# Microbial co-cultures for biochemicals production from lignocellulosic biomass: A review

Mercedes Llamas, Silvia Greses, Jose Antonio Magdalena, Cristina González-Fernández, Elia Tomás-Pejó

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Mercedes Llamas, Silvia Greses, Jose Antonio Magdalena, Cristina González-Fernández, Elia Tomás-Pejó. Microbial co-cultures for biochemicals production from lignocellulosic biomass: A review. Biore source Technology, 2023, 386, pp.129499. 10.1016/j.biortech.2023.129499. hal-04264232

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# Microbial co-cultures for biochemicals production from lignocellulosic biomass: A review

Mercedes Llamas<sup>a</sup>, Silvia Greses<sup>a</sup>, Jose Antonio Magdalena<sup>b,c</sup>, Cristina González-Fernández<sup>a,d,e</sup>, Elia Tomás-Pejó<sup>a,*</sup>

## HIGHLIGHTS

* Lignocellulosic biomass conversion within biorefineries is key to develop bioeconomy.

* Innovative pretreatments are needed to enhance organic matter/sugars availability.

* Microbial co-cultures can boost biochemicals production from the sugar platform.

* Tuned mixed cultures are crucial to exploit the carboxylate platform.

## GRAPHICAL ABSTRACT

<figure><img src="image_2.png" /></figure>

## ARTICLE INFO

### Keywords:

Lignocellulosic biomass

Bioproducts

Co-cultures

## ABSTRACT

Global reliance on fossil oil should shift to cleaner alternatives to get a decarbonized society. One option to achieve this ambitious goal is the use of biochemicals produced from lignocellulosic biomass (LCB). The inherent low biodegradability of LCB and the inhibitory compounds that might be released during pretreatment are two main challenges for LCB valorization. At microbiological level, constraints are mostly linked to the need for axenic cultures and the preference for certain carbon sources (i.e., glucose). To cope with these issues, this review focuses on efficient LCB conversion via the sugar platform as well as an innovative carboxylate platform taking advantage of the co-cultivation of microorganisms. This review discusses novel trends in the use of microbial communities and co-cultures aiming at different bioproducts co-generation in single reactors as well as in sequential bioprocess combination. The outlook and further perspectives of these alternatives have been outlined for future successful development.

<footnote><sup>a</sup> Biotechnological Processes Unit, IMDEA Energy, Madrid, Spain</footnote>

<footnote><sup>b</sup> LBE, Univ Montpellier, INRAE, 102 avenue des Étangs, F-11100 Narbonne, France</footnote>

<footnote><sup>c</sup> Vicerrectorado de Investigación y Transferencia de la Universidad Complutense de Madrid, 28040 Madrid, Spain</footnote>

<footnote><sup>d</sup> Department of Chemical Engineering and Environmental Technology, School of Industrial Engineering, University of Valladolid, Dr. Mergelina, s/n, Valladolid 47011, Spain</footnote>

<footnote><sup>e</sup> Institute of Sustainable Processes, Dr. Mergelina, s/n, Valladolid 47011, Spain</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: elia.tomas@imdea.org (E. Tomás-Pejó).</footnote>
# 1. Introduction

Society finds itself at a non-turning point in which a low-carbon future is mandatory (Intergovernmental Panel on Climate Change, 2018). The International Energy Agency (IEA) highlighted that to achieve this objective a deep transformation in the energy production and consumption systems needs to occur by 2050: rising CO₂ prices, decreasing the reliance on fossil sources, increasing shares of renewables and implementing strict policy actions (International Energy Agency, 2021).

Renewable resources, like biomass, are good contributors to meet net zero carbon emissions. Biomass is the most widely abundant feedstock with a worldwide production of around 181.5 billion tons/year (Dahmen et al., 2019) and it can be exploited in biorefineries mimicking the traditional petrochemicals refineries. In the specific case of lignocellulosic biomass (LCB), biorefineries valorize cellulose, hemicellulose and lignin into valuable biofuels and biochemicals (Usmani et al., 2020). Indeed, more than 200 marketable products have been reported to be produced from LCB (Isikgor and Becer, 2015).

The organic nature of LCB makes it susceptible to be valorized by two main biotechnological conversion routes: i) hydrolysis of sugars and fermentation, and ii) production of carboxylic acids by anaerobic fermentation (AF). When LCB is valorized via the sugar platform, glucose is easily converted into biofuels and biochemicals (e.g., bioethanol (EtOH), lactic acid, etc.) by most fermenting microorganisms. In this platform, research efforts have been directed to the valorisation of the rest of sugars (e.g., xylose) (Zhu et al., 2020; Ndubuisi et al., 2023; Qiu et al., 2023). Regarding the carboxylate platform, research especially targets the optimization of the traditional anaerobic bioprocesses devoted to biogas or the most innovative approach devoted to carboxylates production (Hashemi et al., 2022; Holtzapple et al., 2022). In this manner, two platforms, namely sugars and carboxylates, can be envisaged as mediating molecules for the productions of biochemicals.

The use of LCB to obtain different products (e.g. biofuels such as EtOH/biodiesel or biochemicals such as oils) can be conducted in sequential bioprocesses or implementing co-cultures and mixed cultures in a single reactor (Farzad et al., 2017). The use of sequential reactors allows a tailored fermentation in which a specific microorganism strain at optimum operational conditions maximizes the production of one specific compound in each reactor. However, under this approach, the overall process cost, technical complexity and required reaction times are higher. Alternatively, the use of co-cultures or microbial consortia in a single reactor (instead of one single strain), can be a promising option for the simultaneous generation of multiple bioproducts (Diender et al., 2021). This strategy allows maximum LCB exploitation in one single reactor taking advantage of the specific metabolism of different microorganisms. In this later case, the challenge relies on setting up the right environmental conditions (e.g., pH, temperature) for the different microorganisms to get the highest conversion yields.

Overall, tapping the full potential of LCB within the biorefinery context needs further improvements in specific areas such as the pretreatment step to reduce the recalcitrant nature of LCB, the presence of inhibitory compounds, the avoidance of by-product formation or the co-utilization of sugar mixtures to ensure maximum resource bioconversion efficiencies.

The interest in the co-cultivation strategy aiming at different products is increasing as proven by number of recent review articles on this topic (Padmaperuma et al., 2018; Rosero-Chasoy et al., 2021). These works often give a general overview on natural consortia and suggest different approaches engineer microbial co-cultures. Besides, some works have also tried to give some insights on the interaction mechanism models of microbial consortia (Jiang et al., 2019; Qian et al., 2020) but the information on how this co-cultivation systems can be applied for LCB valorization is still scarce. Thus, this review discusses novel trends in the use of microbial communities and co-cultures aiming at different bioproducts generation in single reactors as well as in sequential bioprocess combination putting the focus on LCB use.

# 2. LCB: Sources and structure

LCB refers to plant-based materials rich in polysaccharides, which arise as a strategic feedstock to be used in a bio-based society. Besides being a renewable material non-competitive with food crops, LCB includes waste streams. This fact remarkably reduces the production costs associated to the feedstock, thereby contributing to the economic feasibility of processes based on this material.

The LCB annual worldwide generation is currently estimated to be around 1.3 billion tons (Ashokkumar et al., 2022). Its global availability, makes LCB an appealing alternative to fossil sources. The most common LCB materials include forestry woody residues (e.g., switch grass, spruce, eucalyptus or birch), agricultural residues (e.g., cereal straw, sugarcane bagasse or corn stover), industrial wastes (e.g., brews' spent grains or paper and lumber mill residues) and municipal solid wastes. Within this classification, Asian countries lead the world cereal and cocoa straw waste generation; United States generates corn stover and wheat straw; Brazil produces large amounts of sugarcane bagasse; Russia stands out for forest wood residues; and Mediterranean countries are well known for the generation of olive processing residues (Zhao et al., 2022).

Although LCB presents a variable composition depending on its nature, it is made of three major elements: cellulose (40–50 % w/w), hemicellulose (25–30 % w/w) and lignin (15–20 % w/w). Other minor components such as extractives or inorganic ash can be also commonly found. Cellulose is a water insoluble linear biopolymer of D-glucose linked via 1,4-glycosidic bonds and it is decomposed over 180 °C. On the other hand, hemicellulose is a heteroglycan with a variable composition of branched C5-C6 sugar (D-xylose, L-arabinose, D-mannose, D-glucose and D-galactose) linked by β-1-4 bonds. Hemicellulose presents higher accessibility and is easier to hydrolyze than cellulose. Lastly, lignin is an aromatic amorphous biopolymer consisting of coniferyl, sinapyl and ρ-coumaryl alcohols. Lignin forms a three-dimensional network highly recalcitrant toward degradation, conferring the robustness required for this type of feedstock.

