ABE 501, 12/15/2019

A Review of Advancements in Microalgae Biofuel

Manufacture
Robert Adams
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Abstract: The proceeding review will discuss several scientific articles published in the
Journal Bioresource Technology and the Journal Chemosphere. These scientific inquiries all
targeted thraustochytrids, mostly the genus Aurantiochytrium. They all are seeking to pieces to
the question of: is microalgae the ideal biofuel feedstock, and, if so, how must it be utilized for
optimum efficiency and economic viability. First, an investigation into optimal feeding strategies
will be reviewed. Then, an evaluation of harvesting methods will be discussed. Next, the cellular
development and intracellular lipid body accumulation is studied. Lastly, Lipase-catalyzed in-
situ biosynthesis will be reviewed.
Introduction: Ethanol began being used more widely as a fuel source in the 1970s, when
“petroleum-based fuel became expensive and environmental concerns involving leaded gasoline
created a need for an octane. Corn became the predominant feedstock for ethanol production
because of its abundance and ease of transformation into alcohol.” (Gustafson, n.d.) In more
recent history, the perceived increasing scarcity of serviceable petroleum deposits has been
driving investigations into fuel energy sources. The goal of all biofuel research is into a few
different broad categories: find the ideal feedstock, efficiency of fermentation and make the first
two economically viable to produce fuel ethanol to replace the non-renewable fuels the world
currently uses. In the U.S., corn is most widely used as the feedstock because of its available and
its carbohydrate content can be readily converted into substrate sugars yeast can ferment into
ethanol, in large scale processes. However, is not the idea ethanol feedstock. Corn ethanol
production has a few major flaws: ethanol greenhouse gas emissions, when used as a fuel source,
are “typically 30–90% lower than those for gasoline or diesel fuels”, but the indirect emissions
from corn ethanol manufacture can be cause higher total emission production, (Raadal et al.,
2011), “Increasing bioenergy crop cultivation poses risks to ecosystems and biodiversity” (IPCC,
2014), competition for land versus food crops, potential water scarcity, and nitrogen pollution
possibilities through fertilizer use. (Stecker, 2014) The world’s petroleum supply will all run dry
eventually. Optimistically, a biofuel feedstock that lacks the negative aspects of corn ethanol
production will be found, and the following is a review on investigations into novel aquatic
feedstock sources, microalgae.
Review: “A novel fed-batch process based on the biology of Aurantiochytrium sp.
KRS101 for the production of biodiesel and docosahexaenoic acid” was conducted by Kyochan
Kim, Eun Jung Kim, Byung-Gon Ryu, Soojung Park, Yoon-E Choi, and Ji-Won Yang at
Universities and research laboratories across the Republic of Korea. The importance of co-
products and by-products in biofuel manufacture was the motivation behind this research. The
corn ethanol dry grind process produces the co-products of DDGs and corn oil. They are used for
ruminant, cattle, and as an additive to poultry feed, respectively. With current technology, the
economic viability of a feedstuff’s utilization for biofuels is heavily dependent of the what other
commodity can be retrieved and be sold after it has been fermented. The authors target
polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA), as the co-product
of manipulation and interest because the current commercial PUFA source has two major
limitations. “Fish oils are the main source of omega-3 PUFAs, including DHA, but further
commercialization is limited due to their unpleasant smell and/or taste. Moreover, purifying
DHA from fish oils requires extensive purification procedures, due to the complex forms of fatty
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acids in these oils.” (Kim et al., 2013) Additional confidence in microalgae as a superior PUFA
source is offered. In the resulting biomass, after DHA removal, “contains a plethora of proteins
and carbohydrates, which could be further utilized for new biomass production.”. (Kim et al.,
2013) The target organism species is important in production of both target products.
