Other interesting option for producing biodiesel from feedstocks with high concen­tration of impurities such as water and FFA is the transesterification of triglycerides with supercritical methanol, which is receiving a lot of attention [17, 58, 59]. This process is catalyst-free and it is able to obtain full conversion of the triglycerides in a matter of minutes [58], with the possibility of continuous operation mode [26].

The operation is also simpler, as the transesterification of triglycerides and methyl esterification of fatty acids occurs simultaneously without using any cata­lyst. Because no catalyst is used and has to be recovered, the downstream process­ing is much simpler, and soap-free glycerol can be obtained [89] . Other advantage this process presents is the insensitivity to the presence of impurities in the vegeta­ble oil, such as water and FFA [59]. The presence of moisture is not only negligible, but it can also be advantageous in this process [6] .

The reaction is carried out in supercritical methanol (or ethanol), in which feedstocks react with the alcohol under conditions of high pressure (above 100 atm) and high temperature (more than 276°C). At these conditions, the alcohol is in a supercritical gaseous state and the triglycerides are somewhat dissolved in a single phase. The reasons for this behaviour are not yet fully understood, but are certainly related to the high solubility of triglycerides in supercritical alcohol and solvent effects [65]. Also, some authors have observed a dependence on the type of alcohol and triglyceride used [4].

Notwithstanding its clear advantages over other processes, significant hurdles remain for the full scale implementation of supercritical production units. First of all, high temperatures and pressures are necessary to ensure that the alcohol is in super­critical state, requiring the utilization of special equipment designed to support these conditions. This will lead to high equipment and operational costs, making the pro­cess economics not so attractive when compared to other options. Also, the excess of methanol used in the reaction is much larger when compared to the conventional pro­cess; normally an alcohol/oil molar ratio of 42:1 is used, which needs to be recovered and recycled back to the reactor this way complicating the process design [58].

Alternatively, Cao et al. [23] proposed the supercritical methanol process, using propane as co-solvent, which decreases the reaction temperature and pressure, as well as the alcohol to oil molar ratio. This is because propane decreases the critical point of methanol allowing the supercritical reaction to be carried out under milder conditions than those of 424 atm and 350°C reported by Kusdiana and Saka [58]. In this case, the optimal reaction conditions are a temperature of 280°C, a pressure of 126 atm, an alcohol to oil molar ratio of 24:1, and propane to oil molar ratio of 0.05:1. At these conditions, 98% of oils are converted to biodiesel for a reaction time of 10 min. Kasteren and Nisworo [53] performed an economic analysis of this pro­cess, considering the industrial production of biodiesel from waste frying oil and concluded that it can compete with the existing alkali and acid-catalyzed processes.

Centrifugation is the preferred method for harvesting algal cells [2, 20, 21]. However, centrifugation can be extremely energy intensive, especially when considering large volumes. Centrifugation involves the application of centripetal acceleration to sepa­rate the algal culture into regions of greater and lesser densities. Once separated, the algae can be removed from the culture by simply decanting the supernatant spent medium. Filters can also be used during centrifugation to enhance the separation of the supernatant from the medium. Mohn [20] compared the appropriateness of dif­ferent makes and brands of centrifuges for dewatering of microalgae. Key parame­ters included in the study consisted of the concentration factor produced, energy consumption, relative cost, operation mode and reliability.

In ascertaining whether the microalgal biodiesel production process is carbon neu­tral or carbon negative (absorbs carbon dioxide) or carbon positive (releases carbon dioxide) a life cycle assessment (LCA) is carried out. The LCA is based on ISO 14,040 standards [11].

1.7.3 Life Cycle Assessment: Goal and Scope

The LCA is based on a solid understanding of the GHG neutrality of the process. The LCA will be conducted on the entire process, from the cultivation stage to the final processing stage (gate-to-gate assessment).

