Continuous-batch hybrid process for production of oil and other useful products from photosynthetic microbes

A process for bettering photosynthetic microbes containing Closed Systems for continuous cultivation and Open Systems for batch cultivation, in which (a) the Closed System Area occupies no more than 20 percent of the entire Land Area of the farming centre; (b) batch cultures in the Open Systems are initiated with an inoculum in the Closed Systems comprising a mobile biomass of no less than 5 percent of the carrying capacity of said Open System; (c) the diminishing rate of said photosynthetic microbe is no less than once every 16 hours; and (d) the residence time of the batch culture in said Open System is no longer than a span of 5 times.


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Countless species of photosynthetic microbes have been routinely cultivated at comparatively small scale in the laboratory, in culture vessels ranging from several milliliters up to a few hundred liters in power. But, attempts to cultivate atlarger scales, normally necessary for industrial production, have demonstrated effective for fewer than 10 species– even despite a global campaign that has lasted half a century and consumed billions of dollars.

There are two standard types of culture vessels that were employed in the effort to cultivate photosynthetic microbes in commercial scale: (1) Photobioreactors (Closed Systems), and (two ) Open Systems.

(1) Closed Systems are characterized primarily by the provision of means to control access into the atmosphere. Gas exchange with the atmosphere is permitted to occur under controllable conditions. Carbon dioxide enters the culture vessel as afuel for growth, and oxygen, the gaseous waste product of photosynthesis, is permitted to escape the culture vessel. (The carbon is assimilated into plant biomass, and also the”dioxide”–oxygenis expelled.) However, gas exchange happens through filtrationmechanisms which are made to prohibit entrance into the culture vessel of any species of photosynthetic microbe aside from the one which has been preferentially cultivated there.

Closed Systems are usually also designed to permit for the control of other ecological problems. The supply for control of environmental factors like temperature, pH, nutrient concentrations, and mild makes it possible to optimizegrowth conditions for various species of microbial plants, which, like terrestrial plants, possess distinct preferences for unique combinations of such factors.

For a given set of environmental requirements, all species of photosynthetic microbes develop in their highest speed within a narrow selection of cell concentrations. Thus, some Closed Systems are designed to operate as”turbidostats,” wherein theoptical land of turbidity (opaqueness), which can be a function of cell concentration, is monitored by way of programmable sensors which measure the optical density of the medium. The operator can specify a desirable range of acceptable cellconcentration, between a very low value and a high value. The very low value corresponds directly to a specific reduced optical density (the”low set stage”), and the high value to a specific high optical density (the”high set stage”). The optical density detector isthen programmed accordingly. When optical density reaches a value that exceeds the designated upper set point, the turbidostat activates a control mechanism that provides for removing (harvesting) that a fraction of the civilization and replacing it withcell-free nutrient medium, thus diluting the cell concentration to give a value of nitric oxide which matches the designated lower set point. The cells then increases, increasing in concentration until optical density reaches a value which onceagain surpasses the upper set point, at which time the cycle repeats.

Preferably a Closed System culture vessel is constructed chiefly of transparent substance, such as glass or plastic, that permits the transmission of photosynthetically active radiation (visible light), but that otherwise separates the culturemedium from the atmosphere. Culture vessels can take many different shapes, but they all share in common one spatial dimension that restricts their functionality, and that’s their depth relative to incident light intensity. This feature arises from a basicproperty of photosynthesis, namely, that photosynthetic rate is limited by light intensity. Consequently, in any given light intensity, the rate of photosynthesis of a mobile culture is improved as a function of the Lighted Area, i.e. the area of the culturemedium that’s subjected to light (not the Surface Area, which may include areas of the culture vessel that are not exposed to light). Consider two culture vessels, both outside and exposed to sun. One is in the form of a rectangularpond with strong sides and bottom, and the next is in the form of a translucent cylinder, placed horizontally in addition to the ground. For the rectangular pond the Surface Area involves the top, bottom, and sides of the pond, but just the very best area of theculture moderate is subjected to sun. For this kind of outside pond, then, the Lighted Area is equal to less than half of the Surface Area. By contrast, for the translucent cylinder, the Surface Area is the entire surface of the cylinder and, regardless of whatthe time of day, half of the Surface Area will always be immediately illuminated by sun. Therefore, for the cylinder, the Lighted Area is equal to half the Surface Area

Another factor affecting the relationship between photosynthesis and light will be that the cell concentration in the culture medium. The larger the cell concentration in a moderate, the less the depth to which light may penetrate, because lightpenetration decreases roughly exponentially as a function of cell concentration. In other words, if the cell concentration increases at a constant pace, light fades quicker and faster. At some depth at a cell culture, subsequently, light willactually reduction to zero.

