Renewable and Sustainable Energy Reviews 80 (2017) 1089–1099

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Renewable and Sustainable Energy Reviews

journal homepage: www.elsevier.com/locate/rser

Microalgae culture enhancement through key microbial approaches MARK

⁎
Puja Tandon, Qiang Jin
School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China

A R T I C L E I N F O A BS T RAC T

Keywords: The demand for biofuels is rising with the growing population and decreasing reserves of petroleum. Indeed the
Microalgae culture rising population and increasing level of greenhouse gases not only contribute for global warming, but also
Biofuels imparts serious effects on humans and the environment. There is a constant urge to find other alternatives to the
Biomass petroleum-based products. Of the three generations of biofuel feed stocks i.e. food crops, non-food crops and
Enhancement
microalgae-derived biofuels; it is discussed that microalgae can be the best source of feed stock in terms of food
Renewable energy
Carbon dioxide
security and for reducing the harmful impact on the environment. This paper serves as base to discuss the
benefits of using microalgae as a source of biomass for biofuels and the methods that can be used to enhance the
microalgal biology in order to improve their production. It reviews how the use of optimized nutrient
concentration and culture conditions increase the yield, cell density and lipid productivity. It further reviews the
role of bacteria in enhancing/inhibiting the microbial biomass and lipid productivity in microalgal-bacterial
biological associations. The final part of the paper lays emphasis on the importance of improving microalgal
biology by the use of genetic engineering and metabolic engineering in order to obtain desired results. Overall
stress is placed upon unfolding and unraveling the gaps present in producing microalgal biomass, such as
inefficient and reduced production, poor penetration of light in dense cultures, low oil content and higher
harvesting costs. Overcoming of these major bottlenecks by ecologically engineering species specific valuable
microalgal-bacterial mini consortium might lead to specific and noteworthy breakthrough in the production of
clean, green, renewable, sustainable and high yielding microalgal biofuel.

1. Introduction
The world's growing population is moving towards Malthusian
catastrophe expanding at an incredible rate of 83 million annually, or
1.18% per year [1,2]. It is estimated that by the year 2050 the world's
population will reach 9.72 billion. The demand for petroleum based
products and energies are rising with the growing population.
However, nearly half of the reserves of fossil fuel have been depleted,
thus challenging the overall growing demand for fuel worldwide. This
has led to the political and scientific impetus related to apprehensions
about global energy security and supply of petroleum based products
[3]. Almost 85% of the crude oil is being utilized by the transportation
industry for the production of gasoline, petrol, diesel and 10% for
production of chemicals in industries. This undoubtedly is an area of
concern and the reason why production of alternative renewable energy
sources such as biofuels attracts worldwide attention [4].
Green House Gases (GHG) not only contribute to global warming
(GW) but also impart adverse effects on the environment and human
life [5]. The largest amounts of GHG are injected to the biosphere by
the transportation sector. Compatible mitigation strategies are re-
quired in order to neutralize the excess of carbon dioxide that is not
removed by natural processes [6]. The incidences of the formation of
regional haze - air pollution in the atmosphere, is increasing due to the
presence of fine particles with aerodynamic diameter less than 2.5 µm,
PM2.5 (Particulate Matter 2.5), [7]. The PM2.5 is composed of organic
carbon, elemental carbon and water soluble inorganic ions that
adversely affect public health and environment [7]. All together the
increasing levels of greenhouse gases (carbon dioxide, water vapour,
methane, nitrous oxide etc.), presence of fine PM 2.5 (haze) in the
atmosphere and the consequential global warming, have contributed
intense implications towards the environment. In order to reduce the
consumption of traditional fossil fuel and their impact on the environ-
ment, sustainable and renewable energy resources such as wind power,
solar energy, tidal energy, hydropower, geothermal energy and biomass
energy have to be explored extensively [8].
Biomass for renewable energy, specifically biofuels, has heavily

Abbreviations: CA, Carbonic Anhydrase; CO2, Carbon Dioxide; DGGE, Denaturing Gradient Gel Electrophoresis;; FACS, Fluorescence Activated Cell Sorter;; GHG, Green House
Gases; GW, Global warming; KEGG, Kyoto Encyclopedia of Genes and Genomes;; N, Nitrogen; O2, Oxygen; P, Phosphorous; PGPB, Plant growth promoting bacteria; PM, Particulate
Matter; RSM, Response Surface Methodology; TAGs, Triacylglycerols
⁎
Corresponding author.
E-mail address: qiang.jin@outlook.com (Q. Jin).

http://dx.doi.org/10.1016/j.rser.2017.05.260
Received 7 July 2016; Received in revised form 9 May 2017; Accepted 29 May 2017
1364-0321/ © 2017 Elsevier Ltd. All rights reserved.
P. Tandon, Q. Jin Renewable and Sustainable Energy Reviews 80 (2017) 1089–1099

contributed towards upliftment of energy, environment, expansion and Table 1

economics. It consists of varied organic matter that originates from Comparison of different feed stocks from first generation, second generation and third
generation used for biofuel production.
green plants and microalgae and transforms sunlight into biological
material through the process of photosynthesis [8]. Biomass for Crop Seed oil Oil yield Land area Water Energy
biofuels have received much attention lately as they can be stored (% oil (L /ha required (m2 footprint (Gj/ ha/
and directly used in the existing vehicle engines, thus proving to by wt) year) year/kg (m3 /GJ) a)
become a significant resource for transportation fuels. The long biodiesel)

emerging necessity for sustainable resources of transportation fuels 1st Generation