1.7.4 Life Cycle Assessment: System Boundary

The LCA system boundary is based on the physical boundary of the entire plant. Basically, a cordon is placed around the entire plant and an audit is performed on the inflow and outflow of GHGs and any energy input and output. However, as the process is developed stage-wise, the boundary is enclosed around the individual stages to simplify the audit. By including all possible factors which may affect the carbon neutrality of the process, the goal is to ensure the carbon audit is a true rep­resentation of the actual emissions from the process. Conducting such an extensive and in-depth audit allows an accurate analysis of whether such a process is feasible in reducing the GHG emissions.

Several kinds of processes for microorganism cultivation have been developed for different applications. These processes may be conveniently classified according to the chosen operation mode: batch, fed-batch, continuous, semi-continuous (repeated batch), and their variations. In a batch operation, neither substrate is added after the initial charge nor the product is removed until the end of the process. Some phar­maceutical products are produced by this process, but generally, batch operation is not commercially attractive [50], where its main application is in food and beverage production. Conventional batch process can lead to inhibitory concentrations of substrate in reactors or even to formation of undesired products through direct metabolic pathways of the organism, decreasing the yield and/or productivity of the system.

The fed-batch process has been used in the cultivation of baker’s yeast since 1920 [47]. However, Yoshida et al. [117] were the first to use the term “fed-batch process” in cataloging. Whereas the whole substrate is added at the beginning of cultivation in a batch process, in the fed-batch process one or more nutrients are added to the fermentor during cell growth, while cells and products remain in the fermentor until the end of operation [14].

The fed-batch process does not require any special piece of equipment in addi­tion to equipment required for batch fermentation. It is only characterized by a little longer overall time cycle that is certainly acceptable by industrial practice where at present very effective procedures for sterilization and preventing contamination are commonly utilized [59] .

The better operating procedure for this system is to start with small amounts of biomass and substrate, and to add more substrate when the greatest part of the initial substrate is already consumed by the microorganism [42] . The inlet substrate feed should be as concentrated as possible to minimize dilution and to avoid process limitation caused by the reactor size. According to Lee et al. [56], the feed control strategies are: simple indirect feed-back (single-loop) methods; nutrient feeding according to inferred substrate concentration or specific growth rate; and predeter­mined feeding strategies.

Two cases of fed-batch process can be considered: the production of a growth — associated product and the production of a non-growth-associated product. In the first case, it is desirable to extend the growth phase as much as possible, minimizing the changes in the fermentor as well as the specific growth rate, production of the product of interest, and avoiding the formation of by-products. For non-growth- associated products, the fed-batch process would be carried out in two phases: a growth phase in which cells grow up to the required concentration and then a pro­duction phase in which carbon source and other requirements for production are fed to the fermentor [59] .

In the fed-batch cultivation, volume variation may happen depending on the sub­strate concentration added and the rate of evaporation in the system [14]. In the fixed volume fed-batch process, the limiting substrate is fed without diluting the culture. The culture volume can also be maintained practically constant by feeding the growth limiting substrate using a very concentrated solution. Alternatively, the substrate can be added by dialysis or, in a photosynthetic culture, radiation can be the growth limiting factor without affecting the culture volume [32] . Contrarily, the variable volume fed-batch process is one in which the volume changes throughout the cultivation time due to the diluted substrate feeding in the system.

Fed-batch culture has been widely employed for the production of various bio­products including primary and secondary metabolites, proteins, and other biopoly­mers. It is used aiming to overcome different difficulties in cell cultivation. When a nutrient with high viscosity is used, this process usually reduces the viscosity of the medium, and toxic effects of components can also be limited by dilution [113].

The advantages of this process include: deviation of cell metabolism via the formation of the desired product; prevention of catabolite repression; prevention of formation of toxic substances in microbial metabolism; and control of the specific growth rate [116]; the main advantages are the possibilities of controlling both reaction rate and metabolic reactions by substrate feeding rate [36].

The fed-batch process has been also used to prevent or reduce substrate-associ­ated growth inhibition by controlling nutrient supply. Since both overfeeding and underfeeding of nutrient is detrimental to cell growth and/or product formation, the development of a suitable feeding strategy is critical in fed-batch cultivation. Fed — batch fermentations can be the best option for some systems in which the nutrients or any other substrates are only sparingly soluble or are too toxic to add the whole requirement for a batch process at the start. This process is particularly important in A. platensis cultivation. In the next items, the factors that affect A. platensis growth are discussed, aiming to correlate them with the employment of the fed-batch process.

