cellulose and hemi- have been investigated. Those have included cheese whey, rape
cellulose was achieved (Dogaris et al., 2012). It should be noted, seed oil, whey permeate, date syrup, banana extract, maize zea,
however, that higher solid loading was used for pretreatment orange processing waste, yam bean starch, rape seed oil, carob
(16%) and fermentation (8% w/v). N. crassa has also been studied pod, and root crops (Angumeenal and Venkappayya, 2005). More
for production of ethanol from alkali-pretreated brewer spent recent research insights regarding citric acid yields and productiv-
grain where 36% of the theoretical yield based on glucose and ities from different substrates using A. niger strains are presented
xylose was obtained. No negative effect of pretreatment- in Table 3. Dhillon et al. (2011b) reported an increase of citric acid
originated inhibitors such as acetic acid of lignin phenolic com- production both in SSF and SmF of apple pomace and its sludge,
pounds was found on the fungus performance since no accumula- respectively, when 3% (v/v) of ethanol and 4% (v/v) of methanol
tion of glucose and xylose was observed (Xiros et al., 2008). Zhang were added to the media in comparison to the absence of these
et al. (2008) have proved the link between xylose fermentation and alcohols. Methanol (3% (v/v)) was also shown to increase the pro-
supply of oxygen in N. crassa. Intracellular enzyme activities of duction of citric acid by about 40% when A. niger was grown in
xylose reductase, xylitol dehydrogenase and xylulokinase, the first apple pomace ultrafiltration sludge. The addition of methanol also
three enzymes in xylose metabolic pathway, decreased with the induced pellet growth whilst A. niger presented filamentous
increase in oxygen limitation, leading to a decreased xylose uptake. growth when the alcohol was absent. Rheological studies were car-
Nair et al. (2015) have cultivated N. intermedia in dilute-acid pre- ried out where the former gave rise to less viscous non-Newtonian
treated wheat bran in separate hydrolysis and fermentation where broths, whilst the latter gave rise to a medium with non-
95% of the theoretical yield based on wheat bran glucan was Newtonian pseudoplastic behaviour (Dhillon et al., 2013b). In addi-
achieved using a 7.5% (w/v) solid loading. N. intermedia has also tion, several optimisation research studies have been carried out
been evaluated for production of ethanol from residuals from the that took into account the influence of different parameters on
1st generation bioethanol process namely whole stillage and thin the production of citric acid by A. niger strains grown in different
stillage, waste streams originated after ethanol distillation (Bátori substrates. Dhillon et al. (2013a) reported optimum conditions
et al., 2015). When grown in 15.6% solids whole stillage, an ethanol for citric acid production from apple pomace using a rotating drum
concentration of around 9 g/L was obtained with addition of cellu- type solid-state bioreactor regarding methanol concentration (3%
lase, which was around the double of ethanol produced when no (v/v)), agitation frequency, aeration and incubation time. The
enzyme was added. However, further studies have shown that authors also reported optimum conditions for extraction of citric
the addition of cellulase helped the most at converting left saccha- acid (294 g/kg dry weight) regarding extraction time (20 min), agi-
rides, in the liquid fraction, to monomers. Therefore, a pretreat- tation rate (200 rpm), and extraction volume (15 mL). Bari et al.
ment step is hypothesised to be needed in order to improve the (2009) reported the optimised conditions for citric acid production
ethanol production (Bátori et al., 2015). When grown in 9% solids using empty fruit bunches, generated by palm oil industries during
thin stillage, the liquid fraction after whole stillage centrifugation, extraction of oil, regarding sucrose, minerals and inoculum concen-
N. intermedia could produce around 5 g/L of ethanol enzymatically trations; a maximum production of 338 g/kg of dry empty fruit
un-aided (Ferreira et al., 2014). When set under continuous culti- bunches was achieved. Karthikeyan and Sivakumar (2010)
vation at 0.1 h 1 dilution rate using a 26 L airlift bioreactor, the reported the maximum citric acid production (170–180 g/kg dry
same ethanol concentration was achieved (Ferreira et al., 2015). weight) using banana peel after optimisation of the moisture, pH,
Taking into account that the addition of enzymes to the process temperature, spore concentration and incubation time.
of ethanol production from lignocellulosic materials still consti- Barrington et al. (2009) have optimised citric acid production in
tutes a relevant economic burden, it is expected that the research a semi-continuous process using a column bioreactor where they
using filamentous fungi with relevance for CBP will continue at studied the aeration rate, bed depth and temperature.