Aurantiochytrium limacinum, “has a high fatty acid content, with increased biomass and DHA
yield”. (Kim et al., 2013) The oil extracted from the microalga contains high levels of palmitic
acid (PA), 46% of the total fatty acid content, and, “PA, which can be used to produce high
quality microalgal biodiesel due to its high cetane number, low iodine content and high oxidation
stability”. (Kim et al., 2013) They identified an optimized strategy of fed-batch cultivation has
not yet been designed. Their design increased biomass, DHA, and PA (2-4 fold) compared to
current established methods. To establish a new optimized method two experiments on initial
nutrient concentration effect were performed, stage specific standards were mathematically
derived, and the preceding results were synthesized and tested to identify the best method. To
test initial concentrations of glucose and yeast extract concentrations and initial salinity
concentration effects on growth they first performed fed-batch cultivations, “200 mL modified
basal medium (40 g/L glucose, 10 g/L yeast extract, 9 g/L KH 2PO4, 20 g/L sea salt and 10
mg/L tetracycline ) in 500-mL baffled flask for 7 days.” (Kim et al., 2013) These conditions
were also set at 28 °C, pH 7, 200 rpm, and 8 ppm oxygen. Throughout this cultivation period
glucose and yeast extract stock were added once a day, and amounts depended on experimental
conditions. To analyze the biomass, mL microalgal suspensions were obtained from
centrifugation, 8000 rpm for 5 mins. Supernatant was washed with PBS, pH 7.4 and freeze dried
for 3 days. It was weighed to be able to calculate lyophilized biomass. Lipids and FA
composition was obtained from the Folch and Lees method for lipid extraction and
transesterification to obtain fatty acid methyl esters (FAMEs). FAMES were analyzed by gas
chromatography using a flame ionized detector and an INNOWAX capillary column. Lastly,
“The quantities of individual fatty acids were estimated from the peak areas on the chromato-
gram using a C19:0 fatty acid (nonadecanoic acid) as an internal standard”. (Kim et al., 2013)
The effect of initial glucose and yeast extract concentrations on cell growth and lipid product
was: for a given yeast extract added (2, 6, and 10 g/L), an increase glucose would cause
increased biomass, DHA, and PA. However, for a given glucose concentration added (20, 40, 60
or 80 g/L), DHA and biomass increased with more glucose, but PA was inversely correlated.
From this data, the authors hypothesize nitrogen concentration determines the relative
composition of fatty acids in the lipids of the cells. They experimentally determined, “medium
supplemented with 40 g/L glucose and 10 g/L yeast extract resulted in the highest lipid and DHA
yields”. (Kim et al., 2013) Additionally, increased cell lysis was observed in fresh water and
dependent on environmental conditions. As nutrient levels decreased, cell rupture incidence
increased. They determined that as sea salt increased, rupture decreased; this is consistent with
the previous set of observations. They determined the optimum is between 0-3.5%. They offer
2% salinity as the target because of the species origin, coastal waters such as mangrove regions.
Salinity does not affect lipid concentrations, therefor the limiting addition should be that which
reduces lysis the most. Next, they calculated yield coefficients (YX/C) for carbon and nitrogen to
determine optimum levels and growth limiting rates. Glucose showed a linear positive curve.
Showing positive correlation throughout the cells life. Nitrogen shows a sharp uptake after
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400mg/L. The authors concluded that this is due to the cells requiring nitrogen early on in life
cycle for initial growth and division, but glucose is used for biomass growth throughout a cells
life. Lastly, they tested four different batch-fed scenarios: yeast extract alone, glucose alone,
yeast extract for 1 day and glucose beginning on day 2, and batch cultivation for 7 days. The best
results came from the yeast extract for 1 day and glucose beginning on day 2 system. This was
seen as a confirmation Aurantiochytrium requires nitrogen early and carbon is required
throughout. They succeeded in making the microalgae more efficient, “reduced the costs of the
primary nutrients, glucose and yeast extract, by 66% and 37%, respectively”. (Kim et al., 2013)
The work that needs to be done in the future is preventing and minimizing cell lysis. Cell lysis
creates a floating layer consisting of lipid bodies, and it is impossible to harvest. Both of the
target products are lipids; therefore, reducing lysis will increase yield and profitability, since
“floating layer was impossible to harvest by centrifugation or any available method, resulting in
a significant loss of biomass.” (Kim et al., 2013) Next, I will discuss harvesting techniques.
Through another collaborative effort made previous authors, efficiency has been further
added to the microalgae utilization processes in biofuel production. “Evaluation of various
harvesting methods for high-density microalgae, Aurantiochytrium sp. KRS101” describes their
effort to test five technologies, and their efficacies for lipid extraction from the cells. The authors
performed the consequent experiments because, “Harvesting, however, is not well studied and a
technology with sufficient efficacy has not yet established; and it is more so because the final
cell density is much higher than the photoautotrophic cultivation.” (Kim et al., 2015) The five
methods evaluated were coagulation, electro-flotation (EF), electro-coagulation–flotation (ECF),
centrifugation, and membrane filtration. First, coagulation was performed using FeCl3 as the
coagulant at five different doses. 3mL were then collecte4d to measure harvesting efficiency. It
was only effective above .5 g/L, with retrieval at 81.9%, 90.3% and 98.8% for 0.5, 0.75, and 1
g/L, respectively. The high requirement was assumed to be due to the high biomass in samples.