Ultrasound-assisted extraction (UAE) is also widely considered as an advanced extraction technique. This technique uses high-frequency sounds, usually higher than 16 kHz and a limited amount of solvent in order to produce an effective extrac­tion of the compounds of interest in the solvent employed, increasing their mass transfer and solubility, by disrupting the food matrix being extracted. As in PLE, the selection of the suitable solvent for extraction by UAE will be made depending on the compounds of interest. For instance, a mixture of dichloromethane/methanol (2:1) was employed to extract lipids from microalgae using UAE [140]. For more polar compounds, such as chlorophylls, methanol was demonstrated as a more effective solvent [175]. This technique has the advantage of providing faster extrac­tion processes compared to conventional techniques. UAE was compared to other solvent-based extraction of pigments and fatty acids from several algae samples. It was demonstrated that UAE was simple, allowed extraction of interesting com­pounds and did not produce alteration or breakdown products [197]. However, when this technique was directly compared to SFE for the extraction of carotenoids from

D. salina, it was shown that SFE was more effective for the extraction of these low polarity compounds, above all in terms of selectivity [98]. At certain conditions, in which a complex sample is being extracted containing the interesting compounds as well as other polar compounds, SFE was demonstrated to be more selective than UAE [98]. UAE has been also employed to extract polysaccharides derived from

Chlorella pyrenoidosa [171].

When sonicating the samples for a given period of time, an increase in the tem­perature of the sample can be observed as a result of the vibration of the molecules. For this reason, considering that most of bioactives are thermally labile compounds, it is common to proceed in a temperature controlled environment. For instance, pig­ments and fatty acids were obtained from algae at -4°C using 35 kHz and 80 W for 90 min [197]. The use of temperatures below 4-5°C allows a better preservation of the extracted compounds, that otherwise, could be degraded.

The last advanced extraction technique also used for bioactives extraction from algae is MAE. In MAE, the sample is heated by using microwaves, at typical pow­ers of 700 W for a short time. Compared to traditional extraction techniques, the use of microwaves allows the decreasing of extraction times significantly limiting also the amount of solvent needed. Again, the temperature will be an important param­eter to be controlled. Once selected the extraction solvent for the extraction of bio­actives from algae, the microwaves power as well as the extraction time has to be defined. Experimental designs can be useful in determining the best extraction con­ditions. For instance, response surface methodology was employed to optimize the MAE of astaxanthin from H. pluvialis [203]. By using this statistical approach, the microwave power (141 W), extraction time (83 s), solvent volume (9.8 mL), and number of extracting cycles (4 cycles) were optimized. At present, MAE has not been extensively applied to extraction of bioactives from algae, although given its success in the extraction of plant materials, it can be easily inferred the great pos­sibilities for its application to algae samples.

The oxidation-reduction potential (ORP) generally is a measurement of a sub­stance’s affinity to either gain or lose electrons. In AD it reflects the availability of oxidants, such as oxygen or nitrate ions or of reductants such as hydrogen. A high ORP (>50 mV) indicates the presence of free oxygen in the anaerobic environment. An ORP between 50 to -50 mV is characteristic of an anoxic environment with nitrates and nitrites, the most favorable electron acceptors. At ORP lower than -50 mV, the environment in the digester is strongly reducing. If sulfate ions are present and the ORP is in the range from -50 to -100 mV, sulfate reducing micro­organisms can outperform methanogens for hydrogen and acetate since sulfate is a more thermodynamically favorable electron acceptor. The most favorable ORP for fermentation and acid production is from -100 to -300 mV, which indicates that the strongest oxidant available is found in different organic compounds that can be reduced to a mix of acids and alcohols. Methanogenesis requires the ORP <-300 mV when carbon dioxide is used as an electron acceptor and methane is formed [76].

The main goals for improving the ADP are increasing the conversion efficiency while simultaneously decreasing capital and operational costs.