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esour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.03.018
 J.A. Ferreira et al. / Bioresource Technology xxx (2016) xxx–xxx 9

5.2.2. Gluconic acid manganese (around 10 ppb) (Li et al., 2012). A doubled productiv-
D-Gluconic acid, a polyhydroxycarboxylic acid, is used in food, ity in itaconic acid has been achieved when both enzymes aconi-
feed, beverage, textile, pharmaceutical and construction industries. tase and cis-aconitate decarboxylase were expressed in the
The worldwide demand is fulfilled mostly by submerged fermenta- mitochondria of A. niger in comparison when both enzymes were
tion with A. niger which reach yields of 98% using glucose (Singh expressed in the cytosol (Blumhoff et al., 2013). In another study,
and Kumar, 2007). Similar to production of citric acid by yeast, Li et al. (2013) observed an increase in itaconic acid production
the use of bacteria for production of gluconic acid has also been by A. niger at low DO levels (10–25%) after overexpression of the
investigated, but it is limited by the side production of oxogluconic haemoglobin domain. By other A. niger strains, Li et al. (2013) have
acids (Ramachandran et al., 2006). In fungi, gluconic acid is pro- also increased the amount of itaconic acid by deleting the produc-
duced via dehydrogenation of D-glucose catalysed by glucose oxi- tion of oxalic acid. Expression of the A. terreus itaconic acid cluster
dase (Singh and Kumar, 2007). Singh and Kumar (2007) have consisting of the cadA gene (encoding a cis-aconitate decarboxy-
reviewed the main aspects of gluconic acid production namely lase), mttA gene (encoding a putative mitochondrial transporter)
the influence of carbon sources, concentration of salts, inorganic and the mfsA gene (encoding a plasma membrane transporter)
nitrogen sources, aeration, modulators (vegetable oils, H2O2, and results in A. niger strains producing over twenty-fivefold higher
starch), pH, and cultivation type (Smf and SSF). Further research levels of itaconic acid and a twenty-fold increase in yield compared
for gluconic acid production with A. niger has included other sub- to a strain expressing only CadA (van der Straat et al., 2014). Some
strates namely cheese whey, grape and banana must, waste office medium optimisation studies have also been carried out. Li et al.
paper, tea waste and sugarcane molasses where product yields (2012) reported that copper was positively correlated with
within the range 0.60–0.92 g/g were obtained (Table 3). The need improved itaconic acid production and that the optimal conditions
of substrate clarification (removal of heavy metals and presented for itaconic acid clearly differ from conditions for citric- and oxalic
in Table 3 as rectified medium) that can impact the final medium acid production. Other studies reported optimised production of
nutrients is viewed as a constraint for feasible application (Singh itaconic acid based on concentration of sago starch hydrolysate,
and Kumar, 2007). The influence of oxygen was clearly shown by glucose, corn steep liquor and salts as well as pH and temperature
(Ikeda et al., 2006) since an increase of four times in gluconic acid values (Dwiarti et al., 2007; Kuenz et al., 2012). Rao et al. (2007)
was achieved when pure oxygen was used instead of air in a tur- also reported production of 24.5 g/L itaconic acid from Jatropha
bine blade bioreactor. Sharma et al. (2008) reported a maximum seed cake.