These three biopolymers are unevenly placed in plant cell walls. Cellulose polymer gathers to form robust microfibers acting as cell wall skeleton. On the other hand, amorphous hemicellulose is found embedded and covering the cellulosic bedrock. Lastly, the lignin-linked material fills the gap between cellulose and hemicellulose. Based on that, it can be stated that LCB is a heterogeneous feedstock with high structural complexity. Because of this recalcitrant structure, an efficient utilization of the sugars contained in LCB still remains a challenge for biotechnological applications.

# 3. Challenges in LCB utilization

Despite all the positive aspects mentioned above, there are many challenges to overcome when using LCB as a feedstock for biochemicals generation, such as the low biodegradability, the lignin recalcitrance or the release of inhibitory compounds during pretreatment that can affect microorganisms involved in subsequent bioconversion processes.

## 3.1. Pretreatment

When LCB is devoted to a biological conversion process, the organic matter must be available for an efficient biochemicals generation. To make organic matter readily available for fermenting purposes, the first challenge is associated to the complex nature and structure of LCB. As mentioned in Section 2, in addition to the lignin content, cellulose crystallinity and the available surface area are also key factors affecting LCB decomposition. Hence, a pretreatment step to increase the accessibility to cellulose and hemicellulose fractions, whilst breaking down the lignin, is crucial to boost LCB bioconversion in biorefineries.

Traditional LCB pretreatments mainly include physical, chemical, physico-chemical and biological methods. Physical pretreatments (e.g., mechanical, ultrasonic and thermal pretreatments) aim to disrupt the
rigid structure of LCB, increasing the surface area and reducing the
polymerization and crystallinity degree of cellulose (Bichot et al., 2020).
However, since lignin is not degraded during physical pretreatments, it
is often combined with other methods. Chemical pretreatments (e.g.,
acid, alkali, oxidative and organosolv) employ specific chemical re-
agents to promote the delignification of LCB, and solubilization of
hemicellulose (Nazli et al., 2023). Despite being one of the most efficient
methods, chemical pretreatments entail high capital inputs and envi-
ronmental problems. Biological pretreatments (e.g., fungal, bacteria and
termite pretreatments) are based on specific microorganisms or enzymes
activities employed for lignin and hemicellulose degradation (Andlar
et al., 2018). So far, these are the most cost-effective pretreatments, but
the treatment time and low solubilization yields hinder their applica-
tion. In general, these single pretreatment technologies do not succeed
to achieve effective solubilization of sugars but rather open up the
feedstock physical structure. On the other hand, physico-chemical pre-
treatments (e.g., steam explosion, alkali-heat, ammonia fiber explosion
and extrusion pretreatments) exhibit more favourable lignin removal
and cellulose crystallinity reduction efficiencies (Kumar et al., 2020).

Some cutting-edge pretreatment techniques (e.g., biochemical, ionic liquid, deep eutectic solvents or supercritical fluid pretreatments) have arisen with high development potential since they exhibit high performances while being environmentally friendly and compatible with subsequent bioprocess (Roy et al., 2020; Tang et al., 2023). Despite all their benefits, the high cost and energy consumption are the main drawback to be addressed for their practical application (Zhao et al., 2022).

3.2. Presence of lignin

Lignin does not only impede LCB decomposition and sugar release but also acts as a physical barrier hindering the activity of the hydrolytic bacteria thus hampering LCB conversion by both sugars and carboxylate platforms.

The harsh pretreatment conditions applied to achieve a high
delignification degree to enable cellulose and hemicellulose accessibility
might result in the formation of inhibitory by-products (Moreno et al.,
2015). Additionally, lignin can unspecifically bind cellulolytic enzymes,
reducing hydrolysis yields (Maheswari et al., 2023; Dong et al., 2023).

Different tools can be deployed to overcome the negative effects
associated to lignin presence (Sharma et al., 2023). For instance, the
addition of solvents during pretreatments (e.g., alkali, surfactant or
laccases enzymes) can assist lignin modification enhancing its hydro-
phobicity and weakening the lignin-enzymes nexus (Suman et al., 2022;
Tang et al., 2023). The addition of blockers (e.g., soybean protein, BSA,
Tween80) on lignin-enzyme links have also been used to reduce the
enzymes adsorption on lignin surface improving the overall enzymatic
activity rates (Huang et al., 2022).

3.3. Inhibitors

Despite being crucial to boost bioprocess yields, some pretreatments release a wide range of inhibitors that significantly affect the subsequent bioprocesses (Bhatia et al., 2020). Although these pretreatments were initially developed for EtOH production via yeast fermentation, they are also being used to produce other valuable biochemicals and energy streams and even applied for anaerobic digestion (AD) of LCB (Monlau et al., 2014; Hashemi et al., 2022; Ma et al., 2022). More than 40 compounds resulting from LCB pretreatments have been reported as potential inhibitors, among them furan aldehydes, phenol compounds and weak acids are highlighted.

3.3.1. *Furan aldehydes*

Furan aldehydes, mainly furfural and 5- hydroxymethyl furfural (5-
HMF), are principally generated from the degradation of the pentose and
hexose sugars during some pretreatments. Furan derivative compounds

have been identified as inhibitors of microorganisms growth due to the
blocking of several enzymes involved in the carbon central metabolism
(Yang et al., 2022). Among the furans, furfural was shown to be more
toxic than 5-HMF, causing DNA damage (Guo et al., 2022). Likewise,
previous studies also identified furans as important inhibitors of
fermentative bacteria due to their low molecular weight and water-
repellent behavior (Zhai et al., 2018), being more detrimental to bac-
terial activity than weak acids or phenolic compounds. Regardless of the
furan aldehyde compound, researchers agreed that fermentative mi-
croorganisms are more sensitive to these inhibitors than those involved
in anaerobic fermenters (De Bhowmick et al., 2018; Ferraro et al., 2020).

In pure cultures of yeasts, 5-HMF was identified as less harmful than furfural (Liu et al., 2021). Additionally, most of *Saccharomyces cerevisiae* strains have been proven to convert furan derivatives into less inhibitory compounds (furfural to furfuryl alcohol and 5-HMF to 5-(hydroxymethyl)furfuryl alcohol) which makes furans less challenging compounds for yeast than phenols or weak acids.

3.3.2. *Phenolic compounds*

Phenolic compounds are commonly derived from the degradation of
lignin (Monlau et al., 2014), being mainly constituted by guaiacol,
vanillin, guaiacylacetone, acetovanillone and syringaldehyde (Singh
et al., 2017). Similar to furan aldehydes, these inhibitors negatively
affect the activity of hydrolytic enzymes and integrity of microorgan-
isms' cells, provoking membrane disruption (Cámara et al., 2022).
Nevertheless, the inhibition levels of phenolic compounds are notably
higher than those determined for furans in bioprocesses using open
mixed microbiomes, single bacteria or yeast strains (Cubas-Cano et al.,
2020b; Basak et al., 2021). Li et al. (2017) also determined that
S. cerevisiae inhibition by phenols was significant even at very low
concentration. Indeed, these authors classified the inhibitors from the
strongest to the weakest effect as: phenol > vanillin > syringaldehyde >
furfural > 5-HMF > formic acid > levulinic acid > acetic acid.

3.3.3. *Weak acids*

High concentrations of weak acids, mainly comprising acetic, levu-
linic, and formic acids are also present in pretreated LCB (Wang et al.,
2019; Bhatia et al., 2020). These acids can enter the cell and inhibit the
microbial growth due to an increase of H⁺ that provokes the generation
of reactive oxygen species. The toxicity of weak acids has been mainly
reported in yeast fermentation for EtOH production, and their effect on
other bioprocesses remains to be further explored (Ndukwe et al., 2020).
Whereas a co-culture yeast-bacteria might be negatively affected by
weak acids, open mixed cultures such as those used to perform anaerobic
bioprocess (i.e., AD, dark fermentation (DF) or AF) can tolerate high
concentrations of these metabolites. Furthermore, some acids (e.g.,
propionic acid) exhibit higher toxicity than others (e.g., acetic acid) in
particular microorganisms, such is the case of yeast (Hemansi et al.,
2022). Thus, not only acids concentration, but their distribution is
relevant when assessing their inhibitory effect on microorganisms.