has created a convincing exploration in technologies that assist biofuel Corn 44 172 66 50 75
production [3]. The use of biomass has helped to mitigate climate Soybean 18 446–636 18 383 26
change, reduce the dependence on fossil fuels, decrease risks to life and Sunflower 40 952– 11 61 31
1070
land, and has provided a safe and competitive energy source [9].
2nd Generation
However, the points to be taken into consideration in selecting a Jatropha 20–60 1892 15 396 62
technically and economically viable biomass for biofuel are that it Coconut 65–75 2689 11.8 49 78
should be competitive or cheaper than petroleum fuels, should utilize Oil palm 36 5366– 2 75 192
5950
less land, should be able to decrease greenhouse gas emission (e.g.
3rd Generation
carbon dioxide (CO2) sequestration) and should need minimal water Microalgae 30–70 24.4– 0.1–0.2 < 379 793–
[6,10]. Biomasses from different sources such as forestry, agricultural 136.9k 4457
and aquatic sources have been vigorously explored as the feedstock for
the production of various types of biofuels such as bioethanol, biogas,
bio-hydrogen, biodiesel and bio-oil. However, biomass from these of biofuel feed stocks i.e. food crops, non-food crops and microalgae-
sources when burnt might have a detrimental effect on the environ- derived biofuels, it is assessed that microalgae can be the best source of
ment, almost similar to that of combustion of fossil fuel that might end feed stock in terms of food security and for reducing the harmful
up disturbing the carbon balance (conversion of fixed carbon into impact on the environment. Table 1 represents the comparison of
carbon dioxide). Adding to it, the diminution of the existing biomasses certain feed stocks from first generation, second generation and third
such as wood without proper replacement might intensify severe generation that have been used for biofuel production [5,9,14–17].
environmental concerns such as deforestation, negative ecological These feed stocks are compared with each other in respect to their oil
impacts and loss of biodiversity [11]. The net energy balance might content, biofuel yield, land area required, water footprint and energy
be affected by the energy costs of transporting the feed-stocks and use produced. For example, the oil content and yield within a microalgal
of nitrogen fertilizers from the agricultural resources. These concerns species depends upon the selection of a specific culture conditions and
need to be addressed before choosing the specific source for biomass. the microalgal species. Although, the oil content is almost similar
Microalgae are simple unicellular and multicellular photosynthetic between the crop plants and microalgae, however there are huge
micro-organisms. They can be either prokaryotes (cyanobacteria) or differences between overall biomass productivity, energy produced
eukaryotes that can grow and live in harsh conditions because of its and resulting oil yield (24,355–136,886 L ha−1 a−1) with strong ad-
simple structure [5]. All existing earth ecosystems comprising both of vantage for microalgae. In the present scenario, it is clearly reflected
aquatic and terrestrial elements are composed of them. Sunlight is used from various studies that the production rate (L ha−1) of oil extracted
by them to convert carbon dioxide to prospective biofuels through the from microalgae (91%) is substantially higher than other feed stocks
process of photosynthesis similar to that of plants. However, they have for example, palm oil (3%), corn (1.5%), avocado (1.4%), rapeseed/
been considered more effective and efficient in converting sunlight into canola (1%) and peanut oil (less than 1%) [18]. In terms of land area
biochemical energy or biomass [12] as compared to the crop plants required for mass production, microalgae (0.1–0.2 m2 year kg−1) fol-
with the photosynthetic efficiency in the range of 10–20% or higher lowed by palm oil (2 m2 year kg−1) utilize minimum arable land
[13]. Microalgae biomass, mostly consist of lipid, carbohydrate and compared with other crop plants. However, the cost involved for
protein. It has the capacity to occupy between 1% and 3% of the total meeting the water and energy requirements for microalgae need
cropping area of U.S. to significantly meet 50% of transport fuel needs further refinement. After analysing the table it seems that microalgae
as compared to oil crops [14]. They alone appear to be capable to meet have the potential to replace the other feed stocks in terms of utilization
the world-wide demands of biofuel production as compared to crop of less arable land and high oil yield compared with first and second
plants. The present study begins with a review of various benefits of generation crops. The biofuels produced from microalgal biomass will
using microalgae biomass as a source of clean renewable energy hence not compromise production of food, fodder and other relevant
reserve. This is followed by discussion on how optimized nutrient products derived from first two generation crop plants. Hence in total,
concentration can effect microalgal growth, specifically lipid produc- we can summarize the potential advantages of using microalgae culture
tivity. The next section throws light about different types of microalgal- for biofuel production over crop plants as below [5,6,12,19,20].
bacterial biological consortium and it latter focuses on the advantages The microalgae culture can:
of establishing an artificially engineered species specific microalgal-
bacterial (plant growth promoting nitrogen fixing bacteria; decrease a) reproduce anywhere using sunlight and simple nutrients. It can
release of greenhouse gases) mini consortium. The final section almost double their biomass in 24 h or within 3.5 h during the
describes the merits of exploiting the genetically/ metabolically mod- exponential growth phase using less cropping area. Thus the
ified microalgal species in creation and streamlining of endless valuable competition for arable soil is reduced.
processes. b) easily cultivated even under harsher environments and it requires
less water in comparison with crop plants.
2. Why choose microalgae culture? c) tolerate high CO2 content in gas streams that permits productive
CO2 mitigation. They can easily remove CO2 from industrial flue
The microalgae culture has the infinite potential to be used as gases by bio-fixation thus reducing the emission of GHG of an
primary producers of biomass in coming years and eventually they will industry while producing biodiesel.
turn out to be a most significant energy source. They can act as d) reduce the release of nitrous oxide in the air that is one of the
feedstock for several types of renewable fuels such as ethanol, greenhouse gases
methanol, biodiesel, methane and hydrogen. Of the three generations e) yield high cell density and high lipid content as compared with crop

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P. Tandon, Q. Jin
Table 2
Comparison of different metabolisms found in microalgae after utilizing different carbon and energy sources.
Source: adapted from [25].
Cultivation condition Microalgae species
Phototrophic Botryococcus braunii UTEX 572, Chlorella emersonii
CCAP 211/11N, Chlorella minutissima
UTEX 2341, Chlorella vulgaris
#259, Scenedesmus obliquus
FCTU Coimbra, Scendesmus actutus
Heterotrophic Chlorella protothecoides, Chlorella vulgaris, Chlorella
vulgaris
#259
Mixotrophic Chlorella vulgaris #259, Scenedesmus obliquus
Photoheterotrophic Micractinium reisseri YSW05), Chlorella vulgaris ESP6,
Chlorella vulgaris ATCC 13482
plants (50 times more than switchgrass that is estimated to be the
fastest growing crop plant; [21]).
f) yield biodiesel per hectare almost 80% more than that of crop
plants. The selection of potentially high oil producing species will be
beneficial as the oil production of microalgae culture greatly
exceeds the oil productivity of the crop plants.
g) end the battle of scarcity in production and use of food, fodder and
other value-added products derived from crop plants.
h) be more cost-effective as compared to conventional farming
[19,20]. They can be practically grown and harvested all year
round, thus thereby increasing the production of biomass and
eliminating problems linked with storage.
i) produce many value-added biological derivatives with plenty of
commercial applications in a number of areas including biofuels,
therapeutics (pharmaceuticals), food additives, cosmetics, pollution
prevention etc.
j) reproduce energy by burning biogas as a source of electricity or
power for the production and separation of microalgal biomass.
3. Limitations of microalgae culture that need to be resolved
Certain disadvantages of using culture of microalgae for production
of biofuel can restrict its further use. They are production of low
concentration of microalgae biomass, poor penetration of light into
dense cultures because of mutual shading by the microalgal cells and
small size of microalgae cells. The above mentioned disadvantages have
increased the cost of harvest of microalgal biomass [19,22] which has
thereby increased the overall cost of production. Harvesting of dilute
suspensions of microalgae culture is not energy-efficient.
Methodologies need to be developed for recycling water, nutrients
and energy using environmental footprint calculations. The enhance-
ment in microalgae culture proves to be a very important probable
source for a potential renewable energy future. The higher content of
water in the microalgal cells requires more energy for harvesting and
drying, thereby increasing the energy and cost of total production. The
higher capital cost and intensive care required by microalgal farming
compared to conventional agricultural farming hampers the biofuel
production of microalgae for its commercial application. The econom-
ics of producing microalgal biofuel need to be enriched extensively to
compete with petroleum based products. However, all of these
disadvantages can be overcome and diminished by utilizing microbial
metabolic and genetic engineering coupled with a biochemistry under-
standing of microalgae culture which can lead to production of the
fourth generation of biofuels [23]. Optimized growth conditions and
use of productive strains can yield microalgal cells with a concurrent
high lipid productivity and growth rate [22]. More strategies that could
be worked out in order to enhance microalgal culture/biomass either
through mutualistic interaction (microalgae-bacteria symbiosis), opti-
mization of growth medium and other growth parameters will be
Renewable and Sustainable Energy Reviews 80 (2017) 1089–1099
Energy source Carbon source Cell density Reactor Type Cost
Light Inorganic Low Open pond or Low
photobioreactor
Organic Organic High Conventional fermentor Medium
Light and Inorganic and Medium Closed photobioreactor High
organic organic
Light Organic Medium Closed photobioreactor High
discussed in the next section.
4. Enhancing or optimization of microalgal biomass
productivity
Medium composition and cultivation conditions play an important
role in the cell growth and lipid accumulation of microalgae. Several
parameters influence the microalgal growth, such as abiotic factors and
biotic factors. The abiotic factors consist of light (quantity and quality),
nutrient concentration, CO2 etc. The biotic factors consist of pathogens
(bacteria, fungi, viruses) and competition with other microalgae. The
operational factors consist of the shear stress produced by bioreactor
design, mixing/turbulence, harvest frequency, hydraulic retention
time, dilution rate and depth [5], for example, high liquid velocities
and degree of turbulence produced by mechanical mixing or air bubble
mixing might damage microalga cells because of shear stress. The
optimum level of turbulence (above which cell death occurs) was strain
dependent and should be investigated in order to avoid decline in
productivity [5,24]. Proper equilibrium between all these factors is
necessary and vital for microalgae growth.
The effect of different cultivation factors (abiotic) on microalgal
growth has been examined by several researchers, however in the
current review paper we will be discussing the following parameters:-
4.1. Optimization of growth medium and culture conditions
Microalgae have the capability to forage their ambiances for
resources, to stow them, or increase their competence by accurate
resource utilization. They can grow photo-autotrophically by utilizing
light as a solitary energy source and inorganic carbon i.e. CO2 as the
carbon source in order to form chemical energy via the photosynthetic
reactions at an optimum temperature ranging between 20 and 30 °C
[25]. This is the most common method of microalgae cultivation.
Table 2 represents comparison of different metabolisms found in
microalgae after utilizing different carbon and energy sources. It can
also assume other types of metabolisms such as heterotrophic (utilize
only organic compounds as carbon and energy source), mixo-trophic
(light and organic compounds as energy source; inorganic and organic
compounds as carbon source) and photo-heterotrophic (light as energy
source and organic compounds as carbon source), however they are
capable of a metabolic shift in response to changes in environment [5].
For example, Chlorella vulgaris #259 can vary its metabolism type
from heterotrophic growth, to mixo-trophic or photo-heterotrophic
growth depending upon which cultivation condition i.e. energy and
carbon source it is utilizing [25]. Table 2 also depicts different
microalgal species that can perform hetero-tropic and photo-auto-
trophic growth as independent and simultaneous methods. However,
certain microalgal species will grow preferably either photo-autotropi-
cally, example Scenedesmus actutus, or hetero-tropically Chlorella
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quirements of microalgae [26]. Table 3 represents chemical biomass

