Category Archives: Advanced Biofuels and Bioproducts

Challenges and Opportunities

The Electrofuels approach to biofuels represents a significant departure from photo­synthetic strategies, and it is important to consider both the merits and challenges inherent to a system built around a chemolithoautotrophic platform. Here, we con­sider the relative advantages and disadvantages of an Electrofuels vs. a photosyn­thetic approach for each of the important drivers of the system, specifically the required resources and the economics of at-scale production.

3.1 Land Resources

The biochemical conversion of solar photons to liquid fuels involves many steps, each with the potential to produce losses of both energy and carbon. Plants harness energy from only a portion of the solar spectrum, and only during the growing sea­son, which in temperate climates ranges from roughly 180 to 250 days per year. Under many conditions, plant growth is limited by access to resources other than light, in particular water, trace nutrients, and CO2. Even the most prodigious dedi­cated energy crops produce harvestable biomass encompassing roughly 1% of the incident solar radiation; additional losses from agriculture and conversion significantly reduce the usable portion of the captured energy. The primary consequence of this low efficiency is that enormous land resources are required to collect sufficient solar energy for US liquid fuel production (Table 2). Several advanced photosyn­thetic biofuels approaches under development claim higher solar photon utilization efficiencies, some as high as 7.2% [49] , although none of these approaches have been demonstrated at scale.

Chemolithoautotrophic approaches to biofuels require land only for solar radia­tion collection, and can thus use nonarable land for fuel production. Any of several currently deployed technologies, for example concentrating solar or solar PV, can capture >20% of incident solar radiation energy. Coupling one of these technologies with a microorganism, up to 13% of total available solar energy could plausibly be captured in a liquid fuel (Supp Calc 4). This solar radiation can be captured year round, significantly increasing the solar yield from a given land area when com­pared to seasonal crops. Further, it is possible to intersperse farmland with renew­able resources such as wind so that fuel production no longer competes with food production.

И-Butanol

я-Butanol has favorable properties as a gasoline blending agent and provides a valu­able target to validate R. eutropha as a host for synthetic biology [14]. The PHB synthesis pathway in R. eutropha proceeds through 3-hydroxybutyryl-CoA, which is also an intermediate in the я-butanol synthesis pathway. Our strategy will be to divert the flux from 3-hydroxybutyryl-CoA to PHB and to redirect it to я-butanol. Recent work has demonstrated that high titers of я-butanol can be produced in E. coli by choosing heterologous genes judiciously and maximizing reducing equiv­alents available for я-butanol production [2]. Therefore, a detailed understanding of the expression of heterologous genes for я-butanol production in R. eutropha and the metabolic flux in R. eutropha PHB — mutants will be essential in achieving high titers of я-butanol.

Although я-butanol can be used directly as a gasoline replacement due to its higher energy content and lower water solubility and corrosivity relative to ethanol, it would only address short-haul ground transportation, since it could not be used to power aircraft or long-range rail and trucks. Dehydration of butanol to butylenes (C4) and oligomerization affords C8, C12 and C16 olefins with some disproportion­ation to non-oligomer C9 , C10, C11, C13, C14, and C15 olefins. These olefins can undergo double-bond isomerization, skeletal isomerization, cyclization and/or aro — matization, forming isoalkenes, cycloalkenes, and/or aromatic products. Hydrogenation of this mixture may provide hydrocarbons suitable for use as jet fuels. We will transform the я-butanol obtained from H2/CO2 cultivation of engi­neered R. eutropha to these hydrocarbon mixtures and evaluate them as replacement for jet fuel. We are currently exploring novel catalysts for the dehydration and oli­gomerization of butanol to hydrocarbon mixtures that resemble jet fuel.

Tubular Reactors

The straight, looped or coiled transparent glass or plastic tubes of tubular reactors are usually arranged in horizontal or vertical arrays [18, 28]. Liquid flow is induced by pumping of the liquid volume or by airlift circulators [24].