Overall, the presence of inhibitors has promoted the development of
several strategies to diminish their negative effect on microbial activity
such as forcing a robust microbiome by applying evolutionary engi-
neering (Tomás-Pejó et al., 2010) or implementing detoxification pro-
cesses (Xiros and Olsson, 2014). The production of these inhibitory
products can be also taken as an economic advantage due to their high
economic value. In this regard, recent studies reported the trans-
formation of furanic compounds into formic and levulinic acids that
have high industrial interest (Bhatia et al., 2020; López-Lorenzo et al.,
2023), while the development of downstream technologies to recover
these chemicals are being simultaneously investigated (Jia et al., 2023).

4. Co-cultures for production

Monocultures have been traditionally used in fermentation bio-
processes to produce a wide range of bioproducts (Shahab et al., 2020).
However, single-strain cultures may not be the most appropriate strategy when it comes to the use of complex substrates like LCB because the required traits are often not included in one single microorganism (e.g., robustness, utilization of different carbon sources, etc.). Metabolic engineering has been applied for decades to overcome the bottlenecks associated with the use of complex feedstock. However, when introducing new metabolic routes, co-factors imbalance, by-product generation and undesired chemical reactions can result in poor fermentation yields and productivities.

In nature, different microorganisms work collaboratively to perform complex tasks. Bioremediation, AD, and composting are some examples of this type of relationship. The benefit of collaborative microorganisms is that these communities are able to respond more efficiently to environmental fluctuations and process disturbances (Qian et al., 2020). These bioprocesses rely on natural microbial communities comprising bacteria, fungi, and archaea (González et al., 2021). However, the mechanisms through which microorganisms interact during co-cultures remain unknown in most cases.

When co-cultures are involved in a bioprocess, there are many special requirements that have to be considered. For example, to avoid competition of microorganisms and prevent overgrowth of one specie over the others, process conditions may be carefully tuned. Besides, changes in abiotic conditions (pH, dissolved oxygen, etc.) may be also taken into account to get a stable co-culture.

In this sense, a complete understanding of the advantages and interactions in microbial co-cultures could allow the design of microorganisms' controllable co-cultivation systems which would definitively be beneficial for the bioconversion of LCB.

## 4.1. Sugar platform-based bioprocesses aiming at different biochemicals co-generation

The most traditional way of biological LCB valorization implies the release of sugars from the complex matrix and further conversion of the sugars by fermenting microorganisms. Both hydrolysis and fermentation are crucial steps for the microbiological conversion of LCB into biochemicals. While fungi and bacteria may be primarily involved in the hydrolysis step, LCB sugars fermentation can be accomplished by fungi, bacteria and yeast. Thus, several strategies of co-cultivation of microorganisms may result in the production of LCB sugars and simultaneous conversion to biochemicals in one single production system (Fig. 1). As shown in Table 1, the interest in the simultaneous production of LCB

sugars, biofuels, and other biochemicals by co-cultivation of fungi, bacteria, or yeast has been increasing in the last few years. For an efficient co-cultivation of microorganisms, the ratio of the involved strains has to be adjusted to avoid competition. Likewise, abiotic conditions may fulfill the requirement of all involved strains. Changes in oxygen, temperature, pH, etc. may be used to adjust the prevalence of one or other microorganisms depending on the targeted products. Thus, fermentation conditions should be carefully fine-tuned to favor the most appropriated microorganisms for the consortia giving rise to a stable co-culture.

### 4.1.1. Simultaneous production of sugars and biochemicals

Complete hydrolysis of LCB requires the action of different enzymatic activities and it is commonly achieved by the addition of different commercial enzymatic cocktails (e.g., Celluclast 1.5L; Cellic CTec2; Novozyme, etc.). Enzyme production has great impact on the overall process economic viability and big efforts are being addressed to reduce enzyme costs. In this sense, the integration of enzyme production, hydrolysis and fermentation in a single process (Fig. 1) could also entail great potential to reduce operational costs. To achieve this in a co-cultivation strategy, the most commonly studied approach has been the combination of hydrolytic fungi with fermenting microorganisms.

In this sense, the fungi *Trichoderma reesei* has been efficiently co-cultivated with two fermenting yeasts (i.e., *S. cerevisiae* and *Scheffersomyces stipitis*) to simultaneously produce sugars and EtOH from non-detoxified wheat straw slurry reaching a 67 % EtOH yield (Brethauer and Studer, 2014). The same cellulolytic fungus was also co-cultivated with lactic acid bacteria (*Lactobacilli* sp.wt) producing up to 19.8 g/L (85.2 % of the theoretical maximum) of lactic acid from non-detoxified steam-pretreated beech wood (Shahab et al., 2018). The white-rot fungus *Phlebia* sp MG-60-P2 was co-cultured with *Clostridium saccharoperbutylaceticum* to also aim at butanol production from hardwood (Tri and Kamei, 2020). In this case, the pyruvate decarboxylase gene from MG-60-P2 was knocked out to avoid EtOH production and promote high accumulation of sugars which was ultimately translated in higher butanol concentration. EtOH from cedar wood has been also targeted with co-cultivation of 3 different wood-rot fungi (Horisawa et al., 2019). More specifically, *Bjerkandera adusta* and *Fomitopsis palustris* (lig-ninolytic and cellulolytic fungi, respectively) were co-cultivated with the EtOH producer *Schizophyllum commune* NBRC 4928.

Apart from hydrolytic fungi, some cellulolytic bacteria have been also applied in combined hydrolysis and fermentation processes. Several

<figure><img src="image_3.png" /><figcaption>Fig. 1. Sequential and combined microbial co-cultivation strategies for biochemicals production from LCB via carboxylates and sugars platforms.</figcaption></figure>
<table><caption>**Table 1**
Examples of simultaneous production of sugars and biochemicals from LCB via co-cultivation of microorganisms.</caption><thead><tr><th>Lignocellulosic material</th><th>Microorganisms</th><th>Bioproducts</th><th>Titers</th><th>Reference</th></tr></thead><tbody><tr><td>Rice straw</td><td>C. thermocellum ATCC 27405</td><td>Acetic acid, EtOH, sugars</td><td>5.91 g/L, 8.08 g/L, n.r.<sup>a</sup></td><td>(Chi et al., 2018)</td></tr><tr><td>Rice straw</td><td>C. thermobutyricum ATCC 49875</td><td>Butyric acid</td><td>33.9 g/L</td><td>(Kiyoshi et al., 2015)</td></tr><tr><td>Rice straw</td><td>C. thermocellum NBRC 103400</td><td>Sugars</td><td>n.r.<sup>a</sup></td><td>(Brethauer and Studer, 2014)</td></tr><tr><td>Rice straw</td><td>C. saccharoperbutylacetonicum N1-4</td><td>Butanol</td><td>6.9 g/L</td><td>(Brethauer and Studer, 2014)</td></tr><tr><td>Rice straw</td><td>T. reesei</td><td>Sugars</td><td>n.r.<sup>a</sup></td><td>(Brethauer and Studer, 2014)</td></tr><tr><td>Rice straw</td><td>S. cerevisiae</td><td>EtOH from glucose</td><td>9.1 g/L</td><td>(Brethauer and Studer, 2014)</td></tr><tr><td>Rice straw</td><td>S. stipitis</td><td>EtOH from xylose</td><td>n.r.<sup>a</sup></td><td>(Brethauer and Studer, 2014)</td></tr><tr><td>Rice straw</td><td>T. reesei wt</td><td>Sugars</td><td>19.8 g/L</td><td>(Shahab et al., 2018)</td></tr><tr><td>Rice straw</td><td>Lactobacilli sp.wt</td><td>Lactic acid</td><td>n.r.<sup>a</sup></td><td>(Tri and Kamei, 2020)</td></tr><tr><td>Rice straw</td><td>Pseudomonas sp. MG-60-P2</td><td>Sugars</td><td>3.2 g/L</td><td>(Svetlitchnyi et al., 2013)</td></tr><tr><td>Rice straw</td><td>C. saccharoperbutylacetonicum</td><td>Butanol</td><td>n.r.<sup>a</sup>, 1.8 g/L</td><td>(Svetlitchnyi et al., 2013)</td></tr><tr><td>Rice straw</td><td>Caldicellulosiruptor sp.</td><td>Sugars, EtOH</td><td>n.r.<sup>a</sup></td><td>(Lu et al., 2020)</td></tr><tr><td>Rice straw</td><td>Thermoanaerobacter sp.</td><td>Sugars</td><td>12.5 g/L</td><td>(Lu et al., 2020)</td></tr><tr><td>Rice straw</td><td>T. thermosaccharolyticum M5</td><td>Succinic acid</td><td>n.r.<sup>a</sup></td><td>(Horisawa et al., 2019)</td></tr><tr><td>Rice straw</td><td>A. succinogenes 130Z</td><td>Sugars</td><td>n.r.<sup>a</sup></td><td>(Horisawa et al., 2019)</td></tr><tr><td>Rice straw</td><td>F. palustris NBRC 30339</td><td>Sugars</td><td>1 g/L</td><td>(Kumar et al., 2023)</td></tr><tr><td>Rice straw</td><td>B. adusta 1WA5b</td><td>Sugars, EtOH</td><td>n.r.<sup>a</sup></td><td>(Kumar et al., 2023)</td></tr><tr><td>Rice straw</td><td>S. commune NBRC 4928</td><td>Sugars, EtOH</td><td>40 mg PHB/g Miscanthus (yield)<sup>a</sup></td><td>(Kumar et al., 2023)</td></tr><tr><td>Rice straw</td><td>Streptomyces sp. SirexAA-E</td><td>Sugars</td><td>n.r.<sup>a</sup></td><td>(Kumar et al., 2023)</td></tr><tr><td>Rice straw</td><td>P. megaterium PHB</td><td>Poly(3-hydroxybutyrate) (PHB)</td><td>n.r.<sup>a</sup></td><td>(Kumar et al., 2023)</td></tr></tbody></table>