Fig. 1. Microalgae growth curve in a batch culture (solid lines) and nutrient concentra-
tion (dashed lines) depicting five phases of growth.
Source: adapted from 4.
vulgaris or photo-hetero-tropically example Micractinium reisseri
YSW05. Under suitable climatic and growth conditions (exponential
phase), microalgae grow rapidly and can double their biomass as much
as eight times a day [26]. Fig. 1 illustrates the microalgal growth rate
curve in a batch culture, changing with nutrient concentration and
time. The growth rate curve is divided into five phases of growth
obtained by microalgae under optimum growth conditions, describing
(1) lag phase; (2) exponential phase or log phase (characterizing
maximum yield and high concentration of proteins); (3) linear phase
(characterized by decreasing relative growth); (4) stationary phase
(characterized by high carbohydrate and glycogen content); and finally
(5) death or decline phase. The dashed lines in the graph (opposite
pattern in the graph) represent nutrient depletion with time during
stationary phase and onwards [5]. During the stationary phase the
growth is likely to be limited because of reduction in resources such as
light or nutrients. During each growth phase microalgal cell undergoes
changes in its cell wall structure, composition, cell content and
morphology [27]. In most cases, microalgal cell cultures consist mostly
of proteins in the exponential growth phase, while they consist mostly
of carbohydrates and glycogen in the stationary phase.
Microalgal biomass mainly consists of three components that are
lipid (oil), protein and carbohydrate. Chisti [26] developed the
approximate molecular formula of the microalgal biomass as
CO0.48H1.83N0.11P0.01 after considering the minimal nutritional re-
composition i.e. lipid, protein and carbohydrate of 21 species of marine
and freshwater microalgae [5,9,20,21,28–30] on dry matter basis. It
reflects that the variance in chemical composition in microalgae varies
in between different microalgal species with green algae having
maximum lipid content. The content of lipid of different microalgae
usually falls in between 20–50% of dry biomass which can however
increase to 70% under specific cultivation environments [31]. The lipid
content is defined as the concentration of lipids inside the microalgal
cells without taking into account the biomass production, however the
lipid productivity depends upon the lipid concentration within the cells
and the biomass produced by the cells, acting as more valuable
indicator of the potential costs of liquid biofuel production [18].
Thus lipid productivity could be the most effective criteria for selecting
microalgae species for biofuel production [29,30]. Among different
species of microalgae, maximum lipid productivity of 170 mg/L day−1
was observed for Chlorella vulgaris [22,31].
In order to cultivate microalgae on a large-scale the composition of
the growth medium, specifically nitrogen and carbon sources in
addition to balanced growth environment play a vital role [32]. They
require carbon as a fundamental source of energy to grow. Microalgal
cultivation requires 40–50% of CO2 as a source of carbon and light to
carry out photosynthesis for growth of biomass. However, this growth
mode results in only 8–9% of biomass productivity at maximum
photosynthetic efficiency [33]. Moreover, it also cannot rapidly absorb
and add large quantities of biomass when the concentration of CO2 in
the atmosphere is more or less because it leads to change in pH of the
medium that might inhibit growth. Exogenous supply of CO2 and
proper mix of microalgal species are important for high biomass yield
and high lipid content/productivity [34]. At the minimum 1.83 t of CO2
can yield almost 1 t of microalgal biomass that can be easily fixed from
external industrial sources or atmosphere. Consequently microalgal
production can contribute towards mitigation of anthropogenic carbon
dioxide releases [35]. In large scale production, CO2 cost is nearly 50%
of purchasing cost. Microalgal biomass can be employed as a substitute
to coal-firing flue gas by which electricity can be generated by cost
effective approach. In turn the CO2 produced from power plant can be
used to cultivate microalgae thereby reducing CO2 emission to the
environment [9]. The inorganic phosphates also play a key role in
microalgal energy metabolism. H2PO4− (phosphoric acid) and
H2PO42−(dihydrogen phosphate) are the desired forms of phosphorus
that are incorporated into organic compounds through process of

Table 3
Chemical Composition (Lipid, Proteins and carbohydrates) of 21 species of microalgae.