for gluconic acid concentration using tea waste after optimisation Altogether, research towards citric acid has kept going at a high
of concentration of molasses, salts and yeast extract, temperature, pace and it is expected to continue in the future, whereas such
pH, inoculum size and aeration rate. Ramachandran et al. (2006) intensity has not been observed for gluconic and itaconic acids.
have used glucose oxidase retained in conidiospores of A. niger This aspect is related to the fact that there is a continuous growth
grown in SSF using buckwheat seeds for conversion of glucose to demand for citric acid whereas such growth has not been observed
gluconic acid. The reaction rate was found to be 1.5 g/L/h with for gluconic and itaconic acids. It is hypothesised that unless other
102 g/L of gluconic acid produced out of 100 g/L glucose consumed. high-value applications for gluconic and itaconic acid are unveiled
The reaction time was 100 h and the molar yield of around 93%. that compensate for the production cost, the research towards pro-
More recently, the production of gluconic acid from A. terreus has duction of these two organic acids is expected to continue at a
been reported from glucose with a conversion efficiency of comparatively lower pace.
0.7 mol/mol of glucose consumed. The strategy was to set the pH
at 6.5, since at low pH A. terreus produces itaconic acid
6. Cell mass
(Dowdells et al., 2010).

Naturally, the feasibility of biorefineries will depend on several

5.2.3. Itaconic acid
factors where the variety of the products contributes to a great
Aspergillus spp., namely A. itaconicus and A. terreus, can also be
extent. The macroscopic growth of ascomycetes into filaments
sources of itaconic acid, an unsaturated dicarboxylic acid with high
leads to an opportunity to produce a second value-added product
potential to be used as a chemical building block. Itaconic acid has
from fermentation when the primary goal was to optimise the pro-
the potential to function as a substitute for acrylic and methacrylic
duction of e.g. enzymes, ethanol or organic acids. The filamentous
acid for production of polymers (Okabe et al., 2009). Itaconic acid
character of ascomycetes biomass allow easier separation from the
possesses two carboxyl moieties which give it the ability to act
medium that can be simply carried out using a sieve-like separator.
as a co-monomer in the manufacture of polymers. Its applications
In contrast, for separation of bacterial or yeast biomass a centrifuge
areas are mainly in the plastics, papers and adhesives industries (Li
needs to be applied increasing the operation costs of the industrial
et al., 2013). Itaconic acid production is provided via fermentation
process. Moreover, based on their biomass composition regarding
more commonly with A. terreus from molasses or glucose with pro-
lipid and protein contents, ascomycetes biomass can be considered
duction normally lower (>80 g/L) than that for citric acid with A.
for animal feed or human consumption (Ferreira, 2015). This sec-
niger (>200 g/L) (Blumhoff et al., 2013). Production of itaconic acid
ond value-added product can play a crucial role for feasible estab-
has been studied in bacteria, yeast, other fungi (A. niger) and potato
lishment of biorefineries using more complex substrates such as
plants but comparatively lower yields were obtained (Klement and
those based on lignocellulose.
