Towards an integrated microbial process to convert lignocellulosic biomass to ethanol: challenges and research opportunities

The literature reviewed in this paper shows that there are microorganisms which are able to catalyse each of the three steps required for the conversion of lignocellulosic biomass into ethanol: lignin hydrolysis, cellulose hydrolysis and glucose and xylose fermentation to ethanol. Therefore, an entirely microbial process converting lignocellulosic biomass to ethanol can, at least in principle, be considered. Compared to the alternative processes which use high-pressure/high temperature conditions for lignin hydrolysis and external enzymes addition for cellulose hydrolysis, an entirely microbial process at ambient pressure and relatively low temperature would clearly give an important reduction in process costs.

An integrated (or single stage or consolidated) process which uses untreated lignocellulosic biomass as feedstock and converts it to ethanol is clearly the most desirable option. This process could be obtained using two alternative approaches: use of an open mixed culture, where many different naturally-occurring microorganisms co-exist and carry out the various steps, or use of a pure culture of a genetically modified microorganism which is able to carry out all the required process steps. However, either approach is still far from becoming reality. In this section the main challenges and research opportunities for the two approaches will be discussed.

4.1. Open mixed cultures

The main challenges to be overcome for the development of a mixed culture process are the following:

- Low rates of lignin and cellulose hydrolysis. The rates of lignin and cellulose hydrolysis reported so far for microbial processes are lower than for chemical-physical or enzymatic processes;

- Control of the anaerobic fermentation of sugars (mainly glucose and xylose) to ethanol. In a mixed culture environment, fermentation of sugars can lead to many different products, in addition to ethanol, i.e. other alcohols, volatile fatty acids (acetic, propionic, butyric, etc), hydrogen or methane;

- Co-existence of lignin- and cellulose-hydrolysing and of ethanol-producing microorganisms in the same vessel. For an integrated process the microbial populations responsible for lignin and cellulose hydrolysis and the ones responsible for sugars fermentation should co-exist in the same vessel. This might be possible or not, depending on the operating conditions of the process and on the growth rate of the various microorganisms.

Some strictly interlinked research opportunities which may address the challenges above are discussed below.

- Enrichment studies: As discussed in section 3.1.1, microbial hydrolysis of lignin is usually considered to be difficult and slow and a factor that may limit the rate of hydrolysis is adaptation of the microorganisms to the substrate. It is possible to hypothesise that once mixed cultures have become adapted to a lignocellulosic substrate and have synthesised the enzymes required for its hydrolysis, then the rate of hydrolysis should proceed faster. Therefore a process can be envisaged, where a mixed culture is previously acclimated to the lignocellulosic substrate (slow process) and then transferred in a continuous reactor, or semi-continuous reactor such a Sequencing Batch Reactor, with a continuous, or semi-continuous, feed of the substrate (fast process). Having been previously acclimated, the microbial culture should be able to remove the substrate at high rate. Investigation of this process at lab-scale is possible but very few studies have been carried out. An example is the study by Haruta et al.,¹⁰² where a stable microbial community able to degrade various cellulosic and lignocellulosic substrates was generated from composting microorganisms by acclimation on filter paper;

- Particle size reduction: reducing the feedstock particle size is expected to give higher rates of hydrolysis, but the quantitative evidence for this effect is rather limited, especially as far as ethanol production is concerned. Lab-scale studies specifically targeted at exploring and quantifying the possible rate increase obtainable by particle size reduction are required;

- Reactor configuration and process parameters: the reactor used for the integrated process can be operated under various configurations, e.g. continuous-flow with or without biomass recycle, Sequencing Batch Reactor, etc. For each configurations, various process parameters have to be specified, e.g. temperature, pH, hydraulic retention time, solids retention time, length of cycle, length of the feed (the latter two only apply to Sequencing Batch Reactors). The choice of these parameters can affect both the hydrolysis rate and the spectrum of product distribution of sugars fermentation. E.g. it can be expected, in principle, that in a Sequencing Batch Reactor the hydrolysis rate should be higher than in a continuous-flow reactor due to the higher substrate concentration at the start of the cycle, which is expected to give a higher reaction rate. However, no experimental proof of this in the context of lignocellulosic biomass hydrolysis has been reported. Similarly, only limited experimental investigation has been carried out regarding the effect of process parameters on products distribution of sugars fermentation, examples are the studies by Temudo et al.^13,97 All these aspects deserve systematic investigation at lab-scale.