Microalgae Habitat Protein Carbohydrate Lipid (L) L productivity Algal group

Anabaena cylindrica Freshwater 43–56 25–30 4–7 5 Cyanobacteria

Aphanizomenon flosaquae Freshwater 62 23 3 – Cyanobacteria
Botryococcus braunii Freshwater 4 20 86 – Green algae
Chlamydomonas rheinhardii Freshwater/Sea water 48 17 21 36.2–83 Green algae
Chlorella ellipsoidea Freshwater 5 16 84 42 Green algae
Chlorella pyrenoidosa Freshwater 57 26 2 76 Green algae
Chlorella sorokiniana Freshwater 21–53 5–25 20–36 44.7–110 Green algae
Chlorella vulgaris Freshwater 51–58 12–17 14–22 11–170 Green algae
Dunaliella salina marine 57 32 6 46–53 Green algae
Dunaliella bioculata marine 49 4 8 – Green algae
Euglena gracilis Freshwater/Sea water 39–61 14–18 14–20 37 Eukaryotic algae
Prymnesium parvum Marine/fresh water 30–45 25–33 22–38 42 Golden algae
Porphyridium cruentum Marine 28–39 40–57 9–14 34.8 Red algae
Scenedesmus obliquus Freshwater 50–56 10–17 12–14 – Green algae
Scenedesmus quadricauda Freshwater 47 – 1.9 35.1 Green algae
Scenedesmus dimorphus Freshwater 8–18 21–52 16–40 57 Green algae
Spirulina maxima Freshwater 60–71 13–16 6–7 11 Cyanobacteria
Spirogyra sp. Freshwater 6–20 33–64 11–21 – Green algae
Spirulina platensis Freshwater 46–63 8–14 4–9 29 Cyanobacteria
Synechococcus sp. Marine 63 15 11 75 Cyanobacteria
Tetraselmis maculata Marine 52 15 3 43.4 Green algae

