**7. Linking metabolic engineering of** *Synechocystis* **to production**

While progress is being made with metabolic engineering for ethanol production and establishing *Synechocystis* as a cell factory there needs also be to an understanding of the production landscape when developing the system at industrial scale. Generation of a viable ethanol producing photoautotroph will necessitate rolling out of a production system to commercial level. Thus, at one level, there are the limitations and possibilities of metabolic engineering which have been discussed above but there is a second level that also needs to be addressed, that of the production environment to realize the potential of metabolically engineered strains. Indeed a fuller understanding of the requirements at this stage can help inform the strategies used for optimal metabolic engineering of potential production candidates.

## **7.1. Overall process life cycle analysis**

Implementation of an industrial process for ethanol production from cyanobacteria will be the next stage of development once the challenges of metabolic engineering have been addressed. Development of the downstream aspects of production will require optimization of several parameters and a more favorable economic outlook. Capital expenditure (CapEx) will be a key driving force with many components needing to be considered. Chief amongst these is the nature of the producing organisms being a recombinant strain. This poses potential safety and containment considerations, which would add to the economics of plant construction and operation. The need for sunlight (which may limit location of production facilities) or continuous LCD exposure again adds costs with either cyclic day exposure in high light climates or continuous growth with added light, which would come with an added energy cost. Equally, calculations of volumes that would be needed suggest large CapEx expenditure on plant, large water requirements and effluent processing costs. Many geographical areas that have high sunlight with marginal land, such as desert areas, at first sight might seem suitable but will suffer from water limitations. Other issues that are related to CapEx relate to the growth of the production strains themselves and the provision of optimal conditions for growth and production. Currently as one diverts photosynthetic intermediates to ethanol, one is affecting the flux to biomass. The more ethanol that is produced the slower the growth and the less biomass that can be produced. This impinges significantly on the growth rate and hence competitiveness of production strains. Given that, growth under sterile conditions in photobioreactors would be economically unsustainable (due to cost and the low value of the product ethanol); competitor contamination would need to be built into the growth cycle. Thus, slow growth of producers would have two major potential consequences that could affect the process. Firstly, there may be mutational selection for faster growers, which have lost the engineered ethanol cassette reducing the yield during production, and secondly given that axenic conditions could not be maintained during aseptic but non-sterile culturing, contaminants could easily outgrow the engineered strains. Strategies that might mitigate this could be the addition of mutualistic consortia, which might stimulate the production strains by providing vitamins or co-factors while limiting the growth of contaminants [68]. Thus, strategies that would aid production at large scale would need to be factored in at the initial stages of metabolic engineering.

#### **7.2. Reactor design for large-scale economic production**

would have this capacity it is important to determine the level of tolerance to ethanol in engineered strains and examine toxic or stress related effects. Proteomic analysis has been used to determine the response of engineered strains [64] with current ethanol production levels and the response of strains with ethanol added up to projected or expected ethanol production levels [65]. In the case of added ethanol incubation with 1.5% (v/v) of ethanol for 24 h reduced growth of *Synechocystis* by 50% with cell aggregation visible [65]. Proteome analysis revealed some 32 unique proteins up-regulated and some 42 down-regulated after 24 h. This number of altered proteins increased after 48 h. incubation. Many of these proteins were demonstrated to be involved in the common stress response such as those associated with oxidative stress [65], transporters, cell-membrane modifying proteins and proteins associated with the photosystems. Many of the altered protein observed in the proteome response in *Synechocystis* were similar to those observed in the tolerance response of *Zymomonas mobilis* [66]. Proteomic analysis has also been observed on metabolic engineered *Synechocystis* producing levels as outlined in **Table 1** [64]. Here some 60–70% of the carbon fixed was converted to ethanol via a single ethanol cassette. At this rate of ethanol production there was no significant stress response observed rather there was a realignment of systems. Some upregulation of carbon concentrating mechanisms were observed, as were enzymes of the Calvin cycle and photosynthesis antennae proteins [64]. Interestingly the t*hi*C gene, encoding phosphomethylpyrimidine synthase involved in thDP synthesis (the PDC-cofactor), was also up-regulated suggesting that even at this low level of ethanol production that co-factor availability was limited [64]. Tolerance has also been examined via transcriptomic analysis following exogenous ethanol addition [67] with 1.2–3% ethanol addition to wild type *Synechocystis*. Addition of 1.5% caused a 50% reduction in growth rate with visible aggregation suggesting stress. Many of the genes up-regulated in the transcriptomic study were associated with energy metabolism particularly photosynthesis. The results observed were broadly in line those observed in the

**7. Linking metabolic engineering of** *Synechocystis* **to production**

used for optimal metabolic engineering of potential production candidates.