5.1.2 Inoculum Source for Anaerobic Digestion of Algae

As discussed earlier, algal biomass has specific biochemical composition and con­tains unique compounds, such as algin, laminarin, and fucoidan. Moreover, marine algal biomass has a high salt concentration that can affect anaerobic microorgan­isms. Isolation and application of microorganisms adapted for digestion of specific algae is labor-intensive but has the potential to improve algal ADP.

Generally, anaerobic sludge from a domestic sewage plant or marine anaerobic sediment is used for startup of the algal ADP. Several authors reported that anaerobic organisms adapt readily to algal biomass as a sole substrate, and the inoculum source has a minor or no effect on the final methane yield and VS reduction [77,159, 368]. On the other hand, addition of an inoculum from marine sediments to anaerobic sewage sludge increased the initial methane production rate (Fig. 13) [77],

and addition of a rumen and sewage sludge inoculum adapted to algal substrate increased biogas production and methane concentration [369]. Application of an inoc­ulum adapted to high ammonia concentration is a possible solution to overcome the problem of ammonia inhibition. The inoculum from a piggery anaerobic pond yielded stable methane production from algae with an added ammonia-N concentration up to 3 g/L [370]. In contrast, a sewage mesophilic digester inoculum showed inhibition in methane production at an added ammonia-N concentration larger than 0.5 g/L.

Table 1 lists several estimates of natural gas, in hydrate form, in the geosphere’s GH stability zone (i. e., the P and T regime within which hydrates are stable). The maxi­mum value (3.053 x 10,8 m3 STP of CH.) of Trofimuk et al. [195] is based on the assumption of GH occurrence wherever a satisfactory P-T regime exists, while the minimum value (2 x 1014 m3 STP) of Soloviev [180] accounts for limiting factors such as CH4 availability, limited organic matter, porosity, regional thermal history, etc.

The Klauda and Sandler [80] estimate in Table 1 has received significant atten­tion, as it is based on a state-of-the-art model that explains most known GH occur­rences and offers plausible reasons for discrepancies from its predictions.

Even the most conservative estimates suggest enormous amounts of gas in hydrated form, the magnitude of which can be appreciated by comparing them to the current rate of 1012 m3 STP of gas-equivalent annual energy consumption in the United States. All estimates are comparatively large relative to estimates of the conventional gas reserves of 1.5 x 1014 m3 of methane [155]. Kvenvolden [97] indi­cated that his estimate of 1.8 x 1016 m3 of CH4 in hydrates may surpass the recover­able conventional CH4 by two orders of magnitude, or be a factor of 2 larger than the CH4 equivalent of the total of all fossil fuel deposits.

There is an increasing need to identify suitable techniques for monitoring hydrate accumulations during production. The feasibility of remotely monitoring GH accu­mulations during production is only beginning to be examined. The type of geo­physical measurement that has been used most successfully for exploration is surface seismic. 2D and 3D surface seismic surveys have been used extensively for mapping the distribution of GH accumulations, delineating their large-scale fea­tures and providing rough estimates of the average SH. Advanced processing tech­niques can provide depth-varying SH estimates over large areas (e. g., [30,96,149]), though their applicability depends on the depth and thickness of the hydrate.

In exploration surveys, little prior information is available, and the primary goal is to determine whether hydrate is present and, if so, how much. In contrast, inves­tigations using geophysical techniques for monitoring production focus on much smaller regions in the vicinity of production wells and require higher resolution and measurement repeatability in order to image small variations in properties.

To convert 2-KIV to isobutyraldehyde, a 2-Kivd enzyme is used. The Kivd enzyme from L. lactis is an uncommon enzyme with a traditional industrial role of aldehyde production for aroma development in cheese [75]. Given its activity with branched chain ketoacid substrates, this enzyme is uniquely suited to the task of bridging the BCAA production pathway to Adh to allow for heterologous IBT production [33]. The enzyme is a non-oxidative, thiamine diphosphate dependent, Mg2+-dependent keto acid decarboxylase [75]. The kivd gene can be expressed in an active form in R. eutropha (Sinskey laboratory, data not shown).