strains of the cellulolytic bacteria *Clostridium thermocellum* have been efficiently co-cultivated with *C. saccharoperbutylacetonicum* and *Clostridium thermobutyricum* to produce butanol and butyric acid, respectively, from delignified rice straw (Kiyoshi et al., 2015; Chi et al., 2018). Chi and co-workers (2018), concluded that sugars released by the action of *C. thermocellum* were efficiently converted to butyric acid by *C. thermobutyricum*. This study also showed that co-assimilation of acetic acid and ethanol by *C. thermobutyricum* contributed to increase butyric acid production from LCB.

*Thermoanaerobacterium thermosaccharolyticum* M5 was also co-cultured with *Actinobacillus succinogenes* 130Z for direct succinic acid production resulting in 12.51 g/L of succinic acid from 80 g/L of untreated corn cob (Lu et al., 2020).

Extremely thermophilic bacteria have also been combined to directly produce sugars and EtOH. In the particular case of *Caldicellulosiruptor* sp. and *Thermoanaerobacter* sp., when both strains were co-cultured, the EtOH production increased 2- to 8.2-fold when compared to cultivation of both bacteria, separately (Svetlitchnyi et al., 2013). Poly-hydroxybutyrate (PHB) production has also been targeted by co-culturing of cellulolytic *Streptomyces* sp. SirexAA-E and *Priestia mega-terium* reaching 40 mg PHB/g miscanthus biomass, demonstrating the potential of co-cultivation of microorganisms to directly convert LCB to desired bioproducts without the addition of external enzymes with the consequent cost reduction.

When using LCB, efficient downstream processing for product recovery is crucial to achieve a cost-effective process (Kawaguchi et al., 2022). On-site sugar production by cellulolytic microorganisms will avoid cost for off-site enzyme production, storage, transportation, etc. and it could also result in reducing the costs of downstream processing (Saini et al., 2022).

Previously mentioned examples are mainly aimed at producing one final bioproduct from produced sugars (e.g. EtOH, lactic acid, butanol) in a co-culture. Thus, downstream processes for product separation should be optimized to target maximum recovery of one single product. This fact would allow the implementation of simpler downstream operations when compared to production of several bioproducts. Furthermore, by means of producing sugars and biochemicals in one single system, downstream processes related with lignocellulosic sugars production and media conditioning before fermentations will also be avoided.

### 4.1.2. Separated production of sugars and sequential biochemicals production

One of the challenges associated with the transformation of LCB is the presence of different carbon sources in the fermentation media. Microorganisms that can use mixed sugars, preferentially consume one sugar first followed by a second consumption phase. The result is the growth in two-stages, also known as diauxic growth. This type of growth results in prolonged fermentation times and reduced product titers and yields. The utilization of different LCB carbon sources by different microorganisms has mainly been studied sequentially from different sugars as shown in Table 2. This sequential strategy implies longer process times and reduced cost-efficiency due to the necessity of two bioreactors.

More specifically, EtOH and lactic acid production have been targeted by sequential fermentation of glucose by yeast, and further conversion of remaining xylose to lactic acid by lactic acid bacteria (Cubas-Cano et al., 2020a; Wang et al., 2018). To attain this, Cubas-Cano et al., (2020a) efficiently adapted a *Bacillus coagulans* strain to cope with up to 40 g/L EtOH. This fact allowed the sequential production of both biochemicals without the need of removing EtOH before lactic acid fermentation avoiding the corresponding operational costs of EtOH stripping at industrial scale.

Similarly, the xylose remaining after 54.1 g/L EtOH production from glucose by *Candida glycerinogenes* UG21 was converted to D-1,2,4-butantriol (BT) by *Escherichia coli* MXW004 (Zhao et al., 2019). However, in this case, EtOH was recovered by vacuum distillation before

<footnote><sup>a</sup> Due to the continuous consumption of carbon source, produced sugars were not reported (n.r.).</footnote>

<footnote><sup>b</sup> Titers were not reported.</footnote>
<table><caption>**Table 2**
Examples of co-utilization of different carbon sources after enzymatic hydrolysis by different microorganism for production of biochemicals from LCB.</caption><thead><tr><th>Lignocellulosic material</th><th>Microorganisms</th><th>Bioproducts</th><th>Titers</th><th>Cultivation type</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="2">Sugar cane bagasse</td><td><i>E. coli</i> LW419a</td><td>EtOH from xylose</td><td rowspan="2">48.6 g/L</td><td rowspan="2">Co-cultivation</td><td rowspan="2">(Wang et al., 2019)</td></tr><tr><td><i>S. cerevisiae</i></td><td>EtOH from glucose</td></tr><tr><td rowspan="2">Gardening residues</td><td><i>S. cerevisiae</i></td><td>EtOH from glucose</td><td rowspan="2">44.0 g/L</td><td rowspan="2">Sequential</td><td rowspan="2">(Cubas-Cano et al., 2020a)</td></tr><tr><td>Evolved <i>B. coagulans</i> A20-EXA</td><td>Lactic acid from xylose</td></tr><tr><td rowspan="2">Corn stalk</td><td><i>S. cerevisiae</i> M3013</td><td>EtOH from glucose</td><td rowspan="2">22.2 g/L</td><td rowspan="2">Sequential</td><td rowspan="2">(Wang et al., 2018)</td></tr><tr><td><i>B. coagulans</i> LA1507</td><td>Lactic acid from xylose</td></tr><tr><td rowspan="2">Sugarcane bagasse</td><td><i>C. glycerinogenes</i> UG21</td><td>EtOH from glucose</td><td rowspan="2">50.5 g/L</td><td rowspan="2">Sequential</td><td rowspan="2">(Zhao et al., 2019)</td></tr><tr><td><i>E. coli</i> MXW004</td><td>D-1,2,4-butantriol from xylose</td></tr></tbody></table>

xylose fermentation. In this study, the deletion of the crr gene in *C. glycerinogenes* UG21 resulted in an increase of BT production from 1.3 g/L to 4.9 g/L.

The efficient simultaneous sugar utilization could be accomplished by the co-cultivation of several microorganisms specialized in the consumption and fermentation of one single sugar. However, due to the challenges associated to the glucose fermentation ability of most of the microorganisms, few studies have reported this strategy. As a matter of fact, Wang et al., (2019) co-cultured a recombinant glucose utilization negative ethanologenic *E.coli* LW419a with *S. cerevisiae* for efficiently producing 48.6 g/L EtOH from both xylose and glucose in sugar cane bagasse.

As previously mentioned, *S. cerevisiae* and *S. stipitis* were also co-cultivated for simultaneous production of EtOH from glucose and xylose, respectively (Brethauer and Studer, 2014). In this case, a single multi-species biofilm membrane was designed and implemented for producing 67 % of the theoretical maximum EtOH from pretreated wheat straw.