Scale-up by increasing the tube diameter is limited with respect to light penetra­tion depth into the culture. However, light is focused in radial direction to the center of the tube. This effect partially compensates for exponential decrease in light inten­sity due to absorption which is described by Lambert-Beer’s law. Diameters in the range of 3-6 cm are common [18, 24]. The occurrence of dark volumes in the center of the reactor does not necessarily lower productivity. Induction of favorable light/ dark cycles can be achieved by high liquid velocities or installation of static mixers and thus increase volumetric productivity [27] . Molina et al. suggested identical light/dark cycle frequencies on different scales as a suitable scale-up criterion for achieving similar productivities [24] .

Since scale-up potential with regard to tube diameter is restricted, tube lengths are increased and modular units are multiplied, e. g., mounted on vertical scaffolds

(Fig. 10).

Gas exchange is crucial for this type of reactor. Aeration and oxygen removal usually take place in specific gassing and degassing compartments, whereas gassing

Fig. 10 Tubular photobioreactor in Klotze, Germany (meanwhile belonging to the Roquette group)

at several points along a tubular track is a conceivable option. The flow regime within the tubes can be regarded as plug flow regime with minimal backward and forward mixing. Therefore, considerable spatial gradients of oxygen and CO2 along tubular axis occur and gain importance with increasing lengths of tubes. Limited availability of carbon dioxide limits cell growth at some point. One should also keep in mind that pH gradients are concurrent with CO2 gradients on account of the car­bonic acid equilibrium [39]. Oxygen removal is another important aspect since oxy­gen supersaturation inhibits growth or even causes oxygen-induced cell damage [3]. Therefore, dimensions of the degassing section, length of tubes, liquid flow rates, and mass transfer must be reasonably adjusted in order to avoid detrimental oxygen concentrations and CO2 limitation as well.

Tubular reactors can attain high productivities, e. g., 1.4 g/L/day in a helical reac­tor [18] or 1.9 g/L/day in an airlift-driven tubular reactor with flat arrangement of solar collecting tubes [25] . However, power supply for tubular reactors is usually much higher compared to the aforementioned alternative design concepts. Power supply in magnitudes ranging from 800 to 3,200 W/m3 [18, 42] is unfavorable for production of biodiesel and hydrogen. Therefore, tubular reactors should rather be used for cultivation of high value products, e. g., for the pharmaceutical market.

Most prominent commercial application of tubular photobioreactors is probably the biggest indoor tubular reactor, set up by the company Bisantech in Klotze, Germany (Fig. 10).

About 500 km of tubes with a total volume of circa 700 m3 are located in a green­house where tubes are arranged in vertically oriented scaffolds to attain maximum areal productivity.

The tubes are not all interconnected because gradients resulting from such an arrangement would necessarily lead to inhibiting accumulation of oxygen and simultaneously to carbon dioxide limitation. Before being recirculated through the tubular system, the culture suspension intermittently enters degassers. In the facil­ity, temperature is actively regulated to adjust to optimal culture conditions [30]. The facility’s output targets the nourishment market sector with production costs of circa 15€/kg [approximately 20 US$/kg] biomass. Productivities of 100 t/ha are attained under mixotrophic growth conditions (Prof. Steinberg, personal communication).

Design Considerations for Tubular Reactors

1.3.1 Airlift Pump

For the algae to grow optimally, the mass transfer characteristics of the photobiore­actor must be optimised to suit the specific strain of algae. Mass transfer is achieved by pumping compressed air and carbon dioxide into the reactor, thus creating flow through the culture that is dispersed within the solar tubing [22]. It is preferred that the aeration and mixing of the cultures in tubular photobioreactors are carried out by an airlift pump [35]. The airlift pump must provide an adequate velocity to circulate the culture through the solar receiver so that the dissolved oxygen build up within the culture can be stripped by the degassing section before it accumulates. Airlift pumps are typically used in algal cultivation instead of standard mechanical pumps. Airlift pumps have been found to cause less damage to algal cells and are less expensive to instal than mechanical pumps [5] .

