New Biotechnology  Volume 00, Number 00  February 2015

RESEARCH PAPER

Jingjing Lia, Ying Liua, Jay J. Chenga,b, Michal Mosc and Maurycy Daroch

Research Paper

Biological potential of microalgae in China for biorefinery-based production of biofuels and high value compounds a

Q1 a

School of Environment and Energy, Peking University Shenzhen Graduate School, Shenzhen 518055, China Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC 27695, USA c Energene sp. z o.o., ul Wroblewskiego 38A, Lodz 93-578, Poland b

Microalgae abundance and diversity in China shows promise for identifying suitable strains for developing algal biorefinery. Numerous strains of microalgae have already been assessed as feedstocks for bioethanol and biodiesel production, but commercial scale algal biofuel production is yet to be demonstrated, most likely due to huge energy costs associated with algae cultivation, harvesting and processing. Biorefining, integrated processes for the conversion of biomass into a variety of products, can improve the prospects of microalgal biofuels by combining them with the production of high value coproducts. Numerous microalgal strains in China have been identified as producers of various high value by-products with wide application in the medicine, food, and cosmetics industries. This paper reviews microalgae resources in China and their potential in producing liquid biofuels (bioethanol and biodiesel) and high value products in an integrated biorefinery approach. Implementation of a ‘high value product first’ principle should make the integrated process of fuels and chemicals production economically feasible and will ensure that public and private interest in the development of microalgal biotechnology is maintained. Introduction This work introduces China’s biological microalgal resources Q2 which could be used for biorefinery-based production of high value products and biofuels in an integrated approach. Expansion of world population and rapid industrialisation of many countries have caused a serious depletion of fossil fuel resources. According to the latest British Petroleum (BP) Statistical Review of World Energy 2014, world oil consumption grew above 1.4 million barrels per day (b/d), from 89.931 million b/d in 2012 to 91.331 million b/d in 2013, while proven oil reserves were estimated as 1687.9 billion barrels at the end of 2013. These reserves are sufficient to meet 53.3 years of global production assuming current consumption trends [1]. These trends however are likely to change with increased fuel consumption in fast-developing Asia. In addition, greenhouse gases (CO2) [2,3], noxious gases (SO2, NO2) [4], Corresponding author: Daroch, M. ([email protected]) http://dx.doi.org/10.1016/j.nbt.2015.02.001 1871-6784/ß 2015 Published by Elsevier B.V.

and PM 2.5 [5] emitted by fossil fuel combustion are the main causes of environmental problems like global warming, acid rain, and haze. These serious environmental issues produce additional indirect effects that are externalised to society with costs that are rarely accounted for. All of these issues render fossil fuels as unsustainable energy resources. Excessive dependence on fossil fuels not only brings serious environmental costs but also causes wars and conflicts because of the pressure for securing crude oil supply resulting from unbalanced global distribution of these resources. Biofuels have long been expected to at least partially relieve these problems and steadily provide energy for more sustainable development of societies worldwide. Biofuels contain energy derived directly or indirectly from geologically recent carbon fixation in living organisms, that makes them nearly carbon neutral; they also have lower environmental impact and emission profiles than fossil fuels [6–8]. Two of the most dominant biofuels, bioethanol and biodiesel, www.elsevier.com/locate/nbt

Please cite this article in press as: Li, J. et al., Biological potential of microalgae in China for biorefinery-based production of biofuels and high value compounds, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.02.001

1

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New Biotechnology  Volume 00, Number 00  February 2015

Research Paper

are seen as substitutes for the petroleum-derived fuels, diesel and gasoline. To date, three generations of crops have been proposed as feedstocks for biofuel production. First generation crops used for biofuel production are corn, soybean, and sugarcane – edible crops with high sugar or oil content. Second generation crops include dedicated lignocellulose crops grown on marginal lands like switchgrass, poplar, or Miscanthus. Third generation feedstocks are microalgae and other aquatic organisms, mostly unicellular or simple multi-cellular, that exhibit a variety of metabolisms, predominantly autotrophic but also facultative or obligate heterotrophic [9]. High growth rates reported for many microalgae [10–12] make them a promising source of biomass that could produce higher amount of biomass per hectare than most terrestrial plants can. Moreover, some microalgae species such as Dunaliella salina [13] are able to grow well in saline water, which makes them more promising feedstock than terrestrial crops that rely exclusively on fresh water. Despite these advantages, technical and economic bottlenecks in cultivation, harvesting, dewatering, and energy conversion efficiency are still main barriers which hinder commercialisation of algal biofuels [14]. To date, neither isolating new strains nor optimising known cultivation and harvesting techniques have been able to successfully demonstrate cost effective production of biofuels from microalgae. In order to make microalgae a viable biofuel feedstock, significant advances need to be made in cost reduction through technological breakthroughs and/or integrating multiple processes. Biorefining, integrated processes for the conversion of biomass into a variety of products, can improve the prospects of microalgal biofuels by combining them with the production of high value coproducts such as polyunsaturated fatty acids like DHA or EPA, carotenoids, antioxidants, proteins and pharmaceuticals [15,16].