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P. Tandon, Q. Jin
phosphorylation [34]. The concentration of nitrogen also plays a
significant effect on lipid accumulation in photo-autotrophic and
heterotrophic metabolism, with lower nitrogen content in the medium
resulting in higher lipid content in microalgal cells, however at the
expense of lower biomass growth [33,36,37]. The possible reason for
the same is explained as follows: high content of nitrogen in culture
medium favors the synthesis of starch in microalgae cells to support
their growth. The starch synthesis pathway is blocked under nitrogen
limited medium and thus photosynthetically fixed carbon is directed to
fatty acid, causing higher lipid accumulation in microalgae cells [38].
After some time, starch which is a carbon and energy source for
microalgae cells will be consumed and thus, the photosynthetic
efficiency will be reduced. This will lead to significantly retarded
growth and less biomass. Add on the overall lipid productivity might
be even less in the case of nitrogen deficient condition because of low
biomass productivity. This will limit the overall synthesis of bioethanol
from microalgae residue as starch synthesis in cells has been inhibited
[39]. For example, up to 55.2% of increase in the lipid content was
noted in heterotrophically grown Chlorella protothecoides species
(approximately 4 times of autotrophic cultivation) with reduction of
nitrogen source and addition of carbon source [40]. Similarly increase
in the lipid content (up to 63%) by dry weight respectively was noted in
all five strains of Chlorella with reduction of the nitrogen in the growth
medium [5]. In culture medium in order to confirm the simultaneous
utilization of both nitrogen and phosphorus, the ratio of N/P should be
in the proper range. This ratio indicates the removal rate of nitrogen
compared to the removal rate of phosphate. This optimal ratio of N/P
varies among different species of microalgae. Wan et al. [41] have
discussed the effects of nitrogen concentration and media replacement
on cell growth and lipid production of oleaginous marine microalga
Nannochloropsis oceanica DUT01. They found out that highest lipid
content reached 64% in nitrogen diminished f/2 medium
(NaNO3=37.5 mg/L) in combination with 1/5 fresh medium replace-
ment. However, in case of BG11 medium (NaNO3=1.5 g/L) in combi-
nation with 1/5 fresh medium replacement as well, the highest lipid
productivity (31 mg/L/d) was achieved, corresponding to maximum
biomass production (1.4 g/L). The results indicate the importance of
high biomass accumulation for efficient lipid production. Stable
carbohydrate content and decreased protein content was observed to
be coupled with increased lipid production for the evaluation of
biomass compositions throughout the culture. The effect of different
organic carbon sources such as glucose, galactose, fructose, sucrose,
maltose, lactose, and starch on the growth and biochemical composi-
tion of Chlorella pyrenoidosa was studied by Zhang et al. [36]. They
noted that the addition of both sugar and starch to the growth medium
increased the growth of microalga. The results under different nitrogen
conditions proved that monosaccharides displayed stronger stimulative
effect on microalgal growth and biochemical composition as compared
to disaccharides. Thus, in order to achieve the highest performance of
the biomass and lipid production, the appropriate carbon, phosphorous
and nitrogen sources for a particular microalga and their optimal
concentration in the medium should be calculated.
Optimized cultivation conditions are critically significant in im-
proving the production of biomass and lipid. In a similar way,
augmentation of the cultivation conditions is proved to be the best
direct and effective strategy to obtain high density of biomass.
Furthermore, it can contribute towards improvement in the efficiency
and productivity of cultivation of microalgae [42,43]. Han et al. [43]
studied growth-state-based schemes for cultivation optimization (new
method of cultivation by changing conditions) in order to stimulate the
growth rate of microalgae Chlorella sp. for lipid production, compared
with the usual optimization studies proved to be beneficial. However, in
this case further research needs to be carried out to understand the
real-time effect of adjustment of cultivation by changing conditions for
further improvements [43]. Response Surface Methodology (RSM) can
be used to optimize the growth medium composition with the resolu-
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Renewable and Sustainable Energy Reviews 80 (2017) 1089–1099
tion of augmenting the productivities of biomass and lipids [44].
Nevertheless, the problematic situation of low algae biomass concen-
tration still exists in most of the microalgae culture methods and
biomass production processes that lead to inefficient and considerably
less production. The advancement and enhancement are a very
important requirement in order to develop the microalgae industry to
an extent of using it as biofuel. For example, the limited light
penetration in the photo-bioreactors is one of the major reasons
resulting in difficulty to grow high density microalgae at a commercial
level (or capacity) which can be partly resolved by utilizing hetero-
trophic microalgae that will eliminate the use of solar radiation as they
only require certain concentration of organic compounds as nutrient
source. Moreover, use of Phagotrophic microalgae (Ochromonas
danica) that can grow easily in light limiting circumstances as their
growth depends on ingesting bacteria [45] can also resolve the limited
light condition in the reactors. The exploitation of mutualistic symbio-
tic associations (discussed in detail in next section) between specific
microalgal and bacterial (nitrogen-fixing; plant growth promoting
bacteria) species under optimized environmental conditions can also
be used to minimize the cost of nutrients (Nitrogen and Phosphorous)
in the optimized growth medium.
4.2. Microalgal-bacteria biological association
Symbiotic associations contribute towards industrial microbiology
by playing an integral part in environmental ecosystems. Some of the
well-known symbiotic associations have been found between termite-
enterobacterium, mycorrhizal fungus-plant, lichen symbiosis and rhi-
zobia-legume [46]. A greater insight into these types of associations in
between microalgae and other micro-organisms, especially bacteria
(inhibiting and enhancing) in natural ecosystems is a very important
aspect that has not been rigorously addressed. Bacteria and microalgae
have lived together since the time of evolution, thereby transforming
living organisms on earth in various characteristics [47,48]. They
together interact and influence the biomes ranging from deep seas
(sea sponges) to mycorrhizal fungus/lichens in all conceivable modes of
symbiotic associations, stretching from mutualism to parasitism [48].
These associations, specifically Roseobacter-microalgae interaction
collectively mark an impact to the global carbon cycle and other
biogeochemical cycling processes; especially carbon and sulfur, by
oxidizing greenhouse gas carbon monoxide and producing dimethyl-
sulfide and thereby lay profound effect on the global environment.
Fig. 2 displays a typical microalgae-bacterial biological association in
aquatic ecosystem. The mechanism of exchange of different compounds
between microalgae and bacteria is species specific as the mini
environment around every microalga is dissimilar and also environ-
ment driven [49]. Carbon, macro- and micronutrients appear to play a
dominant role in the mechanism established so far. Studies reflect that
during these interactions, micronutrients such as vitamins [50], trace
elements, macronutrients such as nitrogen and carbon [51,52], and
phytohormones [52] are usually exchanged between bacteria and
microalgae [47,53] that leads to overall enhancement in microalgal
productivity and quality. In the underlying process microalgae provides
photosynthetic oxygen to the bacteria which in replacement creates a
suitable environment for microalgal hydrogen production [48]. A
recent research demonstrated that the growth of Chlorella vulgaris
(common microalga used for wastewater treatment) was significantly
enriched in presence of a bacterium Azospirillum brasilense that
produce a plant growth promoting hormone indole acetic acid (IAA)
[54]. Ectosymbiotic heterotrophic bacteria primarily known as decom-
posers of organic matter are also known to play integral part in
microalgal growth promotion through specific complex communication
mechanisms and nutrient exchange, establishing symbiotic associa-
tions [48]. These bacteria that are mostly associated with green algae
and plants consist of core group of genera termed as plant growth
promoting bacteria (PGPB), earlier known as plant growth promoting
P. Tandon, Q. Jin
Fig. 2. A typical microalgae-bacterial biological association in aquatic ecosystem.
Rhizobacteria (PGPR) that was latter extended to other bacteria [55].
Similar type of studies depict that both microalgae and bacteria modify
their metabolism to accomplish each other's need and this type of
association is most prevalent in aquatic ecosystem. Some of the
common type of microalgal-bacterial interactions and their mode of
action are discussed below:-
4.2.1. Mutualistic microalgal-bacterial association
Mutualism is a type of biological interaction in which two or more
partners of different species benefit each other. Table 4 illustrates one
important example of mutualistic relationship between physiologically
healthy cells of Emiliania huxleyi and Phaeobacter gallaeciensis.
Herein the microalga fixes carbon dioxide via photosynthesis and
produces cysteine, methionine and dimethyl sulphoniopropionate
(DMSP) [48,56]. The roseobacteria, Phaeobacter gallaeciensis traps
DMSP and produces growth promoting factors such as promoters and
antibiotic molecules to safeguard microalgae against other bacterial
pathogens [57]. In other example bacteria, Azospirillium sp. acted as
microalgae-growth promoting bacteria (MGPB) that stimulated the
polluted water purification capacity of microalgae Chlorella vulgaris
and C. sorokiniana [58]. In mutualistic biological interaction bacteria
stimulate microalgal growth by:-
(1) Crafting a favorable environment through eradication of excess of
photosynthetic oxygen and production of carbon dioxide;
(2) Providing vitamins (B12, B9, B7/H or B1), inorganic nutrients and
trace elements necessary for its growth (microalgae); and
(3) Creating growth promoting factors, chelators and plant growth
promoting hormones (Auxins, Indole acetic acid) [49].
Typically vast numbers of studies have been carried out that depict
that heterotrophic bacteria influence positively on microalgal growth,
biomass composition and other related processes such as flocculation
or cell aggregation [48,51]. Another study validated that ecologically
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Renewable and Sustainable Energy Reviews 80 (2017) 1089–1099
engineered microalgal-bacterial consortium would stimulate microal-
ga, Chlorella vulgaris lipid productivities and biomass through ex-
change of carbon with growth enhancing bacteria such as
Flavobacterium and Rhizobium [59]. Chlorella has been most widely
studied microalga in respect to its interactions with other micro-
organisms such as bacteria and viruses. Past studies by Cho, et. al
have shown that both Azospirillium sp. (MGPB, rhizosphere dwelling,
N2 fixing bacteria) and Bacillus sp. have been associated in growth
promotion of unicellular microalgae Chlorella vulgaris by influencing
the cell morphology, lipid content and pigment production [59]. The
lead author constructed artificial bacterial consortium and studied their
growth patterns and mechanism of interaction with algae. The re-
searchers studied cell growth on 10 days old BG-11 medium and
focused on understanding the specific growth rate, cell density, lipid
content and flocculation efficiency of axenic cultures of Chlorella
vulgaris, indicating that the bacteria influenced microalgal growth
and metabolism. They further studied the effect of dominant bacterial
strains that were able to enhance the growth of microalgae while some
bacterial strains proved to be algicidal as they inhibited the growth of
alga e.g. Exophiala strain. The microalgae growth promotion in
artificial microalgal-bacterial consortium might be because of the
elimination of harmful growth inhibiting bacteria as well as the
synergistic effect of all growth promoting dominant bacteria on
microalgae [59,60]. The underlying mechanism behind the change in
microalgae physiology upon its cultivation with bacteria, mutually
affecting growth and physiology of each other was studied. The BG-
11 medium used to grow microalgae is devoid of an external carbon
source, thus unable to support any bacterial growth. The study revealed
that the selective invasion of growth promoting bacteria in phycosphere
of microalgae will result in an increase in biomass and productivity of
microalgae [59]. The microalgal associated bacteria play a crucial part
in the flocculation of Chlorella vulgaris by increasing the floc size and
thus causing easy sedimentation of microalgae [61]. This was con-
firmed by denaturing gradient gel electrophoresis (DGGE) analysis of
16S rRNA gene of xenic Chlorella vulgaris culture which revealed the
presence of bacteria such as Flavobacterium sp., Terrimonas sp.,
Sphingobacterium sp., Rhizobium sp. and Hyphomonas sp. as the
bacteria associated with microalgae. The Rhizobium sp., is part of a
community of bacteria that enhance and promote the growth of
microalgae and are commonly referred to as plant growth promoting
rhizobacteria (PGPR) [62]. In the presence of bacteria the flocculation
activity of microalgae culture was confirmed to be increased to 94% and
this was decreased to 3% when these bacteria were eliminated from the
microalgae by the use of fluorescence activated cell sorter (FACS). The
bacterial extracellular substances might be the reasons behind the
increasing flocculation of the microalgae. The axenic and xenic culture
of C. vulgaris when examined under the microscope had different sizes,
the size of axenic flocs was calculated about 20 µm or less., However,
the size of xenic floc was calculated to be 100 µm or more that resulted
in higher sedimentation rates and thus high flocculating activity [61].
When pH was increased it conferred to provide a negative charge both
on microalgae and associated bacteria that resulted in more efficient
and effective aggregation during impact with flocculants. Similar
studies by other researchers have also been carried out to understand
the role of microalgal-bacterial association in flocculation. These
studies suggest the role of light and formation of transparent exopo-
lymer particles as the adhesive for particle clumping and production of
floc [63,64]. Guo et al. [65] discussed the relevance and importance of
microalgal-bacteria consortium in flocculation. The bacteria have a
direct role in flocculating the microalgae using the association between
them. The dominant bacterial groups consist of both hydrophobic and
hydrophilic cells. The hydrophobicity is mostly consisted of the extra
cellular protein cause of the inactive hydrophilicity of microbial
extracellular polysaccharide. The Flavobacterium sp. among other
bacteria was identified as the most significant (by FACS and DGGE
analysis) floc-forming bacteria.
P. Tandon, Q. Jin Renewable and Sustainable Energy Reviews 80 (2017) 1089–1099

Table 4
Examples of microalgae-bacteria interactions having positive, neutral and negative effects on microalgal growth and accumulation of valuable compounds.
Source: adapted from [48].