Pump

Fig. 1 Different configurations of microalgae cultivation system. (a) Raceway pond (RP) [5]. (b) External loop tubular reactor (ELR) [21]. (c) Horizontal tubular reactor (HTR) [5]

1.3.2 Degassing Column

The degassing section of the reactor plays an integral role in the success of the cul­tivation. The degasser is designed to remove accumulated dissolved oxygen and extricate gas bubbles from the culture. An excessive amount of bubbling within the tubing can hinder light absorption. Therefore, gas-liquid separators are employed [5]. It is required that the culture spends the least amount of time in the dark regions of the reactor. The degassing zone is considered to be optically deep in comparison with the solar receiver. It is a dark zone and hence it is unsuitable for growth. It is a necessity that the degassing section’s volume is much lower than the volume of the solar receiver to allow the culture to spend longer periods in the section of the reac­tor that is optimal for biomass proliferation. The airlift pump controls the liquid
velocity in the solar tubing. The velocity required by the system will depend on the configuration of the tubing and gas holdup in the riser and downcomer regions within the airlift operation. Once the tubing geometrics are selected, the height of the airlift section and the appropriate areas for the downcomer and riser portions of the airlift can be determined.

The Economic Model

The economic analysis undertaken in this report puts the process technology into fourmajorproduction stages: cultivation, dewatering, extraction and transesterification. The integration of these individual production stages was used to determine the total production cost of biodiesel from microalgae. To allow a greater comparability between the results of the analysis, the economic model applied a consistent approach to each production stage. This approach estimated the total production cost using a number of components, which included major equipment costs; individual fixed capital costs and fixed capital investment (FCI); annual costs; and running costs.

1.7 Major Equipment Costs

The first element necessary in estimating the total cost of each alternative unit opera­tion technology was to determine the major equipment costs of each scaled-up pro­duction option. The primary source of costing information regarding standard process equipment such as pumps, tanks and compressors was provided in Peters et al. [26], whilst similar economic studies were employed to estimate the costing of more specialised equipments such as the raceway ponds [4], HTRs [10] and ELRs [21].

In the dewatering stage, the major equipment cost of centrifuge was based on information provided in Peters et al. [26], whilst the costs of the chamber, suction and vacuum filters were each estimated as a proportion of the centrifuge cost, using the ratios reported in Mohn [20]. In the esterification stage, costing for all equip­ment was scaled-up from a study completed by Sakai et al. [29]. All major equip­ment costs were scaled to current prices using appropriate indexing from the Chemical Engineering Plant Cost Index (CEPCI).

Goal and Scope Definition

A life cycle analyst’s motives for carrying out an LCA can have important implications for the results of a study. These “zero order” assumptions are often rooted in the type of LCA being performed. LCAs fall broadly into one of two categories. Attributional LCAs are those in which all of the environmental impacts associated with a product or process are compiled and reported [1]. Consequential LCAs are those that evaluate the impacts of making a particular change to a process or prod­uct, or compare two technologies with related functions. Consequential LCAs are often more straightforward to perform because they permit for the canceling of unit processes or systems that are common between the technologies of interest. In the case of algae, most published LCA studies are attributional since there are few tech­nological systems existing to which algae-to-energy can be compared. There is, however, an important role for consequential LCA as this field moves forward; since they can help identify and quantify what impacts might arise from evolving algae technologies. The decision to undertake an attributional or a consequential LCA is manifest most notably in decisions about the system boundaries and func­tional units of the study. System boundary decisions include all the elements associ­ated with geographic areas, natural environments, time horizons, and others. The functional unit is the quantitative basis for the life cycle comparison and differs depending on the processes to be compared. Both are explored here.

Chlorophyll Fractionation and Purification on Chromatographic Adsorbents

Once recovered from microalgal biomass, the extracted chlorophyll mixture will have to be fractionated in order to separate out the different chlorophyll types and to remove unwanted components (neutral lipids, polar lipids, other pigments) that have been inevitably co-extracted. Chromatographic techniques are traditionally used to fractionate chlorophyll mixtures. The three types of chromatography that have been widely used are paper chromatography, thin layer chromatography (TLC), and high pressure liquid chromatography (HPLC) [1, 23, 31, 43].