The biorefinery approach will help to close the economic gap between current costs of microalgae cultivation and processing and costs of fossil fuels. China, the world’s most populated country and third largest economy after EU and US, has faced an enormous energy shortage in recent years. In 2013, China remained the largest energy consumer while its oil reserves account for 1.1% of world’s reserves [1]. Releasing the potential of biofuels in China is therefore essential for maintaining its continuous development. China also suffers a shortage of arable land and freshwater resources. Therefore development of biofuels should exclusively focus on second and third generation feedstocks. This paper reviews microalgae resources in China and their potential in producing liquid biofuels (bioethanol and biodiesel) and high value products in an integrated biorefinery approach.

Microalgae resources in China Biodiversity of microalgae in China China’s huge area, complex topography, diversity of climates, and aquatic habitats results in a great diversity of microalgae resources that can be explored. Microalgae are widely distributed in freshwater lakes, rivers, and coastal seawater. Bundles of literature have reported the abundance and distribution of microalgae in various water bodies in China, including South China Sea [17,18], Pearl River Delta [19,20], Bohai Sea [21,22], among others. According to a survey of the phytoplankton community of central Bohai and its adjacent waters [22], approximately 432 species of phytoplankton, dominated by diatom and dinoflagellates, have been found. In the Pearl River estuary [23] there were 239 species identified, among which 72.4% belong to diatom, 23.8% to Pyrrophyta, and 3.8% to others. Table 1 summarises phytoplankton diversity and abundance in a typical water body

TABLE 1

Phytoplankton diversity in typical water area in China. Area

Number of phytoplankton species

Reported highest abundance

Changjiang estuary

208

1277.88  103 cells/dm3 4

3

Normalised abundance (103 cells/L)

Dominant species

Reference

1277.88

Chaetoceros, Coscinodiscus, Thalassiosira

[84]

Pearl river

239

2.767.1  10 cells/m

27

Thalassiothrix, frauenfeldii, Nitzschia delicatissima Thalassiosira subtilis

[23]

Yellow river

114

303.35  104 cells/m3

3.03

Bacillariophyta, Chlorophyta

[85]

4

3

Bohai

432

535.45  10 cells/m

5.3

Coscinodiscus, Excentricus, Ceratium fusus

[22]

South China Sea

150

Summer: 6001.78  103 cells/dm3 Winter: 37.52  103 cells/dm3

Summer: 6001.78 Winter: 37.52

Trichodesmium thiebautii, Thalassionema nitzschioides Pseudo-nitzschia delicatissima, Gymnodinium spp. Thalassionemafrauenfeldii, Chaetoceros messanensis

[86]

East China Sea

144

1158.6  103 cells/dm3

1158.6

Bacillariophyta, Pyrrophyta

[87]

Yellow Sea

379

Summer: 4137.1  103 cells/m3 Spring: 3940.4  103 cells/m3 Winter: 3010.6  103 cells/m3 Autumn: 340.8  103 cells/m3

Summer: 4.137 Spring: 3.940 Winter: 3.010 Autumn: 0.34

Spring: Thalassiosira pacifica, Skeletoema spp., Chaetoceros cinctus Summer: Chaetoceros debbilis, Chaetoceros pseudocurvisetus, Chaetoceros curvisetus Autumn: Thalassiosira curviseriata, Alexandrium catenella, Ceratium fusus Winter: Paralia sulcata, Phaeocystis sp., Bacillaria paradoxa

[88]

2

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NBT 756 1–9 New Biotechnology  Volume 00, Number 00  February 2015