Microalga Bacterium intermediaries from Microalgae intermediaries from Bacteria

mutualism Emiliania huxleyi Phaeobacter gallaeciensis Dimethylsulphonio-propionate Promoters and antibiotics

Botryococcus braunii Rhizobium sp. acyl-homoserine lactones (ACL; signal
molecule)
Lobomonas rostrata Mesorhizobium loti Vitamin B12
Scrippsiella trochoidea Marinobacter Organic molecules Vibrioferrin
Scrippsiella trochoidea Roseobacter Organic molecules Vibrioferrin
Neochloris oleoabundans Azotobacter vinelandii Siderophore
Scenedesmus sp. Azotobacter vinelandii Siderophore

Accumulation of fatty acids and lipids

Chlorella vulgaris Azospirillum brasilense Siderophore mediated nitrogen fixation

Heterotrophic accumulation of starch and carbohydrates

Chlorella vulgaris Azospirillum brasilense Siderophore mediated nitrogen fixation
Chlorella sorokiniana Azospirillum brasilense Siderophore mediated nitrogen fixation

Photoautotrophic accumulation of starch and carbohydrates

Chlorella vulgaris Azospirillum brasilense Siderophore mediated nitrogen fixation
Chlorella sorokiniana Azospirillum brasilense Siderophore mediated nitrogen fixation

commensalism Chlamydomonas reinhardtii heterotrophic bacteria organic carbon not used by bacteria Vitamin B12
Chlorella vulgaris Microbacterium sp. phycosphere/essential nutrients
Scenedesmus acutus heterotrophic bacteria organic carbon

Parasitism Chlorella vulgaris Exophiala sp. phycosphere/algicidal

Cochlodinium polykrikoides
Microcystis aeruginosa
Anabaena variabilis
Alexandrium tamarense
Micrococcus luteus SY-13 secondary metabolites/algicidal bacteria
Rhodococcus sp. KWR2. extracellular algicidal substance
Rhodococcus sp. KWR2. extracellular algicidal substance
Alteromonas sp. and Thalassobius phycosphere β-glucosidase and chitinase
aestuarii sp.

4.2.2. Commensalistic microalgal-bacterial association
Commensalism is a type of biological interaction that is strictly
neutral for one of the micro-organism and positively benefiting for the
other. Commensals basically consist of non-interacting partners.
However, the research studies suggest that there is a very thin line
difference between mutualism, commensalism and parasitism (dis-
cussed in detail in next section i.e. Section 4.2.3) in microalgae-
bacterial interactions. The shift in the mechanism arises mainly
because of change in environmental factors, describing these interac-
tions to be continual and not as discrete interface [47,66]. Table 4 also
illustrates an important example of commensalistic relationship be-
tween Chlamydomonas reinhardtii and heterotrophic bacteria, where-
in the microalga utilizes vitamin B12 (cobalamin; acts as cofactor for
B(12)-dependent methionine synthase enzyme) supplied by bacteria,
however the bacteria does not utilize the organic carbon released by
microalgae [53]. Another study demonstrated that the relationship
between heterotrophic bacteria and phytoplankton Scendesmus acutus
varies according to the balance of light and nutrients. When the
microalgae and bacteria were grown together in different combinations
of light and phosphorous (P) concentrations (high N: P ratio of 80:1).
The synthetic medium consisted of inorganic nutrients so that in this
case bacteria depend solely upon the organic matter excreted by
microalgae as a source of carbon (C). The microalgal densities were
low at low light intensity irrespective of P concentration. The bacteria
became C limited at low light intensity demonstrating a commensal
relationship between microalgae and bacteria [48,67]. However, when
the light intensity was increased at low P concentration still the
bacteria suffered from low organic carbon supply because of low
microalgal concentration. With medium supply of P and light inten-
sities, the bacterial growth was less because of limited supply of P
rather than C. In this case the supply of microalgal C exceeded P supply
comparative to bacterial demand. Herein increase in P concentration
released both microalgae and bacteria from P limitation. This demon-
strated competitive interaction for P between both species at medium
supply of P. The experimental results demonstrated that there is a
modification of interaction between commensalism for C and competi-
tion for P with change in nutrient concentration and light intensity [67]
for both microalgae and bacteria [68].
4.2.3. Parasitic microalgal-bacterial association
Parasitism is a type of biological interaction in which one partner
benefits at the expense of the other and exerts negative effects on it. In
most of the cases the parasite is minute in size and requires a living
host. However, microalgae are also parasitic mostly to taxa above them
or in few cases to their counterparts [47]. Nonetheless, 10% of known
species of multicellular red microalgae are parasites of the free living
red microalgae (their ancestors), and almost 80% of these share the
common ancestor with the hosts [69]. Only few studies lead to
understanding of microalgal parasitism on bacteria and vice-versa,
wherein microalgal/bacterial cell lysis occurs via a mechanism that is
parallel to plant-pathogen interaction. The enzymes such as chitinases,
glucosidases and cellulases that assist in breakdown of plant cell wall
are also involved in microalgal cell wall lysis [47,70]. Table 4 also
illustrates one important example of parasitic/ predatory relationship
between Chlorella vulgaris and Microbacterium sp./ Exophiala sp., a
growth inhibiting bacteria living together in ecologically engineered
bacterial consortium. The bacteria living in microalgal phycosphere
proved to be algicidal [59]. Another example is of a bacterium
Rhodococcus sp. KWR2. that inhibited the growth of Microcystis
aeruginosa and Anabaena variabilis, two harmful bloom forming
cyanobacteria by the release of extracellular algicidal substance by the
bacteria [71]. In parasitic microalgal-bacterial association, bacteria
inhibit microalgal growth by:-
(1) Killing microalgal cells via direct attack or indirectly via liberating
lytic compounds;
(2) Altering the microenvironment of phycosphere or in which micro-
algae live;