Paper chromatography was used extensively during the early development of chromatographic techniques (1950s and 1960s). The method was able not only to separate chlorophyll into its fractions (a, b, and c) but also to effectively fractionate other pigments, such as pheophytins and carotenes [22]. However, the inception of TLC has resulted in the decline of paper chromatography usage. This later tech­nique was preferred due to the ease in recovering pigment fractions from its adsor­bent [31,41]. Additionally, TLC requires less sample, is less laborious, and produces chromatograms with sharper resolutions [27, 41]. Organic adsorbents, such as sucrose and cellulose, were found to be the most effective stationary phases for use in two dimensional TLC. Even though the use of silica gel as a stationary phase was effective in separating all plant pigments (except for some minor components), it was found to promote the formation of chlorophyll degradation products.

HPLC is superior to TLC because it requires even less sample for analysis, is faster and can be easily coupled with an automatic detection system [48, 57]. In addition to these, HPLC is more precise and has a higher degree of sensitivity. Reverse phase HPLC is preferred to normal phase as the latter does not separate polar compounds effectively. An additional drawback to normal phase HPLC is its lack of compatibility with aqueous samples. Several HPLC configurations have been employed, each being able to separate pigments to variable extent and differ­ent resolution [24]. There are different types of detectors that may be used to mea­sure the concentrations of separated pigments as they exit the chromatographic column. The most commonly used detectors rely on fluorescence and absorbance analyses. Jeffrey et al. [24] found fluorescence detection to be more sensitive and more selective than absorbance detection especially when used to analyze chloro­phylls amongst carotenoids. Table 4 summarizes previous studies on chromato­graphic fractionation of phytoplankton pigments. It is noted that the use of chromatographic techniques to purify recovered chlorophylls, albeit very effective on a laboratory scale, is not commercially applicable due to the high installation and operating costs associated with the techniques. Investigating a cost-viable, energy — efficient purification technology that can be retrofitted to industrial-scale chloro­phyll production is a current research endeavour.

Bioactive Volatiles

In another chapter of the present book, volatile compounds from algae and microalgae are studied as an energy production source. Biogeneous hydrocarbons of the marine system, alkenes (mono, di, and cyclic) were originated from algae. One characteris­tic of crude oils that distinguishes them from biogeneous hydrocarbons is their con­tent in cyclo alkenes and aromatic compounds [32]. But hydrocarbons are not the only volatile compounds that can be found in algae and microalgae. In fact, there is a huge number of secondary metabolites with proved antimicrobial and therapeutic activities while some of these volatile compounds have been also related to climate modifications.

When attacked by herbivores, land plants can produce a variety of volatile com­pounds that attract carnivorous mutualists. Plants and carnivores can benefit from this symbiotic relationship, because the induced defensive interaction increases for­aging success of the carnivores, while reducing the grazing pressure exerted by the herbivores on the plants. Steinke et al. [185] reviewed whether aquatic plant use volatile chemical cues in analogous tritrophic interactions.

In general, naturally produced volatile and semivolatile compounds play an essential role in the survival of organisms for chemical defense and food gathering, but high amounts of volatile compounds could produce tremendous environmental actions. Marine algae produce several classes of biogenic gases, such as nonmethane hydrocarbons, organohalogens, ammonia and methylamines, and dimethylsulfide. These gases can transfer to the air, affect atmospheric chemistry, and are climati­cally important. Grazing increases dimethylsulfide and ammonia concentrations, and it is possible that other environmentally relevant volatiles are also produced during this process.