While progress is being made with metabolic engineering for ethanol production and establishing *Synechocystis* as a cell factory there needs also be to an understanding of the production landscape when developing the system at industrial scale. Generation of a viable ethanol producing photoautotroph will necessitate rolling out of a production system to commercial level. Thus, at one level, there are the limitations and possibilities of metabolic engineering which have been discussed above but there is a second level that also needs to be addressed, that of the production environment to realize the potential of metabolically engineered strains. Indeed a fuller understanding of the requirements at this stage can help inform the strategies

Implementation of an industrial process for ethanol production from cyanobacteria will be the next stage of development once the challenges of metabolic engineering have been addressed.

proteomic studies [64].

210 Fuel Ethanol Production from Sugarcane

**7.1. Overall process life cycle analysis**

The need for significant scale up of photoautotrophic ethanol production in a high light environment can add significantly to initial CapEx. Within the reactor system itself, several components may need significant attention. It is impractical for low value ethanol products, at least in comparison to current fuel costs, for growth and production to be carried out in sterile photobioreactors (PBRs) with full control over light, and key physiological conditions. Although the technologies for such photobioreactors are well developed their practicality can reasonably only be considered suitable for high value products [69]. In addition to containment issues, there are issues with inoculum development for non-axenic culturing to insure that initial inoculum is stable, productive and clonal. Depending on the plant size, this may require significant CapEx.

The most frequent types of PBRs proposed are non-sterile horizontal tubular or vertical flat panel PBRs, which have several limitations including: (a) cost, which have been estimated at €2400 m2 for small scale, reducing in cost slightly with scale [70]. This would result in a cost of some €12.6 kg−1 [71], (b) High energy consumption [72] from mixing, CO<sup>2</sup> supply, pumping, separations, cleaning, and (c) Maintenance, cleaning and labor costs [70], (d) The reactor design must be able to withstand photo-oxidation, prevent evaporative loss of product, while maintaining axenic conditions as long as possible. Given the generally slow growth, rates of cyanobacterial species, largely because of the photoautotrophic lifestyle, need to manufacture most of their metabolites, maintain a polyploid genome because of the high sunlight and UV exposure the design of PBRs suitable for low value ethanol production from cyanobacteria is a challenge.

54 W.m<sup>3</sup>

for flat panel PBRs and to 3.7 W.m<sup>3</sup>

**7.4. Ethanol recovery from production media**

**8. Conclusions and perspectives**

may be a key hurdle to overall process efficiency and economy.

ing, hence the current focus on optimizing metabolic engineering for yield.

cant challenges to future implementation of a viable production system.

The basic proof of concept for photoautotrophic ethanol production from model cyanobacteria such as *Synechocystis* has been carried out. Strategies for increasing ethanol yields are currently being investigated but there are challenges going forward. These include the effect of ethanol synthesis on the metabolism of the producing strain, which include understanding and optimizing carbon flux, the tolerance of the organism to ethanol, growth and production rates and the challenges of integrating a production strategy that can inform the metabolic engineering strategy. Indeed the lessons learned from model organisms such as *Synechocysti*s may need to be applied to different candidate strains which grow faster, can be genetically modified, are more robust in non-axenic culture, are more competitive or are more tolerant to the product once yields are increased. It is very much a case of much work done but signifi-

are quite different in the different systems such that in raceway systems there is little mixing, which effects movement of producing cells into light and poor mass transfer limiting overall productivity. Thus, power consumption unless linked to wind or solar in an integrated way