Büchs, 2013). In Aspergillus spp. itaconic acid is most likely pro-
duced via a decarboxylation of cis-aconitate catalysed by a cis-
aconitate decarboxylase (Klement and Büchs, 2013). Several 6.1. Ascomycetes biomass for human consumption
aspects of itaconic acid production including regulation, producing
microorganisms, effects of reactor design, oxygen supply, substrate Several ascomycetes have been widely used for production of
and cultivation mode have been reviewed by Klement and Büchs human food products. Aspergillus spp. have been used for produc-
(2013) and Okabe et al. (2009). The required medium components tion of indigenous Japanese foods and drinks such as sake, shoyu,
for production of itaconic acid are similar to those needed for miso and vinegar (Ferreira, 2015). Moreover, Aspergillus conidia
production of citric acid, including high concentration of glucose are used as starters in the food industry during the first step of fer-
(7.5–15%) and magnesium sulphate, low nitrogen and phosphorus, mentation to digest ingredients such as steamed rice, soybean and
low but adequate levels of zinc, copper and iron, and limited wheat, and its conidiation regulatory pathway has been a matter of

Please cite this article in press as: Ferreira, J.A., et al. Waste biorefineries using filamentous ascomycetes fungi: Present status and future prospects. Bior-
esour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.03.018
10 J.A. Ferreira et al. / Bioresource Technology xxx (2016) xxx–xxx

research (Ogawa et al., 2010). Fusarium venenatum is perhaps one 5% (w/w) protein, consumed by N. intermedia, around 5 g/L of bio-
of the most known filamentous fungi within the food industry mass containing 50% (w/w) protein is obtained (Ferreira et al.,
given its use for production of myco-protein for human consump- 2015). The current process has been scaled up using a 80 m3 biore-
tion sold under the trade name QuornÒ. This myco-protein is pro- actor with similar performance and future prospects include a fur-
duced in 150,000 L pressure-cycle bioreactors in a continuous flow ther scale up using a 1000 m3 bioreactor (Ferreira, 2015). The
process. The final product is prepared by mixing the myco-protein production of A. oryzae biomass has also been studied using whole
paste with a binding agent (egg albumin) to align the mycelia into stillage (stream that gives rise to thin stillage after centrifugation);
a fibrous network with a similar texture to that of meat. QuornÒ around 6 g/L of biomass containing 42% (w/w) protein were
products range from chunks and mince to sausages, burgers, fillets obtained (Bátori et al., 2015). Biomass production has more
and steaks (Wiebe, 2002). Monascus spp. have been used for pro- recently been investigated by our research group using various
duction of red rice with anti-hypertensive effects on humans dairy products. Although it is not surprising to report that A. oryzae
(Seraman et al., 2010). Neurospora is used for production of the could assimilate lactose, N. intermedia has also been found to be
human food oncom (soybean-based presscake), koji (an enzyme able to assimilate this saccharide which has not, to the best of
cocktail which is used as a starter for production of various fer- our knowledge, been reported. Interestingly, when A. oryzae was
mented food such as soy sauce), Iban people in Borneo collect cultivated in fat-rich media such as cream or crème fraiche the
the orange fungus from burnt-down hilly rice fields and also use ascomycete preferred to hydrolyse fat than assimilating lactose.
it as food, to prepare a fermented beverage from cassava, and it This hydrolysis was supported by the amounts of glycerol released.
is normally present in Roquefort cheese in Southern France During a pH screening using cream, the amount of glycerol
(Ferreira, 2015). Altogether, several ascomycetes are well-known increased from 0.2 g/L at pH 4.3 to around 17 g/L at pH 7 (unpub-
microorganisms for humans and due to their massive use for pro- lished results). Further studies are ongoing in order to identify the
duction of human food products they are considered as GRAS (gen- fatty acids released to evaluate their potential for animal feed sup-
erally regarded as safe) microorganisms. This can be an important plementation. Therefore, there is a potential for dairy industries to
property if their biomass is used for animal feed or human produce biomass for feed in addition to their main product build-
consumption. ing so a true biorefinery.
Considering that the biomass of filamentous ascomycetes is rich
6.2. Ascomycetes biomass for feed in proteins and lipids and that essential amino acids and fatty acids
to humans constitute an important fraction of their profiles
Animal feed products are, at great extent, based on soybeans. (Ferreira, 2015), its use for human feed cannot be neglected. Due
However, animal feed products can also be produced as a second to their use for production of fermented human foods since ancient
value-added product from biorefineries. An example is the DDGS times, the biomass of filamentous ascomycetes can constitute a
(distillers dried grains with solubles) originating from drying of potential source of human dietary supplements. Therefore, inten-
solids that are separated after distillation of ethanol in biorefiner- sive research towards production of biomass or of a specific bio-
ies using cereals such as corn or wheat as feedstocks. Examples of mass component for feed or human consumption is expected in
markets for animal feed can include poultry, cattle, chicken, and the future using an increasingly wider range of substrates.