## 4.2. Carboxylate platform-based bioprocesses aiming at different biochemicols co-generation

Microbial communities work collaboratively to degrade organic matter in anaerobic processes. Carboxylates are intermediate metabolites resulting from the fermentative stage of AD. In conventional AD, carboxylates are ultimately transformed into CH₄ and CO₂. However, carboxylates accumulation can be promoted by limiting the methanogenesis and enhancing the AF of LCB. As detailed below, carboxylates can be subsequently transformed into biofuels and biochemicols in a second stage (Holtzapple et al., 2022). Feedstock features and complexity influence the process yield toward carboxylates production, thus making a pre-treatment also crucial to promote process yields (Section 3.1).

The carboxylate profile and concentration obtained in AF is highly dependent on the substrate employed, the operational conditions, the reactor configuration, and the microbial community involved in the process (Luo et al., 2022). For instance, acetic and butyric acids were maximized when AF was conducted at 35 °C and acid pH using rice straw (Soares et al., 2020) or brewery spent grains (Kim et al., 2021), identifying *Clostridium* and *Enterobacter* as main involved bacteria. By contrast Greses et al., (2022a) digested vegetables and fruits at 55 °C to obtain a 51.3 % of organic matter bioconversion into carboxylates (65 % butyric acid), and 157 mL H₂/g volatile solids influent (VSᵢₙ) by maximizing the dominance of *Thermoanaerobacterium* (63.2 % relative abundance) in the microbiome. Additionally, these authors detected other molecules of interest such as 22.8 g/L of EtOH, and they studied the biodegradability of the solid spent through AD obtaining 288.5 mL CH₄/g VSᵢₙ. This multiproduct strategy evidenced the high potential of LCB to be transformed into several bioproducts (carboxylates and EtOH) and energy streams (H₂ and CH₄) through a cascade combination of bioprocesses. In fact, most of the organic matter content of the substrate measured as chemical oxygen demand (COD) accounted for the carboxylates and other soluble organic products, which could be further valorized via subsequent bioprocesses (Fig. 1).

### 4.2.1. Simultaneous carboxylates and EtOH production using open-mixed cultures

AF comprises the first steps of conventional AD, namely hydrolysis and acidification. This process is also carried out by a synergic microbial community capable of performing a wide variety of metabolisms simultaneously, resulting in different metabolites accumulation. In the last decade, carboxylates and H₂ have been studied as the main end products of AF and DF (if devoted for H₂ production), respectively, since they are valuable intermediate metabolites previously observed as conventional AD failure indicators. However, recent studies demonstrated that other added-value compounds can be efficiently co-produced via AF (Table 3). Conventionally, EtOH has been biologically produced through pure cultures of yeasts as a part of the sugar platform, which involves a high economic cost related to the need of axenic conditions. Since ethanologenic pathways have been commonly detected in different phyla (Firmicutes, Actinobacteria, Proteobacteria) present in the open mixed-cultures used as inoculum for AF (Carrillo-Barragán et al., 2021; Greses et al., 2022b), EtOH production via AF is under intense investigation to reduce the costs and simplify the technology. In this regard, Carrillo-Barragán et al. (2021) studied the selective pressures driving ethanologenic metabolisms in open-mixed cultures, identifying inoculum transfer time in batch reactors as a key strategy to enrich the microbiome with *Clostridium* and *Pseudomonas* at pH lower than 5.5. These conditions gave rise to 2.9 g COD/L of EtOH and butyric acid when granular biomass was fed with food waste. Likewise, Tamis et al. (2021) confirmed that granular sludge exhibited high ethanologenic activity that co-produced 0.50 g EtOH/g glucose (both in COD terms), along with 20 % of the COD influent as carboxylates. In this case, pH was identified as a critical parameter. This investigation reported that pH values close to 4 resulted in acetic and lactic acids as unique carboxylates due to the prevalence of *Lactobacillus*. Shi et al. (2021) also demonstrated the feasibility to valorize LCB to co-produce carboxylates (3 g COD/L) and EtOH (4 g COD/L) using pH as the driving factor in a granular microbiome to promote *Brevibacteria*. The ethanologenic activity of conventional sludge used for CH₄ production has been also demonstrated by recent publications. Li et al. (2020) demonstrated that EtOH-type fermentation prevailed at pH between 4 and 5 at 35 °C, whereas Greses et al. (2023) found that pH values close to 6 resulted in notable high EtOH concentration (77 g COD/L) by decreasing process temperature to 25 °C. These authors were able to reach such a high EtOH concentration by boosting *Bifidobacterium* growth.

### 4.2.2. Simultaneous carboxylates and lactic acid production using open-mixed cultures

Similar to EtOH production, lactic acid is another primary intermediate metabolite of AF that can be accumulated in anaerobic bio-processes to maximize the economic revenue of residual feedstocks. Lactic acid has several industrial applications (bioplastics, cosmetics production, chemical industry) and is commonly produced via pure culture of *Lactobacillus* or other lactic acid bacteria (e.g., *Bacillus* sp.), which can metabolize sugars in the absence of oxygen (Cubas-Cano et al., 2019). As a matter of fact, these bacteria comprises one of the main groups of microorganisms identified in AF performed at low pH
<table><caption>**Table 3**
Recent examples of simultaneous production of VFAs, ethanol and lactic acid using open mixed cultures.</caption><thead><tr><th>Feedstock</th><th>Inoculum</th><th>Reactor type</th><th>T (°C)</th><th>pH</th><th>VFAs</th><th>EtOH</th><th>HLact</th><th>H<sub>2</sub></th><th>Microbiome</th><th>Reference</th></tr></thead><tbody><tr><td>Melon waste</td><td>Sludge from WWTP</td><td>Continuous</td><td>25</td><td>6.5</td><td>42.2 g/L (31.9% HAc; 55.6% HBu)</td><td>37.6 g/L</td><td>—</td><td>—</td><td>Open-mixed culture (prevailing Eubacteriaceae and Bifidobacterium)</td><td>Greses et al. (2023)</td></tr><tr><td>Organic fraction of municipal solid waste</td><td>Sludge from WWTP</td><td>Batch</td><td>20</td><td>5.5</td><td>0.45 g HAc/L; 0.93 gHBu/L; 0.11 gHPro/L</td><td>2.8 g/L</td><td>0.23 g/L</td><td>—</td><td>Open-mixed culture (prevailing Pseudomonas, Clostridiales and Ruminococcus)</td><td>Carrillo-Barragán et al. (2023)</td></tr><tr><td>Glucose</td><td>Sludge from WWTP</td><td>Sequencing batch reactor</td><td>30</td><td>4.0</td><td>2.1 g HAc/L</td><td>0.5 g EtOH/g glucose</td><td>2.2 g/L</td><td>—</td><td>Open-mixed culture (prevailing Lactobacillus and Candida/ Pichia genus)</td><td>Tamis et al. (2021)</td></tr><tr><td>Fruit and vegetable waste peptone/ yeast extract/ glucose medium</td><td>Pure culture</td><td>Continuous</td><td>35</td><td>4.4</td><td>3.1 g HAc/L</td><td>1.9 g/L</td><td>—</td><td>—</td><td>Open-mixed culture (prevailing Bacilli and Brevibacteria)</td><td>Shi et al. (2021)</td></tr><tr><td>Melon waste</td><td>Sludge from WWTP</td><td>Continuous</td><td>55</td><td>5.8</td><td>27.3 g/L (41.4% isoHBu. 16.8% HBu. 27.3% HCa)</td><td>27.3 g/L</td><td>0.8 g/L</td><td>1.35 mol-H<sub>2</sub>/ mol-glucose</td><td>E. harbinense YUAN-3</td><td>Li et al. (2020)</td></tr><tr><td>Fruit waste</td><td>Sludge from WWTP</td><td>Continuous</td><td>35</td><td>6.0</td><td>53.3 gCOD/L (44.2% HAc; 30.1% HPro; 9.9% HBu. 7.4% isoHBu. 8.5% HVal)</td><td>—</td><td>—</td><td>395.5 mLH<sub>2</sub>/ gVS<sub>inf</sub></td><td>Open-mixed culture (prevailing Ruminococcus and Lactobacilalles)</td><td>Greses et al. (2021)</td></tr><tr><td>Food waste</td><td>Sludge from WWTP</td><td>Batch</td><td>37</td><td>6.5</td><td>200 mgCOD/ gVS<sub>inf</sub> (79.3% HBu)</td><td>72 mg/ gVS<sub>inf</sub></td><td>—</td><td>—</td><td>Open-mixed culture (prevailing Lactobacillus, Caproiciproducenes, Clostridium, Prevotella and Acidipropionibacterium)</td><td>Li et al. (2022)</td></tr><tr><td>Food waste</td><td>Sludge from WWTP</td><td>Continuous</td><td>29–32</td><td>3.3–4.0</td><td>5 gHBu/L; 2gHAc/L; 2gHSuc/L</td><td>—</td><td>25.3–27.4 g/L</td><td>—</td><td>Open-mixed culture (prevailing Vagococcus and Clostridium)</td><td>Jin et al. (2019)</td></tr></tbody></table>