Lignocellulose based fermentations tend to be more dilute than starch based systems due to the presence of hemicellulose which increases viscosity and the presence of fermentation based inhibitors [75, 76]. This is currently similar in terms of cyanobacterial production of ethanol, which is also dilute and low in terms of yield. Recovery of ethanol from dilute production streams in an energy efficient and economical manner poses significant technical difficulties. Traditionally ethanol is recovered via distillation, however in the case of biofuel ethanol from cyanobacterial production the energy costs of distillation would be far too high particularly from dilute streams. It has been estimated that in a well-integrated lignocellulose to ethanol plant the process would require 4350 MJ.m−3 equivalent to approximately 20% of the energy content of the ethanol produced [76]. Thus, alternatives to distillation are needed to drive economy from cyanobacterial production systems. A number of techniques are available which may be suitable for the recovery of ethanol from cyanobacterial production such as membrane permeation or pervaporation, vacuum stripping, gas stripping, solvent extraction, adsorption and various hybrid processes [76]. However, the efficiency is dependent on the initial ethanol concentration (which is currently low for metabolically engineered *Synechocystis*) and often multiple cycles of processing would be required to achieve optimal yield. Thus, there are currently significant challenges to optimal recovery from dilute streams such as cyanobacterial systems although the potential for energy savings over distillation are possible. This thus implies that the higher the yield initially the better in terms of downstream process-

for raceway systems [74]. However mixing rates

http://dx.doi.org/10.5772/intechopen.77271

213

Metabolic Engineering of the Model Photoautotrophic Cyanobacterium *Synechocystis*…

In production terms once one moves away from a controlled PBR design one halves the production cycle and level of photosynthetic production due to the night-day diurnal cycle and in addition there is less process control over the operation. Many approaches have been taken in an attempt to reduce cost; this has included use of bicarbonate-based systems for supply of carbon following carbon capture [73]. This may have significant cost savings in terms of CO<sup>2</sup> sparging, transport costs and CO<sup>2</sup> loss due to outgassing. Bag type culturing [70] which can be once off or be reusable can offer another potential solution. This may mitigate against some of the limitations of more traditional PBRs. Controlling contaminants in non-axenic culture might be carried out by use of pH as a control mechanism for limiting contamination, however this may necessitate use of more alkaliphilic cyanobacterial species [73]. Indeed adapting the production strain to the process or vice versa may offer a way forward in developing optimal reactor configurations with reduced CapEx. Thus incorporating knowledge of the production cycle, the types of conditions required for growth into a metabolic engineering strategy can be important during initial development of strains and strategies.

#### **7.3. Temperature control, energy and evaporative loss**

By virtue of the fact that ethanol-producing cyanobacteria will be recombinant strains, the current experimental systems tend to be enclosed due to regulatory constraints with GMO's. In geographical locations which are suitable for maximal sunlight and hence photosynthesis, enclosing a facility may raise issues with temperature control unless this is designed into the build. Direct exposure to air circulation or venting may also not be feasible due to safety issues while heat buildup beyond optimal growth temperatures, such as 30°C for *Synechocystis*, may easily occur. In such cases, utilizing a thermophilic or thermoduric strain as a cell factory may be more feasible although this is currently not being done. In addition evaporative loss of the product ethanol may occur given the rather long growth and production rates, thus strategies to constantly remove and collect product during production may be essential, which might mitigate against needing ethanol tolerant strains. These issues illustrate the potential interplay between knowledge of the production system and the metabolic engineering needs and strategies.

Thus to ensure maximal production and recovery of ethanol, systems may need to be engineered to trap and recover ethanol during production which again may add considerably to CapEx. Jorquera et al. estimated, in a comparative analysis of power consumption of different photosynthetic reactors that horizontal tubular PBRs consumed 2500 W.m<sup>3</sup> , which reduced to 54 W.m<sup>3</sup> for flat panel PBRs and to 3.7 W.m<sup>3</sup> for raceway systems [74]. However mixing rates are quite different in the different systems such that in raceway systems there is little mixing, which effects movement of producing cells into light and poor mass transfer limiting overall productivity. Thus, power consumption unless linked to wind or solar in an integrated way may be a key hurdle to overall process efficiency and economy.

#### **7.4. Ethanol recovery from production media**

of some €12.6 kg−1 [71], (b) High energy consumption [72] from mixing, CO<sup>2</sup>

a challenge.