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P. Tandon, Q. Jin
(3) challenging microalgae for nutrients [49].
Even though, that bacterial parasitism of microalgal cells has not
been extensively studied still the role of algicidal bacteria is exclusively
recognized as one of strategic agents in the termination of harmful algal
blooms (HAB) in natural environments [72]. Bacteria interact actively
with HAB species and thereby grow in abundance after inhibiting them.
These types of interactions play no role in enhancement of microalgal
culture, however these interactions can be exploited to build artificially
engineered microalgal-bacterial mini ecosystem wherein microalgae
acts as parasite on bacteria and obtain nutrients from the latter for
growth.
Only a small fraction of information has been revealed till date to
recognize the chemistry behind the microalgae-bacteria association,
thus providing more scope for study in this field. Most of the studies
related to microalgal-bacterial interactions only discuss the microalgal
growth promotions, but not on the underlying role of microalgae in
these interactions [48]. The effect of certain species of bacteria on
growth and physiology of microalgae only have been studied. More
light needs to be shed upon phycosphere, the mini-ecosystem that
surrounds microalgal cell wall [48,51]. Ecologically engineering a
valuable microalgal-bacterium consortium will not only influence the
yield but also avoid regulatory bottlenecks in introducing genetically
modified organisms in commercial production. Moreover, the mini-
ecosystem in the consortium can be explored more extensively in
relation to its role in biogeochemical recycling of carbon and sulfur
(marine ecosystem; oxidizing greenhouse gas carbon monoxide),
bioremediation, in promoting microalgal growth (biofuel production,
formation of secondary metabolites) and inhibiting microalgal growth
(control of microalgal bloom, treatment of wastewater and sewage etc.).
For instance one of the grave concern related to large scale microalgal
biofuel production is whether this process can produce fuel economic-
ally? The most important nutrients required for microalgal growth are
nitrogen and phosphorous and their inputs in culture media increases
the cost. Mutualistic bacterial associations with microalgae have the
potential to fix or revive these vital elements for their microalgal hosts
and thereby lead to reduction in cost of nutrient input.
4.3. Role of genetic and metabolic engineering in improving the
quality of microalgae
Genetically modified micro-organisms are at present date used for
the creation and streamlining of endless products/processes (pharma-
ceutical agents, cosmetics, human therapeutics, probiotics, food pro-
ducts, etc.). Still the potential of the enormous existing expertise has to
be yet fully exploited. While decades of work on microbes have given
enough knowledge of the physiology and pathway engineering of
microbes, such type of information for microalgae also has to be
generated. Genetically modified microalgae that have the competence
to grow under harsh environmental conditions, high CO2 conditions
and able to produce high quantity lipid content will be of great use as a
future feedstock. Mostly the genetic improvements of microalgae are
engrossed in the creation of resources necessary for the pharmaceutical
(medical) and cosmetic fields [73].
Genetic and metabolic studies in microalgae are mostly associated
with the investigation of photosynthetic and metabolic pathways. Little
interest has been placed on the microalgal biology enhancement for
developing biofuels that has great potential for overwhelmingly influ-
encing the capacity of oil production. Genetic and metabolic engineer-
ing taken together are sure to have a paramount effect on enhancing
the economics of microalgal biofuel [12,14,74,75].
Certain parameters that need to be considered for improving the
overall quality of microalgae are listed below:-
a) Enhance the photosynthetic efficiency in order to increase the
biomass yield:
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b) Enhance the overall growth rate of biomass.
c) Enhance the oil content of the biomass so as to decease the external
inputs of nitrogen in growth medium.
d) Enhance the penetration of light into high density cultures of
microalgae for effective photosynthesis.
e) Enhance the carbon capture by the cell.
f) Enhance the quality of Carbonic Anhydrase (CA) to improve post-
harvest combustion of CO2 capture.
g) achieve a controlled autoflocculation of the biomass to expedite
recovery from water.
h) tolerate varying temperature range so as to decrease the cost of
cooling.
i) Grows even at increasing light level, thus does not saturate because
of light.
j) reduces the chance of photo-inhibition which actually reduces
growth rate.
k) decreases vulnerability to photo-oxidation, this actually damages
cells.
Most of the parameters listed above can be resolved to some extent
by using genetic and metabolic engineering, thus both of these
methodologies are crucial for the future expansion of viable microalgal
oil trade [12,76,77].
Genetic manipulations inside a microalgal cell would be easily
carried out as compared to crop plants due to absence of cell
differentiation and the absence of allelic genes (haploid nature of most
vegetative stages), [78]. Only within the last 10 years some work has
been carried out towards adopting this technology for improvements in
microalgae. Typically genomes of only some of the microalgae (eukar-
yotic) have been sequenced which critically delays their genetic and
metabolic engineering [79,80]. Till date more than 30 different
microalgae strains have been efficiently transformed genetically [81].
The effectiveness of transformation is specifically species dependent,
and the transformation method has to be optimized for each micro-
algae [81]. Most of the transformations both at the nuclear and
chloroplast level have been done using the green algae,
Chlamydomonas reinhardtii (model organism). The difficulties in
utilizing genetic engineering techniques are found in the low quantity
of gene expression and low growth rate [82] in microalgae. Thus the
main focus of the recent work is to enhance gene expression. Selectable
markers (based on bacterial antibiotic resistance genes or markers) are
hired for the selection of transforming cells in the culture medium in
order to promote growth. As microalgae have simple structures, thus
recombinant protein purification can be easily performed on them as
compared to the plants. They have faster growth rate than plants and
can achieve high cell densities under high light and aeration.
Microalgae especially the green algae are generally regarded as a safe
(GRAS) category. The estimates of costs of production of recombinant
antibodies are less for microalgae as compared to plants and animal
cells [83]. More microalgal genomic projects could be accelerated in the
near future in order to develop microalgae transformation methodol-
ogies. Some proof-of-concept advances that have already been made
after engineering microalgae are listed in the next Sections (4.3.1–
4.3.7).
4.3.1. Improving light supply to microalgal culture and its
photosynthetic efficiency
By enhancements in photosynthetic efficiency of terrestrial crop
plants substantially improved total biomass yield has been noted [84].
Similar enhancements are undoubtedly possible for microalgae too.
Some researchers have been successful in finding target genes for
improving photosynthetic efficiency of microalgae [76], while others
have reviewed the techniques of genetic engineering of microalgae [85].
Enhanced penetration of light into high density cultures of microalgae
for effective photosynthesis has proved to be possible with the
reduction of the chlorophyll antenna of chloroplast (light harvesting
P. Tandon, Q. Jin
cells) [12,86]. By antenna truncation the photosynthetic efficiency can
be improved up to 3-fold of the microalgal cells that contain chlor-
ophyll [87] but its use is limited (except marine microalgae and
microalgae without chlorophyll). Expanding the solar spectrum used
by photosynthesis i.e. absorbing range of visible light from 400 to
700 nm to infrared light of 700–750 nm will potentially increase the
number of photons required for photosynthesis by 19%. For example,
photosynthesis in case of cyanobacterium Acaryochloris marina that
contains only small amount of chlorophyll a but large amount of
chlorophyll d, the latter gives the cyanobacterium advantage of using
infrared light that cannot be absorbed by chlorophyll a [88] but can be
absorbed by chlorophyll d. Over expression of the photosystems can be
used as another prospective strategy for improving the light capture of
the cells [76].
4.3.2. Reducing photoinhibition
Reduction in microalgal biomass productivity is noticed in bright
sunlight, inducing photoinhibition of photosystem II and thereby
reducing consumption of incident light [89]. This photoinhibition can
be partly decreased by improving the pigment composition, enabling
the cell to produce entirely new or increasing the concentration of more
effective pigments and scavengers of reactive oxygen species (ROS)
[76].
4.3.3. Engineering Calvin cycle enzymes (RUBISCO)
The enzyme ribulose-1,5-bisphosphate carboxylase oxygenase
(RUBISCO) which is utilized for the carboxylation of ribulose-1,5-
bisphosphate (RuBP) in Calvin cycle has the low specific affinity for
CO2 and high affinity for oxygen which can be modified by the use of
molecular level engineering to high affinity for CO2 and low affinity for