WWTP: wastewater treatment plant, HAc: acetic acid, HPro: propionic acid, HBu: butyric acid, isoHBu: isobutyric acid, HVal: valeric acid, isoHVal: isovaleric acid, HCa: caproic acid, HSuc: succinic acid, HLact: lactic acid, EtOH: ethanol, VS: volatile solids, COD: chemical oxygen demand, VFAs: volatile fatty acids.

values to co-produce lactic acid and carboxylates (Greses et al., 2022c, 2021). Bühlmann et al. (2021) found that AF of food waste at acid pH (3.5), short hydraulic retention times (HRTs) (1–3.5 days) and room temperature promoted the dominance of *Lactobacillus* in AF (98 % relative abundance), reaching lactic acid (23.15 g COD/L) and acetic acid as main metabolic products. Likewise, high lactic acid production (41.5 g COD/L) was reached by Acedos et al., (2022), who imposed 55 °C along with similar pH and HRTs in the AF of organic fraction of municipal solid waste.

### 4.2.3. Sequential combination of the carboxylate production and conversion in other bioprocesses

To maximize LCB valorization, carboxylates accumulated during the AF process co-produced with EtOH or lactic acid can serve as a carbon source for subsequent bioprocesses (Table 4).

Coupling carboxylates production with microbial electrolysis cells (MECs) has the potential to produce H₂ from carboxylates achieving a maximum yield of 12 mol H₂/mol glucose compared to the 4 mol H₂/mol glucose using DF and the acetate pathway as the sole technology (Yun et al., 2018). Electroactive microorganisms especially found within the Proteobacteria class (e.g., Geobacter, Shewanella, Enterobacter, Klebsiella, etc.) have the ability to use carboxylates as carbon source and transfer the electrons to the electrode (Castellano-Hinojosa et al., 2022). AF effluents from LCB such as corn stalk (Satinover et al., 2020; Zhang et al., 2022), vegetable leaves (Zhang et al., 2019), paper industry residues (Argun and Onaran, 2016; Marone et al., 2017), or food waste (Magdalena et al., 2023) rich in carboxylates have been evaluated for H₂ production in MECs resulting in H₂ yields ranging 200–1600 mL H₂/g COD<sub>in</sub> and productivities as high as 1–5 L H₂/Ld. Controlling parameters such as the different carboxylates compositions fed into the MECs

(Flayac et al., 2018; Yang et al., 2015), the inoculum source (Ruiz et al., 2014), the start-up method (Li et al., 2017), the electrode materials (Wang et al., 2022), and the operational conditions (Bakonyi et al., 2018) might substantially enhance the overall process performance.

DF to produce H₂ from LCB via carboxylate platform has been also coupled to AD to produce H₂ and CH₄ sequentially (Ravi et al., 2018; Liczbiński and Borowski, 2021; Xu et al., 2021). Several LCB were also evaluated for biohythane production (mixture of H₂ and CH₄) by upgrading the H₂ and CH₄ produced in DF and AD steps, respectively (Rena et al., 2020; Chen et al., 2021). Hydrolytic and fermentative species belonging to Clostridia (C. beijerinckii, C. welchii, C. butyricum and C. pasteurianum), or Enterobacteriaceae (Enterobacter) dominated the DF reactor whereas acetoclastic and hydrogenotrophic methanogens such as *Methanospirillum*, *Methanococcus* and *Methanoculleus*, were highlighted in the AD step (Mozhiarasi et al., 2023). A recent investigation conducted by Rena and co-workers (2020) on LCB for biohythane production highlighted the importance of the LCB composition for these interconnected bioprocesses. The high biohythane values obtained for sugarcane bagasse (53 % v/v biohythane, 41 % CH₄ and 12 % H₂) contrasted with those obtained for rice husk (17 % v/v biohythane, 12 % CH₄ and 5 % H₂), evidencing the key role of LCB components (described in Section 2).

Microalgae cultures can be sequentially performed using the carboxylates and CO₂ produced in DF as carbon source. Due to their mixotrophic metabolism, microalgae can transform these molecules into bioenergy and high-added value compounds (Lacroux et al., 2023; You et al., 2021). Some of the products that can be produced by microalgae from carboxylates include microbial oils (Chintagunta et al., 2021), polyunsaturated fatty acids (PUFAs), vitamins, and pigments such as xanthophylls (lutein, zeaxanthin), carotenoids (astaxanthin,
<caption>**Table 4**</caption>

Recent examples of applications of the carboxylate platform and the microorganisms involved where obtained carboxylates are further valorized in a sequential bioprocess.

<table><thead><tr><th rowspan="3">Substrate</th><th colspan="9">Coupling fermentation and microbial electrolysis cells</th><th rowspan="3">Reference</th></tr><tr><th colspan="3">Dark Fermentation (DF)</th><th colspan="3">Microbial electrolysis cell (MEC)</th><th colspan="3">Main Results</th></tr><tr><th>Feeding mode</th><th>Conditions</th><th>Inoculum</th><th>Feeding mode</th><th>Conditions</th><th>Inoculum</th><th>Anodic biofilm</th><th>COD removal</th><th>mL H<sub>2</sub>/ g CODin</th></tr></thead><tbody><tr><td>Cheese whey</td><td>Batch</td><td>37 °C</td><td>Anaerobic sludge</td><td>Batch</td><td>37 °C</td><td>Anaerobic sediments</td><td>50% of Geovibrio ferrireducens for the reactor fed with fruit juice (best performance)</td><td>50%<br>40-50%</td><td>836<br>754</td><td>(Marone et al., 2017)</td></tr><tr><td>Fruit juice processing WW</td><td></td><td>pH 5.5</td><td></td><td></td><td>pH 7</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sugar production WW</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Coriobacteriaceae family present in all experiments up to 10% of relative abundance</td><td>40%</td><td>396</td><td></td></tr><tr><td>Fruit juice WW</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Concentrated vinasse residue</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Paper mill WW</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cassava starch processing WW</td><td>Batch</td><td>55 °C<br>pH 6.5</td><td>T. thermosaccharolyticum</td><td>Continuous</td><td>55 °C<br>pH 6<br>HRT 2 days</td><td>Mixed culture</td><td>Thermoanaerobacterium sp., Geobacillus sp., Thermacetogenium sp. and Pseudomonas sp.</td><td>40%<br>58%</td><td>256<br>465</td><td>(Khongkliang et al., 2017)</td></tr><tr><td>Food waste</td><td>Batch</td><td>35 °C<br>pH 5.5</td><td>Anaerobic sludge</td><td>Batch</td><td>30 °C<br>pH 7.0</td><td>Anaerobic sludge</td><td>92% of Geobacter</td><td>56%</td><td>873</td><td>(Cardeña et al., 2018)</td></tr><tr><td>Macroalgae S. japonica</td><td>Batch</td><td>22 °C<br>pH 7.0</td><td>DF effluent Bacteroides</td><td>Batch</td><td>35 °C<br>pH 7.0</td><td>MEC effluent</td><td>53% of Geobacter</td><td>67-79%</td><td>236-438<br>mLH<sub>2</sub>/g TS</td><td>(Yun et al., 2021)</td></tr><tr><td>Food waste</td><td>Continuous</td><td>35 °C<br>pH 6.0</td><td>Seed sludge</td><td>Batch</td><td>20 °C<br>pH 7.0</td><td>Anaerobic sludge</td><td>Acetobacterium Geobacter Desulfovibrio Methanobrevibacter</td><td>44%</td><td>0.83 L/Ld</td><td>(Jia et al., 2020)</td></tr><tr><td>Water hyacinth</td><td>Batch</td><td>35 °C<br>pH 7.0</td><td>Anaerobic sludge</td><td>Batch</td><td>35 °C<br>pH 7.0</td><td>Anaerobic sludge</td><td>n.a.</td><td>88%</td><td>565 mLH<sub>2</sub>/g VS</td><td>(Tran and Nguyen, 2022)</td></tr></tbody></table>