Other compounds produced by seaweeds with high importance for environment are halogenated hydrocarbons. Stratospheric ozone depletion and volatile-haloge — nated compounds are strongly connected with each other since the discovery that a massive loss of ozone in the polar stratosphere is catalyzed by halogen radicals derived from chlorocarbons and chlorofluorocarbons. Furthermore, so far unknown natural sources of volatile organohalogens may also contribute to a further destruc­tion of the ozone layer. Marine macroalgae species from the polar regions were investigated [90I for their importance as natural sources of volatile halogenated compounds released into the biosphere. Several different halogenated Ci to C hydrocarbons were identified and their release rates determined. Although, at present, marine macroalgae are apparently not the major source on a global scale, they may become more important in the future due to the influence of changing abiotic fac­tors, such as photon fluence rate, nutrient concentration, temperature, and salinity on the formation of volatile organohalogens.

The release of volatile compounds with defensive functions has been studied in many algae, for example the brown alga Dictyota menstrualis [21]. Although the amphipod Ashinaga longimana preferentially consumes the alga D. menstrualis, its feeding rates can be reduced significantly by high concentrations of diterpe­noid dictyols (dictyol E, pachydictyol A, and dictyodial) produced by the alga. The pattern of variation in the chemical defenses of some seaweed species sug­gests herbivore-induced increases of chemical defenses may be responsible for intraspecific variation in chemical defenses. For example, seaweeds from areas of coral reefs where herbivory is intense often produce more potent and higher con­centrations of chemical defenses than plants from habitats where herbivory is less intense. Their findings suggested that seaweeds are not passive participants in seaweed-herbivore interactions, but can actively alter their susceptibility to herbi­vores in ecological time. Induced responses to herbivory help explain both spatial (i. e., within-thallus, within-site, and among-site) and temporal variation in the chemical defenses of the algae.

As seen above, macroalgae produce volatiles with defensive functions against herbivores, but microalgae also produce defensive volatile compounds. In this sense, it is common in many microalgae to share the ecological niche with bacteria and other microorganism. Therefore, the defensive compounds secreted by microalgae possess antibacterial, antifungal or antiprotozoal activity. The nature of these com­pounds is highly varied. Microalgae have been screened for potential antimicrobial activity, which have been attributed to different compounds belonging to a range of chemical classes, including indoles, terpenes, acetogenins, phenols, fatty acids, and volatile-halogenated hydrocarbons [ 105] . For example, pressurized ethanol and supercritical CO2 extracts of microalgae D. salina were studied for their antibacte­rial activity against Escherichia coli and S. aureus and for their antifungal activity against Candida albicans and Aspergillus niger [56, 111]. Inthe broth microdilu­tion assay, a high antimicrobial activity against C. albicans, E. coli, and S. aureus was observed but not against A. niger. In this work, a GC-MS analysis was per­formed to associate the antimicrobial activity found, it was concluded that antimi­crobial activity of D. salina extracts could be linked to the presence of terpenic (b-cyclocitral and a and b-ionone) and indolic (methyl-1H-indole derivative) com­pounds, Fig. 2.

Terpenoids from algae have also been associated with antiviral activity, for example the above mentioned D. menstrualis produces a terpenoid able to inhibit HIV-1 reverse transcriptase as demonstrated by Souza et al. [181]; or terpenoid derived from plastoquinone that produces Sargassum sp., which acts in the lipid oxidation chain and inhibit cytomegalovirus growing [64].

Short chain fatty acids from microalgae are also volatile compounds associated with antibacterial activity. Santoyo et al. [165] tested, using the broth microdilution assay, extracts obtained from the red hematocysts without flagella (red phase) of

Fig. 2 GC-MS chromatogram of the volatile fraction of Dunaliella salina extract [111]. (1) 3,3-Dimethyl-2,7-octanedione; (2) b-ionone; (3) 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-2(4H)-ben — zofuranone; (4) 4-oxo-b-ionone; (5) neophytadiene; (6) nerolidol; (7) 9-hexadecanoic ethyl ester; (8) hexadecanoic acid; (9) phytol; (10) 9,12,15-octadecatrienoic acid methyl ester; (11) 1H-indole derivative; (12) hexadecanoic acid monoglyceride; (13) neophytadiene derivative; (14) vitamin E. Reprinted with permission from the Journal of Food Protection. Copyright held by the International Association for Food Protection, Des Moines, IA, USA

H. pluvialis microalga. In this work, it was concluded that the presence of short chain fatty acid (butanoic, hexanoic) highly inhibited the growing of gram positive and negative bacteria.