212 Fuel Ethanol Production from Sugarcane

and strategies.

sparging, transport costs and CO<sup>2</sup>

ing, separations, cleaning, and (c) Maintenance, cleaning and labor costs [70], (d) The reactor design must be able to withstand photo-oxidation, prevent evaporative loss of product, while maintaining axenic conditions as long as possible. Given the generally slow growth, rates of cyanobacterial species, largely because of the photoautotrophic lifestyle, need to manufacture most of their metabolites, maintain a polyploid genome because of the high sunlight and UV exposure the design of PBRs suitable for low value ethanol production from cyanobacteria is

In production terms once one moves away from a controlled PBR design one halves the production cycle and level of photosynthetic production due to the night-day diurnal cycle and in addition there is less process control over the operation. Many approaches have been taken in an attempt to reduce cost; this has included use of bicarbonate-based systems for supply of carbon following carbon capture [73]. This may have significant cost savings in terms of CO<sup>2</sup>

be once off or be reusable can offer another potential solution. This may mitigate against some of the limitations of more traditional PBRs. Controlling contaminants in non-axenic culture might be carried out by use of pH as a control mechanism for limiting contamination, however this may necessitate use of more alkaliphilic cyanobacterial species [73]. Indeed adapting the production strain to the process or vice versa may offer a way forward in developing optimal reactor configurations with reduced CapEx. Thus incorporating knowledge of the production cycle, the types of conditions required for growth into a metabolic engineering

By virtue of the fact that ethanol-producing cyanobacteria will be recombinant strains, the current experimental systems tend to be enclosed due to regulatory constraints with GMO's. In geographical locations which are suitable for maximal sunlight and hence photosynthesis, enclosing a facility may raise issues with temperature control unless this is designed into the build. Direct exposure to air circulation or venting may also not be feasible due to safety issues while heat buildup beyond optimal growth temperatures, such as 30°C for *Synechocystis*, may easily occur. In such cases, utilizing a thermophilic or thermoduric strain as a cell factory may be more feasible although this is currently not being done. In addition evaporative loss of the product ethanol may occur given the rather long growth and production rates, thus strategies to constantly remove and collect product during production may be essential, which might mitigate against needing ethanol tolerant strains. These issues illustrate the potential interplay between knowledge of the production system and the metabolic engineering needs

Thus to ensure maximal production and recovery of ethanol, systems may need to be engineered to trap and recover ethanol during production which again may add considerably to CapEx. Jorquera et al. estimated, in a comparative analysis of power consumption of different

photosynthetic reactors that horizontal tubular PBRs consumed 2500 W.m<sup>3</sup>

strategy can be important during initial development of strains and strategies.

**7.3. Temperature control, energy and evaporative loss**

loss due to outgassing. Bag type culturing [70] which can

supply, pump-

, which reduced to

Lignocellulose based fermentations tend to be more dilute than starch based systems due to the presence of hemicellulose which increases viscosity and the presence of fermentation based inhibitors [75, 76]. This is currently similar in terms of cyanobacterial production of ethanol, which is also dilute and low in terms of yield. Recovery of ethanol from dilute production streams in an energy efficient and economical manner poses significant technical difficulties. Traditionally ethanol is recovered via distillation, however in the case of biofuel ethanol from cyanobacterial production the energy costs of distillation would be far too high particularly from dilute streams. It has been estimated that in a well-integrated lignocellulose to ethanol plant the process would require 4350 MJ.m−3 equivalent to approximately 20% of the energy content of the ethanol produced [76]. Thus, alternatives to distillation are needed to drive economy from cyanobacterial production systems. A number of techniques are available which may be suitable for the recovery of ethanol from cyanobacterial production such as membrane permeation or pervaporation, vacuum stripping, gas stripping, solvent extraction, adsorption and various hybrid processes [76]. However, the efficiency is dependent on the initial ethanol concentration (which is currently low for metabolically engineered *Synechocystis*) and often multiple cycles of processing would be required to achieve optimal yield. Thus, there are currently significant challenges to optimal recovery from dilute streams such as cyanobacterial systems although the potential for energy savings over distillation are possible. This thus implies that the higher the yield initially the better in terms of downstream processing, hence the current focus on optimizing metabolic engineering for yield.