<table><thead><tr><th rowspan="3">Substrate</th><th colspan="9">Coupling fermentation and lipid production via oleaginous yeasts growth</th><th rowspan="3">Reference</th></tr><tr><th colspan="3">Dark Fermentation (DF)</th><th colspan="3">Yeast growth</th><th colspan="3">Main Results</th></tr><tr><th>Feeding mode</th><th>Conditions</th><th>Inoculum</th><th>Feeding mode</th><th>Conditions</th><th>Species</th><th>Lipid concentration</th><th>LipidProductivity</th><th>% Lipids in biomass</th></tr></thead><tbody><tr><td>Cyanobacteria Microcystis sp.</td><td>Batch</td><td>35 °C<br>pH 6.5</td><td>Anaerobic sludge</td><td>Batch</td><td>30 °C<br>pH<sub>initial</sub> 7.0</td><td>C. curvatus</td><td>0.32 g/L</td><td>0.15 g/Ld</td><td>14.5 %</td><td>(Cho et al., 2017)</td></tr><tr><td>Microalgae C. vulgaris</td><td>Semi-continuous</td><td>25 °C<br>pH 6-7<br>uncontrolled</td><td>Adapted DF sludge</td><td>Batch</td><td>27 °C<br>pH 6.5<br>5, 10 and 15 g/L carboxylates</td><td>C. curvatus<br>L. lipofer<br>R. toruloides<br>W. saturnus<br>Y. lipolytica</td><td>n.a.</td><td>0.1-0.13 g/g<br>0.02-0.05 g/g<br>0.05-0.06 g/g<br>0.07-0.11 g/g<br>0.05-0.08 g/g</td><td>29.2-36.9%<br>1.4-16.8%<br>19.9-25.7%<br>29.4-33.9%<br>14.5-20.1%</td><td>(Llamas et al., 2020a)</td></tr><tr><td>Food waste</td><td>Batch</td><td>T 37 °C<br>pH<sub>initial</sub> 7</td><td>Mesophilic anaerobic sludge</td><td>Batch</td><td>T 28 °C<br>pH<sub>initial</sub> 6.0<br>T 38 °C<br>pH<sub>initial</sub> 6.0<br>2.5-20 g/L acetate, propionate, butyrate</td><td>Y. lipolytica</td><td>0.55-1.17 g/L<br>0-0.803 g/L</td><td>Up to 0.29 g/g<br>Up to 0.28 g/g</td><td>Up to 31.6%<br>Up to 30.78%</td><td>(Gao et al., 2017)</td></tr></tbody></table>

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<table><caption>Table 4 (continued)</caption><thead><tr><th>Food waste</th><th>Semi-continuous</th><th>T 25 °C<br>pH 5.5-6</th><th>50% anaerobic sludge<br>psychrophilic DF</th><th>Batch</th><th>T 27 °C<br>pH<sub>initial</sub> 6.0</th><th>Y. lipolytica</th><th>Up to 5 g/L</th><th>0.15-0.31 g/g</th><th>Up to 45%</th><th>(Morales-Palomo et al., 2022)</th></tr></thead><tbody><tr><td colspan="11">Coupling fermentation and anaerobic digestion</td></tr><tr><th rowspan="2">Substrate</th><th colspan="4">Dark Fermentation (DF)</th><th colspan="2">Anaerobic digestion (AD)</th><th colspan="3">Main Results</th><th rowspan="2">Reference</th></tr><tr><th>Feeding mode</th><th>Conditions</th><th>Inoculum</th><th>Feeding mode</th><th>Conditions</th><th>Inoculum</th><th>Microbial communities</th><th>H<sub>2</sub> yield</th><th>CH<sub>4</sub> yield</th></tr><tr><td>Rice straw and<br>Pig manure</td><td>Continuous</td><td>37 °C<br>55 °C</td><td>Acid-pretreated anaerobic<br>sludge</td><td>Continuous</td><td>55 °C<br>37 °C</td><td>Anaerobic sludge</td><td>Bacteria in AF<br>Parabacteroides<br>Bifidobacterium<br>Ruminococcus<br>Clostridium<br>Archaea in AD<br>Methanobrevibacter<br>Methanothermobacter<br>n.a.</td><td>3 mL H<sub>2</sub>/g VS<br>16 mL H<sub>2</sub>/g VS</td><td>109 mL CH<sub>4</sub>/<br>g VS<br>197 mL CH<sub>4</sub>/<br>g VS</td><td>(Chen et al., 2021)</td></tr><tr><td>Fruit waste<br>Vegetable waste<br>Sugarcane<br>bagasse<br>Rice husk<br>Wheat straw</td><td>Batch</td><td>Standard</td><td>Cow dung sludge and<br>anaerobic sludge</td><td>Batch</td><td>Standard</td><td>Cow dung sludge<br>and anaerobic<br>sludge</td><td>n.a.</td><td>14-16%<br>27-29%<br>11-13%</td><td>28-29%<br>11-13%<br>39-44%</td><td>(Rena et al., 2020)</td></tr><tr><td>Wheat straw</td><td>Batch</td><td>35-37 °C</td><td>Pretreated digestate from<br>methanogenic reactor</td><td>Batch</td><td>35-37 °C</td><td>Anaerobic sludge<br>from methanogenic<br>reactor</td><td>Bacteria in AF<br>Proteiniphilum<br>vacuoliferans<br>Prevotella<br>Archaea in AD<br>Methanobacterium,<br>Methanosarcina</td><td>4-5%<br>3-4%<br>14.43%</td><td>10-12%<br>25-26%<br>69%</td><td>(Kabaivanova<br>et al., 2022)</td></tr><tr><td>Microalgae<br>C. vulgaris</td><td>Semi-continuous</td><td>25 °C<br>HRT 8 days<br>pH 6.5</td><td>Mesophilic anaerobic<br>sludge</td><td>Semi-continuous</td><td>35 °C<br>HRT 20 days<br>pH uncontrolled</td><td>Mesophilic<br>anaerobic sludge</td><td>Bacteria in AF<br>Clostridiales,<br>Peptostreptococcus,<br>Corynebacterium<br>Archaea in AD<br>Methanobrevibacter<br>Methanosphaera</td><td>16 mL CH<sub>4</sub>/<br>gCODin<br>No H<sub>2</sub> detected</td><td>169 mL CH<sub>4</sub>/<br>gCODin</td><td>(Llamas et al., 2021)</td></tr><tr><td colspan="11">Coupling fermentation and microalgae growth</td></tr><tr><th rowspan="2">Substrate</th><th colspan="4">Dark Fermentation (DF)</th><th colspan="2">Microalgae growth</th><th colspan="3">Main Results</th><th rowspan="2">Reference</th></tr><tr><th>Feeding mode</th><th>Conditions</th><th>Inoculum</th><th>Feeding mode</th><th>Conditions</th><th>Strain employed</th><th>Lipids</th><th>Products obtained and study<br>highlights</th></tr><tr><td>n.a.</td><td>Synthetic DF</td><td></td><td></td><td>Batch</td><td>27 °C<br>pH 6.5</td><td>C. cohnii</td><td>30-50%</td><td>Omega -3 (29.8%)</td><td>(Chalima et al., 2019)</td></tr><tr><td>Corn cob<br>Corn stalk<br>Rice straw<br>Wheat straw<br>n.a.</td><td>Batch and<br>continuous</td><td>36 °C<br>pH 6.5</td><td>Pretreated anaerobic sludge</td><td>Continuous</td><td>25 °C</td><td>Scenedesmus</td><td>30-35%<br>35%<br>25-30%<br>30-35%</td><td>686 mL H<sub>2</sub>/L<br>762 mL H<sub>2</sub>/L<br>425 mL H<sub>2</sub>/L<br>238 mL H<sub>2</sub>/L</td><td>(Ren et al., 2019)</td></tr><tr><td>n.a.</td><td>Synthetic DF</td><td></td><td></td><td></td><td>25 °C</td><td>Polytomella sp<br>E. gracilis<br>C. sorokiniana<br>O. danica</td><td>15-20%<br>15-20%<br>20%<br>15-20%</td><td>Polytomella sp could grow on:<br>- 38 g/L acetate<br>- 18 g/L butyrate</td><td>(Lacroux et al., 2022)</td></tr><tr><td>n.a.</td><td>Synthetic DF</td><td></td><td></td><td>Batch</td><td>T 35 °C<br>30 g/L acetate</td><td>A. protothecoides<br>C. sorokiniana</td><td>30-35%<br>30-40%</td><td>microalgae tolerated up to 30 g/L<br>carboxylates for lipid synthesis</td><td>(Patel et al., 2022)</td></tr></tbody></table>

n.a. not available.
β-Carotene), and phycocyanin (Castillo et al., 2021). Literature on sequential AF and microalgae culture from LCB is scarce, but a recent study assessed the coupling of AF and *Scenedesmus* sp. cultivation for H₂ and lipid production using corn cob, corn stalk, rice straw and wheat straw as substrates (Ren et al., 2019). Results showed a stable H₂ pro-duction rate of 0.81 L H₂/Ld in DF and a lipid accumulation (35 % w/w) in microalgae biomass. Similarly, another study fermenting vegetable garden and food waste showed the growth of *Cryptothecinium cohnii* using acetate, propionate, and butyrate as carbon sources and accu-mulating an omega-3 content of nearly 30 % of total fatty acids (Chalima et al., 2019). In this regard, microalgae species play a key role to metabolize a specific organic acid profile, although in general micro-algae growth is favoured with effluents containing high acetate and low butyrate concentrations (Chiranjeevi and Venkata Mohan, 2017).