Mechanical Pretreatment

Hydrolysis of particulate organic matter is frequently the rate-limiting step during AD [227, 228]. Mechanical pretreatment of lignocellulose materials increased the hydrolysis and methane yield by 5-25% [229] and reduced the digestion time by 23-59% [230] compared to nontreated samples. Disintegration of WAS in an agita­tor ball mill Model LME 50 K with fine sand balls (0.5-0.9 mm of diameter and

2.7 kg/L of density) increased chemical oxygen demand (COD) solubilization by 25-31% and enhanced biogas yield by 38% compared to nontreated WAS [231]. Mechanical pretreatment of WAS in a high-pressure homogenizer (pressure up to 600 bar) increased biogas production by 18% [195].

Ultrasonic treatment has been widely studied as a method for enhancement of WAS solubilization and methane yield. The minimum energy level required to dis­rupt the activated sludge cell wall is in the range of 1,000-3,000 kJ/kg TS [197, 203] or 20-30 kJ/L [197, 232]. Ultrasonic pretreatment (£=40,000 kJ/kg SS) of WAS resulted in a fivefold increase in the amount of organic matter solubilization but had no influence on biogas yield [199]. Another study showed the same beneficial trend as solubilization increased by 127% at 42 kHz for 120 min [201]. Biogas and meth­ane yields were observed to increase by 20% [201] and by 50% (for biogas) at £s = 6,950 kJ/kg TS [197].

Mechanical pretreatment is required prior to AD of macroalgae and includes chopping (>5 mm), milling (1-5 mm), or homogenization (<1 mm). Grinding of the

Ulva did not influence the total methane yield but it increased the kinetics of hydro­lysis and methane production rate [129] . The grinding of the feedstock is likely more important for continuous stirred-tank reactor (CSTR) or semi-continuous reactors where the HRT is an important operating parameter.

One study examined the influence of mechanical disintegration plus ultrasonic treatments on A. maxima organic matter solubilization, VS reduction, and methane yield [233]. Ultrasonic treatment (Polytron generator PT20ST, from Brinkmann Instruments) increased the soluble COD (sCOD) 3.8-fold compared to freshly har­vested cyanobacteria. A. maxima COD solubilization increased from 21.3 to 76.7%. Surprisingly, VS reduction and methane yield were 0.9 and 0.85 times the values from fresh (not pretreated) biomass, respectively. Only hydrolytic bacteria and aci — dogens benefited from the larger amount of readily degradable substrate. The VFA concentration increased from 12 g/L in the digester with fresh algae to 46 g/L in the digester with pretreated algae. The lack of improvement in the methane yield might be due to the inhibition of methanogenic organisms from the high VFA concentration. Ultrasound pretreatment (19 kHz, treatment energy 1-5 Wh/L) had no influence on the methane yield from homogenized red macroalga Polysiphonia [123]. While the amount of biogas increased by 25-28%, the methane fraction dropped from 42-49 to 33-37%.

Algal Production Integrated with Carbon Dioxide Sequestration

An attractive algal cultivation strategy is to use carbon dioxide emitted from power plants for autotrophic biomass assimilation. Such a carbon capture system with microalgae has the following advantages compared to other carbon sequestration technologies [484]:

— Algal system does not require high purity carbon dioxide.

— Algal system produces biofuels that can be used for electric power generation.

— Some flue gas impurities (nitrogen and sulfur oxides) can be removed as well and be used by algae.

A conceptual diagram for algal production and ADP integrated into a scheme with carbon dioxide mitigation and waste treatment processes is shown in Fig. 21.