fish among many others. Ascomycetes can play an important role
either replacing soybean-based feeds or by improving the animal
feeds already produced by biorefineries (Ferreira, 2015). Four asco- 7. Future prospects
mycetes including strains of A. oryzae, F. venenatum, M. purpureus
and N. intermedia were studied for production of biomass from thin The potential of lignocellulosic substrates for production of
stillage, a side-stream from the bioethanol industry which is also chemicals and fuels is expected to dominate research efforts
used for the production of DDGS. During production of ethanol, S. through the years due to their availability as industrial wastes in
cerevisiae is unable to consume all monomers or saccharides from spite of refined sugars. Their recalcitrant structure calls for the dis-
starch hydrolysis or to hydrolyse and consume pentose-based sub- covery of microorganisms with super-potent enzymatic machiner-
strates found in the undegraded bran that end up in the whole stil- ies or the construction of such microorganisms using genetic tools.
lage and so in the thin stillage after centrifugation (Ferreira, 2015). The ideal lignocellulose-based biorefineries would be those
Therefore, due to their enzymatic and pentose assimilation abili- employing consolidated bioprocessing with naturally robust
ties, ascomycetes were used in order to potentially consume those microorganisms and so, bioprospecting will play a crucial role
remaining carbon sources. Biomass ranges of 12–19 g/L containing regarding the discovery of such potent microorganisms. Since such
50–60% (w/w) protein were obtained during shake-flasks cultiva- microorganisms are not available at the present, the successful
tion (Ferreira et al., 2014). N. intermedia was used in another study conversion of monomeric sugars present in lignocellulosic materi-
where the aeration rate and reactor design effect were tested dur- als relies on a pretreatment step increasing though the overall pro-
ing cultivation in thin stillage using 2 m high airlift reactor and cess costs. Thus, a feasible lignocellulose-based industrial process
bubble column of 26 L capacity. The biomass production of around has to represent a true biorefinery producing as many as possible
5 g/L was similar at both reactor designs and increased to 9 g/L as different value-added products. Solid-state fermentation is widely
aeration was increased from 0.5 to 2 vvm. The biomass was found used for biological treatment of lignocellulosic waste materials
to be composed of 50% (w/w) protein and 12% (w/w) lipids. All nine particularly for preparation of enzymatic cocktails. However, stud-
essential amino acids to humans were found in N. intermedia bio- ies are still lacking regarding the use of SSF at larger scales for pro-
mass and accounted to 40% of its amino acid composition; the cess potential clarification. It is constantly stated that cultivation in
amino acid profile was quite similar to that of the DDGS. Moreover, SSF has limitations for scaling up due to the space that would be
its lipid profile was rich in omega-3 and -6 fatty acids. The process needed as well as physical constraints such as reaching sufficient
was also studied in continuous mode using a 26 L bubble column aeration and dissipation of heat and the maximum solid loading
where dilutions rates of up to 0.2 h 1 could be applied without cell possible before decreased performances are observed. The design
wash-out; maximum biomass production rate of around 370 mg/L/ of new bioreactors for solid-state fermentation circumventing
h was achieved (Ferreira et al., 2015). The current process can have these constraints would result in a major research boost. Therefore,
an important impact on the final characteristics of the final animal processes reaching industrial scale using submerged fermentation
feed from the company since from the 15 g/L of solids, containing are expected to maintain their dominance.

Please cite this article in press as: Ferreira, J.A., et al. Waste biorefineries using filamentous ascomycetes fungi: Present status and future prospects. Bior-
esour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.03.018
 J.A. Ferreira et al. / Bioresource Technology xxx (2016) xxx–xxx 11

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Please cite this article in press as: Ferreira, J.A., et al. Waste biorefineries using filamentous ascomycetes fungi: Present status and future prospects. Bior-
esour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.03.018