For the second and third chemical methods, “ECF and EF, harvesting efficiency was found to be
a function of current density supplied.” (Kim et al., 2015) ECF showed to be more efficient than
EF. This is explained by ECF causing increased current density and causing aluminum ion
expulsion, thus causing metal ions to form more rapidly. This means that harvesting can occur
faster and therefore more efficiently. Aurantiochytrium sp. KRS101 was less susceptible to
electrochemical harvesting than other species because of the very traits it is desirable for. Fourth,
centrifugation was deemed effective at the detrimental loss of biomass. There was a loss of
“12.8–15.4% of biomass at all the tested centrifugal forces.” (Kim et al., 2015) The aforemention
floating layer was observed, and the authors hypothesized its presence may not come from
external force alone. Glucose was almost entirely consumed at the point of harvest. This caused a
greater osmotic pressure, which this specie is sensitive too, but higher centrifugal forces caused
more biomass loss. Therefore, it was assumed to be a combination effect. Lastly, another
mechanical harvesting technique was employed, membrane filtration. It was combined with
rotational disks to create rotational speed, the effect of which was first determined. The diet of
this biosystem is an advantage in rapid growth, but in this harvesting technique, without the
rotational forces, it worked against the researchers efforts “Unlike other photoautotrophic
microalgae, high biomass concentration of this heterotrophic microalgae culture rapidly
produced harsh fouling layer on the membrane surface during filtration of microalgae, leading to
more than 97% of flux decline and very low permeate flux.” (Kim et al., 2015) Our opportunity
to feed the organism for faster growth caused inefficiency in this method, before a perturbation
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element was added, “increase in rotation speed of disk decreased fouling resistance notably by
more than 97% from 800 rpm.” (Kim et al., 2015) The first three methods can be sped up with
increased coagulant dose in coagulation or through increasing current density in ECF and EF.
However, these cause increased water in the biomass products obtain. This extra water then must
be removed. The conclude these processes are untenable because it was, “impossible to achieve
sufficiently high harvesting efficiency and at the same time low enough water content of
harvested biomass.” Efficiencies were unviable, as well. EF only recovered half and 90%
recovery through ECF. (Kim et al., 2015) In contrast, centrifugation removed the most water. It
had the driest product at a 10% loss to biomass recovery. Dynamic filtration, membrane filtration
with a rotating disk, is the way forward in harvesting techniques. It was superior in the
departments of, “no biomass loss, low water content in harvested biomass, reasonable energy
consumption, and operation manageability.” (Kim et al., 2015) Moving forward, this technique
can be improved upon. Whether fouling can be prevented in another mechanical fashion to
prevent the decline in flux percent, or, as the authors suggest, perform a synergistic two-step
process. They propose coagulation could be performed first for then performing membrane
filtration, halving the time of the entire harvesting process.
The last article in this review is “Microbial communities of biomethanization digesters
fed with raw and heat pre-treated microalgae biomasses”. Unlike the previous two articles, this
paper deals with anaerobic bacterial fermentation of microalgae species. The bacterial processes
here creates biogas as the end desired product, and the author’s remark it is a much simpler
process than the production of bioethanol or biodiesel, but that doesn’t due diligence in process
isn’t necessary. Pretreatment using thermal, thermo- chemical, ultrasound, microwave or
biological approaches have all been experimented with to drive efficiency into biofuel
production. Specifically, “Thermal and enzymatic pre-treatments have shown to be beneficial to
increase methane yields in batch mode anaerobic digestions, through disrupting/ solubilizing the
polymeric cell structures when applied to the common robust microalgae”. (Sanz et al., 2017)
The experimenters felt there is a lack of information regarding any performance data when pre-
treatment of microalgae is performed when it is feedstock for methane production, using
Continuously Stirred Tank Reactors (CSTRs). Additionally, new community characterization
techniques like pyrosequencing have emerged to more definitively identify microbial specie
constituents within communities. To perform this evaluation, “Two Continuously Stirred Tank
Reactors (CSTR), fed with fresh (CSTR-C) and heat pre-treated (CSTR-T) Chlorella biomass
were run in parallel in order to determine methane productions”. (Sanz et al., 2017) The reactor
with the thermally pretreated microalgae, CSTR-T, was much more productive. 1.5 fold increase
in methane production, and the removal percentages of substrate and volatile solids went up 50%
and 42%, respectively. “This methane yield corresponds to an enhancement of almost 66% with
regard to the untreated biomass” (Sanz et al., 2017) However, the authors used different species,
most likely skewing these results. Relative byproduct production and toxicity levels were about
even; no ammonia inhibition expected. The last several experiments were done to identify the
bacteria present. To do this, they performed DNA extraction, PCR amplification and 454
pyrosequencing on samples collected after 45 days. This resulted in, “A total of 16 different
accepted phyla and 7 candidate divisions were detected in the set of sequences.” (Sanz et al.,
2017) The phyla abundance starting with the most abundant were: “Proteobacteria (13%, 50%
and 6% with respect to I, CSTR-C and CSTR-T), Bacteroidetes (12%, 19% and 22%), Chloro-
flexi (23%, 9% and 25%), and Firmicutes (9%, 8% and 12%).” 94.8% of the innoculum was
from 6 phyla. Proteobacteria dominated the CSTR-C sample, with more than 50% DNA read
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sequences, and Alfaproteobacteria accounted for 37%. The CSTR-T reactor differed sharply. It
only contained Campylobacteraceae, Syntrophaceae and Comamonadaceae families accounting
for 5%. The article goes on to further classify the colonies in each reactor by their constituents.