Microalgae resources for bioethanol production Microalgae have been considered as promising candidates for fermentative production of alcohols, mainly ethanol, because of their cell wall composition and ability to accumulate starch reserves. Unlike those of terrestrial crops, microalgal cell walls are not reinforced with lignin, an aromatic polymer that makes hydrolysis of lignocellulose problematic [25]. Instead, cell walls are largely composed of hemicellulose and to a lesser extent cellulose, both of which can provide valuable sugars for fermentation. Most important, however, many microalgae store their reserves in starch which can be easily converted to simple sugars with mild enzymatic treatment [25]. Starch is currently used as a primary source of carbohydrates in edible and fuel alcohol industries, therefore hydrolysates of algal starch could easily fit in current ethanol production processes without any modifications. Numerous strains of microalgae in China have been assessed as feedstocks for bioethanol production. Species like Mychonastes sp., Chlorella sp., and Scenedesmus sp. have been successfully utilised so far. For example the microalgae strain Chlorella vulgaris FSP-E isolated in Taiwan, China was reported to produce 54.13% (dry weight) carbohydrate under nitrogen-deficient conditions [26] and proved to be a good feedstock for bioethanol fermentation. Another indigenous strain from the Huanghai Sea, Tetraselmis subcordiformis, exhibited starch content of 58.2% and starch productivity of 0.62 g/L/d [27]. The productivity of the strain could reach 1.32 g/L/d under optimised semi-continuous cultivation conditions [28]. Microalgae resources of the Pearl River Delta were also explored for bioethanol fermentation. Two strains, Mychonastes afer PKUAC 9 and Scenedesmus abundans PKUAC 12, were used as a feedstock, and maximal productivity of 0.10 gEtOH/galgae was obtained for the latter strain after fermentation with Saccharomyces cerevisiae [29]. Review of the available literature suggests that very often yeast strains that are not capable of utilising the hemicellulose cell walls of microalgae are used for bioethanol

production. This causes a decrease in ethanol yield [30–32] that usually does not exceed 0.3 gEtOH/galgae [31,33]. These problems can be overcome with application of pentose utilising strains of S. cerevisiae or pentose fermenters like (Escherichia coli KO11 or Zymomonas mobilis) [25].

Microalgae resources for biodiesel production Biodiesel is defined as methyl or ethyl esters of fatty acids obtained by transeserification of triglycerides derived from renewable feedstocks. In recent years microalgae drew attention of researchers as a source of oil, because of their high growth rate, oil accumulation ability, and no direct competition with food crops [34,35]. Research on microalgae resources for biodiesel production in China showed an abundance of species capable of producing high content of lipids composed of favourable monounsaturated fatty acids (Table 2). Oil content of these strains is within the range of 23–58% often under optimal culture conditions without any environmental stress. Application of various stresses can increase triglyceride content in microalgae [36]. These stresses include osmotic, radiation, temperature, pH, heavy metals, and nutrient (e.g. nitrogen, phosphorus) starvation. Nitrogen starvation is the most widely applied method to increase lipid content in most microalgae species. Nitrogen starved cultures divert their cellular carbon flux from the synthesis of proteins and carbohydrates to the synthesis of storage materials like triglycerides. Depending on the strain, the treatment can result in an increase of overall lipid productivity [37] or in its decrease; as higher oil content in nutrient-starved biomass does not compensate for the lower growth rate and biomass concentration [38]. These responses are speciesand condition-specific and should be tested for each strain and cultivation mode. Moreover, various species and even strains of microalgae exhibit different lipid profiles, and these profiles can vary as a response to environmental conditions. All these factors should be taken into consideration when selecting strains for biodiesel production, as the biofuel needs to meet certain quality standards like EN 14214 in the EU, ASTM D6751 in the US or GB/T 30828-2007 in China. Many algal strains of Chinese origin have been found suitable for biodiesel production. The native microalgae strain Nannochloropsis sp. isolated from the coast of Qingdao showed an optimal lipid profile for biodiesel production, containing fatty acids with carbon chain length from 14 to 20 and low degree of unsaturation. A two-step acid-alkali conversion resulted in FAME yield of 70.4%, and physicochemical parameters of purified biodiesel conformed to the Chinese National Standards [39]. Many studies have focused on improving efficiency of extraction and conversion of algal lipids to biodiesel. Cheng et al. produced crude biodiesel with 86.74% FAME content from Chlorella pyrenoidosa in a chloroform-free system. In this process, hexane was used as an extraction solvent to ensure that polar pigments were not present in crude biodiesel, leading to a decrease in nitrogen content [40]. Valuable pigments could then be recovered with alternative treatment to increase the profitability of the biodiesel production process. Other studies focused on an in situ transesterification method that integrates oil extraction and conversion into one step, reducing energy demand and simplifying the entire process. Various co-solvents have been tested to improve the efficiency of the single-step reaction. The toluene/methanol co-solvent system (2/1 volume ratio) maximised biodiesel yield