oxygen [76]. Overexpression of RUBISCO helps to decrease photo-
respiration thus increasing the conversion of fixed carbon to CO2.
Similar molecular level engineering has to be carried out for the other
enzymes in Calvin cycle, preferably during downstream processes of
RUBISCO in order to improve photosynthetic efficiency [12].
4.3.4. Improving lipid production and biomass recovery
Bioprocess methods such as medium optimization (Section 4.1)
have thrown some light on improving the lipid productivity of micro-
algae by altering the composition of growth media (nutrition stress, low
nitrogen concentration) or environmental factors (change in tempera-
ture and CO2) but thereby decreasing biomass growth [33,37]. Other
methods are such as modifying biosynthetic pathway for lipid produc-
tion or engineering enzymes that are involved in lipid synthesis [90].
Boosting growth promoting bacteria in microalgal-bacterial associa-
tions (Section 4.2.1) have proved to increase the biomass productivity
and recovery, for example, flocculation of Chlorella vulgaris was
stimulated by bacteria such as Flavobacterium sp., Terrimonas sp.,
Sphingobacterium sp., Rhizobium sp. and Hyphomonas sp. by in-
creasing the floc size and thus causing easy sedimentation of micro-
algae [59,61]. Furthermore, engineering the auto-flocculation capacity
might also improve the recovery of microalgal cells from water.
4.3.5. Enhancing the quality of Carbonic Anhydrase (CA) to improve
post-harvest combustion of CO2 capture
Microalgae are unable to reproduce on exogenous glucose when
light is a limiting factor. They have to be genetically modified in order
to express glucose transporters to flourish in dark using glucose [91].
The CO2 capture and emission require the presence of an enzyme
Carbonic Anhydrase (CA; fast biocatalyst) that can accelerate the post
combustion capture of CO2. However, CA level is inadequate during
harsh environment conditions. The improvements in CA and the
processes using this enzyme will help in getting the required results
[75]. This includes sourcing CA from other organisms, immobilizing
the enzyme process (both for stabilization and restriction to the coolest
process zones) and utilizing protein engineering techniques to produce
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Renewable and Sustainable Energy Reviews 80 (2017) 1089–1099
thermo-tolerant enzymes.
4.3.6. Genetic modification of cyanobacteria and green algae
The green microalgae have been known to accrue lipids, specifically
triacylglycerols (TAGs), which is often used as the main precursor for
the production of lipids. The metabolic pathway study of green alga i.e.
Chlamydomonas reinhardtii, have explicated certain new metabolic
vital genes which have the capability to improve the lipid production in
the specific microalgae [92]. Lipid metabolism in microalgae is thought
to be complex and diverse, thus the engineering of strains producing
high lipid and moderating the specific pathways for the specific group
of algae is necessary [93]. Kyoto Encyclopedia of Genes and Genomes
(KEGG), [94] and the MetaCyc [95] are the chief resources used
specifically to trace the relevant information for metabolic engineering.
However, genetic modifications of cyanobacteria are much easier than
the eukaryotic microalgae and thus have attracted worldwide attention
for biofuel production. Certain species of cyanobacteria have the ability
to naturally fix nitrogen and grow in alkaline media. Utilizing genetic
engineered cyanobacteria for photosynthetic conversion of CO2 directly
to combustible diesel is discussed to be superior to the use of other
microalgae in similar processes [96].
4.3.7. Other opportunities for genetic/ metabolic engineering
Further advancement in microalgal biomass could be achieved by
using metabolic engineering. Microalgae have vast and differentiated
metabolism that can be easily cultured as single cells. They can be
easily used as a major target for metabolic engineering due to the
production of interesting metabolites [79]. However, use of this
technique in microalgae is said to be limited as specific transformation
tools have to be established for every species for any nuclear or
chloroplast genomes [97]. The metabolites obtained from them pro-
pose a great number of benefits over plants and other microorganisms
specifically in the use of inexpensive culture media. Transgene expres-
sion and protein localization (chloroplast) is necessary for efficient
functioning of many metabolic genes required for production of
biofuel. Mostly this technique is applied in microalgae for the synthesis
of vaccines, hormones or antibodies and recombinant proteins [73]. As
discussed earlier (Section 4.1), limiting nutrients also raises the level of
lipid droplets. While this strategy is very expensive, it is an easy
method where forwarding of metabolic flow occurs, leading to lipid
formation [92]. The major drawback of the latter is slow photosynthetic
activity and growth rate. Commercially algae have been used recently
as a good source for production of carotenoids and omega-3 fatty acid
[98]. At present less information is available about optimization of
carotenoid production using metabolic engineering. Nuclear or chlor-
oplast transformation techniques could be used for carotenoid meta-
bolic engineering as most of the carotenogenic pathways are present in
the chloroplast of the microalgae. The green alga C. reinhardtii has
been frequently used as a model organism for understanding the effect
of genetic engineering in carotenoid accumulation. Yet, carotenoid
metabolic engineering has not proven to be profitable. Maybe the
enzymes that are targeted don’t contain bottleneck stages, or there
consists of lot of rate limiting stages in the trail [97]. The concept of
tropic conversion where in heterotrophy can be carried out in other-
wise obligate phototrophic species has been achieved. For example, the
algae, Volvox carteri was the first green alga transformed with one of
hexose transporter from Chlorella kessleri (HUP1, monosaccharide-H
+ symporter), which helped the strain to last on glucose under dark
conditions [79,99]. However, the degree of conversion to full hetero-
trophy is inconsistent among different species notwithstanding the
glucose conversion. The advancement of a likely novel strain of
microalgae will need laborious changes of the main parent strain.
Different bottlenecks of the production of recombinant microalgae
could pave way to an efficient algal bioengineering only through a
combination of varied approaches. An improvement in this is imme-
diately required to obtain a specific and noteworthy breakthrough in
P. Tandon, Q. Jin
the production of green and sustainable biofuels from microalgae.
5. Conclusion: research needs, challenges and implication
Biofuels have the potential to efficiently substitute the nonrenew-
able fossil fuels. They can be made easily available, affordable,
accessible as per technology, are renewable and biodegradable, non-
toxic and produces low levels of particulates (carbon monoxide,
hydrocarbons) after combustion, easily transported and stored. They
are highly versatile clean energy fuels that can be directly used in
engines, providing both socio-economic and environmental benefits.
The economics of producing microalgal biofuel need to be enriched
extensively in order to decrease its production cost and henceforth
compete with fossil fuels. For example, employing microalgal biomass
as a substitute to coal-firing flue gas in power plants to generate
electricity will prove to be a cost-effective approach and will in turn
reduce CO2 emission to atmosphere. The problematic situation of low
microalgae biomass concentration still exists in most of the microalgae
culture methods, leading to its inefficiency and reduced production, low
oil content, expensive fertilizer utilization, high water-resource utiliza-
tion and higher harvesting costs. While the microalgal biology en-
hancement for developing biofuels has been under represented in
research, it constitutes great potential for influencing the capacity of oil
production and other processes. In order to meet the requirements of
the biomass production a wide variety of novel genetic tools, genome
sequences and manipulation of metabolic pathways are required.
Moreover, a combination of genetic and metabolic engineering meth-
ods might also be exploited in order to obtain a desirable output of
increased growth rate of biomass, lipid productivity, photosynthesis
efficiency, carbon capture etc. Bioengineering of a mini-ecosystem in a
microalgal-bacterial consortium consisting of nitrogen-fixing plant
growth promoting bacterial species (e.g. Roseobacter-microalgae asso-
ciation; heterobacteria-microalgae) can collectively mark an impact to
the global carbon cycle (role in biogeochemical recycling of carbon and
sulfur), decrease the release of greenhouse gases to the environment,
bioremediation, lower the microalgal production cost by minimizing
the input of nutrients in growth medium and aid in faster recovery. The
photosynthetic efficiency and microalgal density can be increased by
the use of metabolic engineering and genetic engineering and by
understanding the microalgal biochemistry. All these improvements
might lead to the production of fourth generation biofuels that has the
capability to produce imperative sources of renewable fuel that will
never compete with food production, available fresh water, or even the
cultivable land.
Acknowledgement
This study was conducted by the financial support from National
Natural Science Foundation of China (21476139).
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