The more important information to note is the relative abundance of bacteria, with respect to
their respiration. “The inoculum and the reactor fed with the heat pretreated biomass had high
levels of anaerobic bacteria, the percentage of aerobic bacteria was relatively high with respect to
the reactor fed with the fresh Chlorella.” (Sanz et al., 2017) The authors hypothesize the
microalgae’s intact protective coating in the untreated reactor is the reason they were not well
metabolized by the inoculum. The work that needs to be done in the future is characterization of
DNA strands that were unreadable, and the taxonomic classification they belong to. A lot of data
from sequencing was unusable due to the sequence being unknown, and it may reveal proteins or
reactions present, previously unsuspected.
Conclusion: The world is running out of oil. It is a finite resource. Governments and
producers have taken small steps towards sustainable and environmentally conscious fuel
production. The United States uses corn ethanol to a small extent, Brazil is using sugarcane for
biofuel, cellulosic biofuel production has a lot of ground to cover before being commercially
viable. However, the recent advancements in microalgae as a biofuel feedstock appear
promising.
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References:

Gustafson, C. (North D. S. U. (n.d.). History of Ethanol Production and Policy. Web Page - The
Energy Information Agency. North Dakota State University. Retrieved from
http://www.ag.ndsu.edu/energy/biofuels/energy-briefs/history-of-ethanol-production-and-
policy
IPCC. (2014). Climate Change 2014: Mitigation of Climate Change. Working Group III
Contribution to the Fifth Assessment Report of the Intergovernmental Panel on Climate
Change. https://doi.org/10.1017/CBO9781107415416
Kim, K., Jung Kim, E., Ryu, B. G., Park, S., Choi, Y. E., Yang, J. W. (2013). A novel fed-batch
process based on the biology of Aurantiochytrium sp. KRS101 for the production of
biodiesel and docosahexaenoic acid. Bioresource Technology, 135, 269–274.
https://doi.org/10.1016/j.biortech.2012.10.139
Kim, K., Shin, H., Moon, M., Ryu, B. G., Han, J. I., Yang, J. W., Chang, Y. K. (2015).
Evaluation of various harvesting methods for high-density microalgae, Aurantiochytrium
sp. KRS101. Bioresource Technology, 198, 828–835.
https://doi.org/10.1016/j.biortech.2015.09.103
Raadal, H. L., Gagnon, L., Modahl, I. S., Hanssen, O. J. (2011). Life cycle greenhouse gas
(GHG) emissions from the generation of wind and hydro power. Renewable and
Sustainable Energy Reviews, 15(7), 3417–3422. https://doi.org/10.1016/j.rser.2011.05.001
Sanz, J. L., Rojas, P., Morato, A., Mendez, L., Ballesteros, M., González-Fernández, C. (2017).
Microbial communities of biomethanization digesters fed with raw and heat pre-treated
microalgae biomasses. Chemosphere, 168, 1013–1021.
https://doi.org/10.1016/j.chemosphere.2016.10.109
Stecker, T. (2014). Biofuels Might Hold Back Progress Combating Climate Change - Scientific
American. Retrieved November 27, 2017, from
https://www.scientificamerican.com/article/biofuels-might-hold-back-progress-combating-
climate-change/