As a practical matter, the best culture thickness for photosynthetic microbes exposed to full sun is usually in the range of 10 to 20 centimeters. No advantage can be gained by providing greater thickness of the culture, since theconcentration of cells each component Lighted Area will remain exactly the same, and deeper cells will not receive enough light. The optimal thickness, then, places a limitation on the standard operating capacity of any culture system, regardless of its Lighted Area. Thisphenomenon is a critically important feature in the design of cultivation systems. Greater amounts require more stuff, at greater cost, but at some point that the growth in quantity provides no growth in productivity per unit Lighted Area.

Cultures of photosynthetic microbes normally require mixing or stirring so as to maintain a homogeneous distribution of cells from the medium. The natural trend of photosynthetic microbes in water that is still would be to produce dense aggregations,in which the properties of this medium are changed to the detriment of the culture. On a microscale, within the aggregation, the availability of the concentration of nutrients and gases get so different from the remainder of the mediumthat growth is constrained. Some species possess appendages called cilia or flagellae which allow them to swim; such motile (“moving”) species knowingly form aggregations. Most non-motile species are heavier than water and will sink, forming a passiveaggregation on the floor. To stop such aggregations, Closed Systems must offer a way for producing turbulence using devices such as airlifts or pumps.

(2) Open Systems differ from Closed Systems in one crucial characteristic, namely that they are open to the atmosphere. This feature is advantageous to both structure and performance, in several ways. To begin with, because the Lighted Area of an OpenSystem is exposed directly to sunlight, there’s no need to use a transparent substance to assemble the culture vessel; this affords broad latitude in the choice of materials. Second, because no substance is used to cover the Lighted Area of theOpen System, the quantity and cost of substance is reduced by about half. Third, Open Systems are normally easier to wash than Closed Systems. Over time the inner surface of any civilization vessel will have a tendency to collect a film of microbial growth. In aClosed System the accumulation of this a movie on the Lighted Area will absorb light; the resultant reduction in light intensity induces a decline in productivity. In both Open Systems and Closed Systems the culture vessel surface can accumulatemicrobial films of undesirable species that could possibly be damaging to growth and production of the desirable species. In any case, the culture vessel surface will require cleaning from time to time. As a practical matter, Open Systems allow a far widerchoice of cleansing methodologies. For example, individuals and large types of mechanical cleaning equipment such as hoses, pressure washers, and scrubbers that cannot enter the confined space of a Closed System can easily input an Open System.

The main disadvantage of the Open System is that, by being open to the air, it is vulnerable to contamination by unwanted species. An individual may start the functioning of an Open System culture with just one desired species of photosyntheticmicrobe. But, undesired species will necessarily be released, whether atmospheric transport or other ways. Any undesired species that develops faster than the desired species in the same environmental conditions will, over time, outcompete thedesired species also will ultimately dominate the culture.

In conclusion, Closed Systems were created specifically to prohibit contamination by undesired species, with the expectation that constant cultivation of a desired species might be potential for a much longer period than would be possible in an OpenSystem. But, Closed Systems are somewhat more complex to construct and operate. Open Systems manage a broader selection of materials for construction, and afford a broader selection of cleansing methodologies. Closed Systems require further operatingpractices, such as the use of sterile technique during fluid transfers, which call for increased time and expertise on the part of the operator.

Theoretical differences involving Closed Systems and Open Systems are borne out in practice. The initial photosynthetic microbe was isolated from nature and grown in pure culture bit over a hundred years back, but it was not till thelate 1930s that sufficiently large volumes of a single species could be cultivated to permit chemical analysis. By the 1940s many species were being grown in lab cultures of about 25 liters, and it was discovered that, by alteringenvironmental states of the culture, possibly the oil or protein content of some species may be made to exceed 60 percent of the total cell mass.

The first efforts at large-scale cultivation started in the 1950s, stimulated by widespread fascination with photosynthetic microbes as a source of cheap protein for meals and animal feeds. The first Open Systems, constructed in Germany, took the contour ofshallow, elongated, recirculating raceways, with flow provided by a paddlewheel apparatus. Nationally-funded programs developed rapidly throughout the world, all following the German”open pond” design. The initial open ponds had capabilities of just a fewthousand liters. From the late 1950s, abilities of nearly 100,000 liters had been reached and, by the late 1960s, nearly 1,000,000 liters. Such increases in capacity attracted economies of scale.

Hundreds of species have been tested in the lab, and efforts were made to grow the ideal protein manufacturers in open ponds throughout the 1960s and 1970s. Only a few species proved to be amenable to sustained cultivation. These few species, suchas Spirulina platensis and Dunaliella salina, went on to become the cornerstone of commercial production, effected in open pond systems covering hundreds of acres. The successful commercial species proved to be”extremophiles,” which flourish in conditions ofunusually high pH or salinity. Most species prefer conditions that prevail in nature, where many species flourish concurrently. For two decades, all attempts to nurture single-species cultures of non-extremophiles in ponds that are open collapsed following lessthan a couple of months because they were infected by other species which thrived under the same environmental conditions.