An interesting alternative to open mixed cultures is the use of selected mixed cultures, where only selected species, responsible for different stages of the lignocellulosic biomass conversion to ethanol, are inoculated in the reactor. A successful study using this approach has been published very recently.¹⁰³ The authors obtained 67% ethanol yield from pretreated (dilute acid) wheat straw using a microbial culture composed of three naturally occurring strains: Trichoderma reesei, Saccharomyces cerevisiae and Scheffersomyces stipitis. The fungus T. reesei was responsible for cellulose and hemicellulose hydrolysis, while the yeasts were responsible for ethanol production from glucose (S. cerevisiae) or pentoses (S. stipitis). The authors utilised a biofilm membrane reactor with the presence in the same reactor of aerobic, microaerophilic and anaerobic conditions, therefore allowing the co-existence of the three different species.

4.2 Genetically modified microorganisms

Similarly to the use of open mixed cultures, the approach of using a single, genetically modified, microorganism to convert lignocellulosic biomass to ethanol is still far from becoming reality.

So far no attempt has been reported to introduce in microorganisms the ability to hydrolyse/break down lignin and this is probably due to the complexity of the genome of the native lignin degrading species. Therefore, so far the concept of metabolic engineering for bioethanol production has been focused on the use of chemically or physically pretreated feedstocks, where lignin has been hydrolysed and cellulose is available for microbial attack. Considering metabolic engineering for cellulose hydrolysis, the ability to hydrolyse pretreated cellulose has been introduced in various microbial strains, but so far very little success has been reported with untreated crystalline cellulose. More success has been reported in the increase of ethanol yield in microorganisms that are naturally able to hydrolyse cellulose, but even in this case the rates are in the majority of cases very low.¹⁰⁴ So far, the main success of genetic engineering for bioethanol production has been the development of microorganisms which are able to convert multiple sugars to ethanol with high yields. While this is an important step forward, the main issues of lignin and cellulose hydrolysis are still far from being solved by means of genetically modified microorganisms.

Interesting research opportunities lie ahead in the following areas:

- Introduction of the lignin hydrolysis capability into microorganisms which are naturally able to hydrolyse cellulose, or, as opposite strategy, introduction of the cellulose hydrolysis capability into microorganisms which are naturally able to hydrolyse lignin;

- Improvement in the ability to hydrolyse crystalline cellulose with microorganisms which are native ethanol producers;

- Increase in the ethanol yield for microorganisms which are naturally able to hydrolyse cellulose.

This paper has reviewed the existing literature on microbial processes for lignin hydrolysis, cellulose hydrolysis and glucose fermentation to ethanol. The main evidence from this study is the following:

- there is a wide range of microorganisms that can perform each of the three steps required for lignocellulosic biomass conversion into ethanol, i.e. lignin hydrolysis, cellulose hydrolysis and glucose, or xylose, fermentation to ethanol;

- while there are many reported fungi species that are able to hydrolyse lignin under aerobic conditions, there is only one recent study in the literature giving clear evidence of lignin hydrolysis under anaerobic conditions. However, many mixed culture studies give an indirect evidence that lignin can be at least partially degraded under anaerobic conditions. In principle, if anaerobic lignin hydrolysis can be achieved, a single-stage process with mixed microbial cultures including lignin and cellulose hydrolysis and glucose fermentation to ethanol can be envisaged;

- cellulose and hemicelluloses hydrolysis can be carried out by many different microbial species, both under aerobic and anaerobic conditions. Interestingly, the literature evidence collected so far indicates no significant differences in the cellulose hydrolysis rate under aerobic or anaerobic conditions;

- regarding anaerobic fermentation of sugars to ethanol, literature studies with mixed cultures specifically targeted at ethanol production have been very limited and they have reported a maximum yield of 0.8 mol ethanol/mol glucose, compared to the 2 mol ethanol/mol glucose which is the theoretical maximum yield;

- metabolic engineering has been successful in generating microorganisms which are able to convert a wider range of sugars to ethanol with high yields, however much more limited success has been obtained by engineering microorganisms in order to combine cellulose hydrolysis and high ethanol yield.

An integrated (or consolidated) process converting untreated lignocellulosic biomass to ethanol can, at least in principle, be conceived according to two different approaches: use of open mixed cultures of existing microorganisms or use of a pure culture of a genetically modified microorganism. Regarding the use of open mixed cultures, the main challenges to be overcome are: low rates of lignin and cellulose hydrolysis, control of the anaerobic fermentation of sugars to ethanol and co-existence of different microbial populations in the same reactor. Possible research areas which can help addressing these challenges are: enrichment studies with microbial adaptation to the lignocellulosic substrate, investigation of the effect of particle size reduction on the hydrolysis rates and investigation of the effect of reactor configuration and operating parameters. Regarding the use of genetically modified microorganisms the main challenges are the development of microorganisms which are able to hydrolyse lignin and crystalline cellulose and convert the produced sugars to ethanol.