Carboxylates might also serve as carbon sources for oleaginous yeasts (Llamas et al., 2020b). Yeast cultivation in carboxylate-rich AF effluent has been demonstrated to mediate the growth of lipid rich biomass. Strains such as *Cryptococcus curvatus*, *Rhodosporidium toruloides*, *Rhodotorula graminis*, *Yarrowia lipolytica*, or *Rhodotorula glacialis* can accumulate intracellular lipids in form of lipid bodies up to 80 % (w/w) (Juanssilfero et al., 2018). Yeast lipid production has been traditionally investigated using the sugar platform from LCB hydrolysates as carbon source (Patel et al., 2015; Probst and Vadlani, 2015; Johnra-vindar et al., 2018; Niehus et al., 2018), whereas studies using carboxylates are novel. Microalgae biomass (Llamas et al., 2021), cyanobacterial biomass (Cho et al., 2017) and food waste (Gao et al., 2017; Morales-Palomo et al., 2022) have been used to produce carboxylates that were converted in microbial oils by several oleaginous yeast. In these cases, lipid concentrations, productivities and biomass yields ranging 0.1–3.2 g/L, 0.1–0.3 g/Ld, and $Y_{x/s}$ of 0.23–0.8, respectively, with a lipid content of 14–43 % w/w were obtained. With regard to LCB, *Y. lipolytica* was grown in carboxylates generated from fruit and vegetable waste reaching lipid levels (w/w) of 26.0 % and biomass yields of $Y_{x/s}$, 0.53 (Gao et al., 2020). Similarly to microalgae, authors highlighted the great influence exerted by the carboxylate profile on both yeast growth and lipid accumulation. Indeed, short-chain carboxylates (e.g., acetate) were preferred over long ones (e.g., butyrate, caproate) as it was pointed out in Section 3.3.3.

## 5. Outlook, future perspectives and challenges

The use of co-cultures appears as a promising strategy for the efficient conversion of challenging feedstock like LCB. The present review demonstrates the possibility of producing different bioproducts from LCB when combining two or more species and bioprocesses. However, the lack of knowledge on the occurring interactions between microorganisms makes the co-cultivation strategies very challenging.

The study of co-cultures may involve large experimental designs, especially when more than two strains are evaluated. This is why high throughput systems could be ideal for these studies. The appropriate selection of the involved microorganisms or the conditions to promote a specific microbiome in open mixed cultures have a large effect on product yields and titters. Furthermore, a better balance in the specific ratios between microorganisms would allow them to efficiently consume mixtures of carbon sources and produce different targeted bioproducts. Not only at biological level, but also new bioreactors design would be beneficial to correctly apply selected co-cultivated microorganisms for optimum LCB conversion. Advanced computational, system and synthetic analysis tools should be developed to better understand species interactions and behaviours.

The development of artificial microbial consortia or co-cultivation systems will open new perspectives in biotechnology. The co-existence of microorganisms may create new environments for strains that may result in the activation of new metabolic pathways not expressed under typical cultivations with single strains (Qian et al., 2020).

As previously discussed, the co-cultivation of microorganisms allows

labor division when the complex LCB conversion into bioproducts takes place, thereby enhancing the overall process efficiency. Main benefits of applying microbial consortia in biotechnology include: i) avoiding contamination of external microbes, ii) reduced process length, iii) emergence of new metabolic pathways resulting in new bioproducts, and iv) higher productivities and efficiency in substrate use (Rosero-Chasoy et al., 2021). In the same line, challenges associated to the use of co-cultures comprise: i) meeting the requirements for all involved microorganisms, ii) process control for stable bioproduction systems, iii) complex and costly bioproducts extraction systems, and iv) application of simulation and analysis tools for complex co-cultivation systems.

Innovative approaches on microorganisms co-culturing included in this review should not be underestimated when addressing the valorization of challenging substrates such as LCB. The combination of the inherent characteristics of individual microorganisms could definitively help overcome main challenges associated to LCB conversion to achieve more efficient and economically viable processes.

Apart from the mentioned challenges, industries using the sugar and carboxylate platforms for bio-based production face other technical challenges. These include feedstock availability, infrastructure limitations, cross contamination and low process yields. Ensuring a consistent and sustainable LCB supply is crucial to avoid potential fluctuations in availability and prices. Building a robust infrastructure and supply chain, including transportation, storage, and logistics, also poses challenges for new companies. Technically, process efficiency and yields, encompassing fermentation and downstream processing, require ongoing research and development for cost-effectiveness.

The use of co-cultures is appearing as a promising strategy to be applied at industrial level to overcome some of the faced challenges. However, the lack of knowledge on metabolic microbial interactions and microorganisms dynamics still hampers the applicability of their full potential at industrial scale (Lima and de Lucas, 2022).

Co-cultures and consortia, for example, can help tackling contamination in industrial systems (Padmaperuma et al., 2018). In the same line, the cost of bioethanol production from LCB at commercial scale is still excessive (Rosales-Calderon and Arantes, 2019) and co-cultivation of lignocellulolytic fungi could significantly reduce the price for enzymes to make LCB conversion more economically feasible (Rosero-Chasoy et al., 2021; Lima and de Lucas, 2022).

Despite the lack of examples of co-cultures applied at industrial level for LCB conversion, Anaergia (Canada) is specialized in converting organic waste (municipal solid waste, wastewaters) into renewable energy (biogas), fertilizer, and carboxylates via microbial consortia in AD. Besides, EcoXtract (France) extracts organic compounds, including carboxylates produced in mixed cultures from various organic waste sources by means of their own technology to be used in different applications such as biofuels and bioplastics.

## 6. Conclusions

Benefits associated to microbial co-cultures can boost the bioconversion of challenging residues such us LCB. Independently of the platform through which LCB is valorized (i.e., sugar or carboxylates), several factors like microorganisms' ratios, inoculation times, optimum growth conditions, nutritional requirements, etc. should be considered. These factors impact the dynamics between the co-cultured microorganisms and affect process outputs. Despite the increasing interest on co-cultures, the knowledge on the mechanisms through which microorganisms interact is still scarce. Thus, it is crucial to gain insights on microorganisms co-cultivation to make their use for production of biochemicals from LCB at industrial scale a reality.

### CRediT authorship contribution statement

**Mercedes Llamas:** Writing – original draft, Writing – review & editing.
**Silvia Greses:** Writing – original draft, Writing – review &
editing. **Jose Antonio Magdalena:** Writing – original draft, Writing – review & editing. **Cristina González-Fernández:** Conceptualization, Writing – review & editing, Funding acquisition. **Elia Tomás-Pejó:** Conceptualization, Writing – review & editing, Funding acquisition.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data availability

No data was used for the research described in the article.

## Acknowledgements

Jose Antonio Magdalena would like to thank the Complutense University of Madrid for the financing of his contract at LBE-INRAE (France), with funds from the Ministry of Universities for the requalification of the Spanish University System for 2021–2023 (Modality 1. Margarita Salas), coming from the European Union-Next generation EU funding. Elia Tomás thanks the Spanish Agency of Research for her Ramón y Cajal Grant (RYC2019-027773-I/AEI/10.13039/501100011033. This work has been also supported by the Spanish Ministry of Science and Innovation through the project RAVIOLIC (TED2021-132809A-100), RESOPLA (TED2021-132024B-C21) and BIOMIO (PID2020-119403RBC21) funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A Way of Making Europe”, “ESF Investing in Your future” and “European Union NextGenerationEU/PRTR”).

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