Renewed interest in large-scale cultivation was stimulated from the 1980s and 1990s from the possibility of producing renewable biofuels using oils from photosynthetic microbes as a feedstock. During this interval government agencies of the USA andJapan, by way of instance, invested approximately $150 million in this kind of attempt. Such programs shared two aims: first, to gather and identify species of photosynthetic microbes that create high concentrations of oil and then to determine the environmentalconditions under which they perform so; and, second, to design and establish the operation of large-scale cultivation techniques for the production of biofuel feedstocks using species that had been developed in the laboratory. Both apps succeeded at thefirst purpose, but failed at the moment.

Laboratory studies measured earlier findings. Culture collections of hundreds of species have been amassed. Research on numerous strains demonstrated that, generally speaking, nitrogen sufficiency (nitrogen is needed for protein synthesis) promoted highgrowth rates and low oil content, whereas nitrogen deficiency resulted in reduced growth rates and high oil content. For many species, it has also been noted that anxiety, caused by factors such as high light intensity or very high temperatures, can inducespecies to shift from protein synthesis to oil synthesis. Species capable of optimal oil production–the maximum oil content in the maximum growth rate–have been chosen for large-scale production trials.

Large-scale production was once again attempted from the late 1980s and early 1990s utilizing open pond systems. Operating results were comparable to those obtained for the three past decades. Promising oil-producing species have been selected from thecollections, and cultures were inoculated to the ponds. However, as in previous experience, single-species civilizations couldn’t be maintained for over a couple weeks or months. The last record of the US program referred to this phenomenon as an”uncertainty together with the character of species control attained.”

From the 1990s the standing of large scale cultivation had not progressed past the point reached in the 1960s. Three sorts of microalgae–Spirulina, Dunaliella and Chlorella–were being cultivated at facilities employing open pond systems coveringmore than 100 acres. Scores of other species had been tried worldwide, but all attempts had failed. The biofuels programs, in particular, had been unable to grow any desirable species in any scale away from the laboratory. Moreover, the biofuelsprograms had concentrated on efforts to demonstrate the greatest potential biomass production rates under nutrient-sufficiency, conditions which are known from laboratory studies to favor non oil content. No efforts were made at large-scale to optimize oilproduction.

Large-scale Closed System technology started to receive significant attention in the early 1990s, once it became evident that cultures of most species exposed to atmosphere were not sustainable. At that moment, the biggest Closed Systems that hadever been used were no longer than a couple million gallons in capacity. Advances in recent years have triumphed at increasing reactor capability by a factor of about 10, to about 30,000 liters. However, this is nowhere close to the speed of increase attained forOpen System capacity that, also more than a decade (in the 1950s to 1960s), improved by a factor of 1,000.

The top limit of Closed System capacity isalso, in substantial part, a direct effect of inherent design requirements. All fundamental Closed System designs in use today were first developed in the 1950s, and may be categorized as follows: (1) verticalbags, tubes, or towers; (2) flat-plate reactors; and (3) flat tubes. Vertical systems are constrained by height limitations. Even when exposed to full sun, most cultures achieve such high cell densities that lighting is practically entirely absorbedat a distance of more than 15 to 20 cm in the Lighted region. This restriction limits the width of the culture vessel to no longer than 30 or 40 cm. To accomplish a capacity of more than 10,000 liters, for instance, a 40-cm diameter vertical system wouldhave to be more than 80 meters (260 feet) high. Such measurements pose clear challenges in structural technology that, even if attainable, become progressively complicated the greater the amount of the system. Among the obvious solutions has been tointroduce an illumination system within the reactor, but experience has shown that this introduces additional problems, of which bio-fouling might be the greatest. Over a relatively short period, the surface of this light source tends to be coated with amicrobial picture, sharply reducing light intensity and therefore defeating the purpose of the light source. Removing the civilization and cleaning the vessel is one alternative, but hardly desirable if the aim is continuing operation. Another frequent anti-foulingoption, making the surface of the light supply toxic to germs, is obviously undesirable. Generally, the usage of inner illumination makes the system more complicated.

Horizontal systems like flat-plate reactors and horizontal tubes eliminate the need for the structural technology required of perpendicular systems. Utilizing the planet’s surface for structural assistance, the possible capacity of these systems mightappear infinite. However, the capacity of systems is generally restricted by the necessity for turbulent flow, if used to maintain sufficient mixing or to fill and empty the culture vessel.

Turbulent flow in a pipe or a channel is described by the Reynolds number, defined as the velocity of the fluid multiplied by the”feature length” of the pipe or channel, and also divided by the viscosity of the fluid. The Reynolds numberdoes have no components, like inches or pounds, and is consequently”dimensionless,” such as”one-half” or”two-thirds”. The feature length of a fluid-filled pipe is its own diameter; the attribute length of a broad channel is its depth. To get a fluidof continuous viscosity, flow will become more and more tumultuous since the velocity of flow increases. Turbulence also increases in proportion to the attribute length; this occurs because pipe and channel surfaces are”sticky.” Surfaces cause frictionthat slows down the flow; the flow rate is practically zero next to the surface, and also increases with distance from the surface. Thus, in a tube or channel with small feature length, the surface friction is going to have fantastic influence on the averageflow. By comparison, in a pipe or channel using large attribute length, the surface friction will probably have little influence on the typical flow, and turbulence will probably be higher.

Surface friction additionally adds up more than space. Imagine a very long pipe through which water is propelled by means of a pump. In the origin, near the pump, the flow is turbulent. The farther the fluid moves down the tube, the more surface it is exposedto and, the more surface it is subjected to, the more its circulation is slowed by friction. At some point from the origin, the accumulated friction has eliminated so much energy in the fluid flow that it ceases to be tumultuous. This occurs when the Reynoldsnumber falls below a value of approximately 2000, and then the flow is believed to be”laminar.”

Laminar flow is not desired in cell cultures because in such conditions that the cells have a tendency to aggregate, either by swimming or sinking. Turbulent flow prevents such aggregations. Imagine, by way of example, the sand particles could rapidlysink to the underside at a still pond, but wouldn’t do this at a large breaking wave or a rapidly moving stream.

In summary, then, turbulent flow is maintained by avoiding quite low fluid velocities, very little feature lengths, and very long stations. The feature length for horizontal Closed Systems for example flat-plate reactors or horizontaltubes is the thickness of the civilization, that, as explained before, has a practical upper limit of approximately 20 cm. One can create turbulent flow within an flat-plate reactor or a horizontal tube with any range of devices like pumps or airlifts. But with increasing distance from the source of the stream, turbulent energy is lost to friction such that, at some finite space flow becomes laminar. In laminar flow conditions that the cells of most photosynthetic microbes will sink to the base of thereactor. This is undesirable for many reasons, not the least of which is that harvesting the cells becomes problematical. One alternative is to supply more tumultuous energy in the origin, but this is okay only to a upper limit where mechanicalshear damages the cells . Yet another alternative is to provide numerous pumps, for example, through the reactor, but this approach introduces additional complexities of both construction and operation.

As a matter of practice, vertical Closed Systems are confined to a power of less than about 1,000 liters, and flat Closed Systems seem to be limited to capabilities less than about 50,000 liters. With the intention of large-scalecultivation of photosynthetic microbes, Closed Systems are much more costly and complicated to build and operate than Open Systems. This is because every individual system demands its own infrastructure: a set of devices or mechanics forproviding turbulent mixing, introduction and elimination of medium, and monitoring and control of variables like pH and temperature. To cover a specified area of land with Closed Systems requires at least 10 times greater infrastructure than covering the samearea of property with Open Systems, rendering Closed System cultivation much more complex.

In practice, every cultivation method for photosynthetic microbes involves a coupling of both Open Systems and Closed Systems at a certain scale. All farming systems, regardless of scale, finally rely for their original inoculum of cells onculture collections regularly maintained around the entire world. All culture collections exclusively keep their cell cultures in Petri dishes, test tubes, or sterilized flasks–all which can be, strictly speaking, Closed Systems. Even large-scaleproduction systems that might be thought to consist”only” of Open Systems should rely ultimately on a Closed System to supply the initial inoculum.

The main technological conundrum for the production of photosynthetic microbes is the fact that Open System technology has advanced to a large scale that is economical and relatively easy to operate, but can’t offer sustainable generation of desiredmicrobes. By contrast, Closed Systems do supply sustainable generation of desirable microbes, but even at their largest scale they are costly and complicated to operate.

Thus, there is a demand for a manufacturing method that provides for sustainable manufacturing by lowering the possibility of contamination and yet doesn’t substantially increase the complexity or cost of construction or operation.

It is therefore an object of the invention to provide an effective way of sustainable production of photosynthetic microbes in large scales that may be easily constructed and doesn’t increase the complexity or cost of building oroperation.

It’s a still further object of the invention to provide a process of manufacturing that’s especially suited to optimizing the production of oils as well as other helpful goods from photosynthetic microbes. Oils and other helpful products can then beextracted and purified from the aggregate biomass by way of a variety of chemical procedures.

IP reviewed by Plant-Grow agriculture technology news