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Research Paper

in China. Bacillariophyta dominated most aquatic habitats followed by Chlorophyta. An abundance of microalgae varies with different geographical locations and seasons. A greater variety of microalgae resources were found in southern China (e.g. Guangdong and Hainan) because of the warm climate, higher photosynthetic activity and abundant water sources. Numerous algae culture collections have been established in China to collect and protect genetic materials in danger of extinction. Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB-collection) in Wuhan is the main freshwater microalgae culture collection, which holds more than 2000 strains belonging to over 120 genera. It also represents China in the World Data Centre for Microorganisms (WDCM, http://www.wdcm.org/) and Network of Asia Oceania Algae Culture Collections (AOACC, http://mcc.nies.go.jp/AOACC/Home.html). The Marine Biological Culture Collection Centre (MBCC) in Qingdao collected more than 600 strains of marine microalgae resources, 70% of which are oil-producing species [24]. Also there are culture collections at individual universities, such as Jinan University in Guangzhou, National Taiwan Ocean University, Guangdong Ocean University, and Peking University Graduate School, Shenzhen. Many of these collections were screened to select promising algae strains for biofuel production.

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New Biotechnology  Volume 00, Number 00  February 2015

TABLE 2

Lipid profiles of microalgal feedstocks for biodiesel production in China.

Research Paper

Microalgae

Location

Habitat

Lipid content (% DW)

SFA (% TFA)

MUFA (% TFA)

PUFA (% TFA)

References

Desmodesmus spp. F1

Taiwan

Freshwater

52.91

29.07

38.65

31.00

[89]

Desmodesmus spp. F2

Taiwan

Freshwater

58.51

27.06

41.12

28.64

[89]

Desmodesmus spp. F5

Taiwan

Freshwater

36.78

35.78

37.60

25.23

[89]

Desmodesmus spp. F18

Taiwan

Freshwater

52.55

26.02

36.99

35.16

[89]

Monoraphidium sp. FXY-10

Yunnan

Freshwater

56.80

23.80

ND

68.00

[90]

Mychonastes afer HSO-3-1

Shandong

Freshwater

51.9

20.80

67.24

11.96

[61]

Ankistrodesmus sp. CJ02

Hainan

Freshwater

26.86

26.78

53.42

19.81

[91]

Ankistrodesmus. gracilis CJ09

Hainan

Freshwater

47.90

37.31

58.12

4.57

[91]

Teranephris brasiliensis DL12

Hainan

Freshwater

27.22

28.29

62.28

9.44

[91]

Ankistrodesmus. gracilis DL25

Hainan

Freshwater

30.66

43.92

54.19

1.90

[91]

Desmodesmus. subspicatus WC01

Hainan

Freshwater

31.10

31.28

49.13

19.60

[91]

Desmodesmus sp. WC08

Hainan

Marine

31.30

33.13

64.21

2.67

[91]

Chlorella vulgaris CJ15

Hainan

Marine

47.39

35.60

39.98

24.42

[91]

Chlorella sorokiniana XS04

Hainan

Marine

36.75

38.00

35.54

26.46

[91]

Chlorella sp. PKUAC 102

Guangdong

Marine

52.80

30.50

28.30

41.20

[92]

Chlorella sp. NJ-18

Hubei

ND

25.03

30.53

17.30

52.17

[93]

Coelastrum sp. HA-1

Tianjin

Marine

43.20

30.13

62.84

7.03

[58]

Shizochytrium sp. PKU#Mn4

Guangdong

Marine

51.50

55.00

0.40

44.60

[94]

Aurantiochytrium sp. PKU#Sed1

Guangdong

Marine

37.90

57.80

0.30

41.90

[94]

Thraustochytrium sp. PKU#SW1

Guangdong

Marine

23.00

63.90

5.20

30.90

[94]

SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; ND, No data.

from Spirulina powder over two reaction cycles [41]. Butanone as a co-solvent was also able to increase the transesterification efficiency [42]. Alternative methods to increase the rate of transesterification reaction were also tested and found promising in lowering energy input required for transesterification. Microwave irradiation was identified as a promising alternative to conventional heating. Combining microwave irradiation with a single-step method increased the biodiesel production rate and yield from C. pyrenoidosa 6-fold and 1.3-fold over a two-step process and conventional heating, respectively [43]. Further efforts on increasing biodiesel yield from microalgae are important for future developments of algal biodiesel.

Valuable compounds production As mentioned previously, valuable compounds are essential in making algal biofuels more cost competitive and to promote further development of algal biotechnology. There is a whole array of products that could be co-produced with biofuels in microalgae. These include polyunsaturated fatty acids (PUFAs), carotenoids, polysaccharides, proteins, and many other compounds with pharmacological or nutraceutical activities.

Polyunsaturated fatty acids (PUFAs) PUFAs are fatty acids containing more than one double bond that have proved to be essential for human health. Two highly valuable PUFAs, DHA (C22:6) and EPA (C20:5), are produced by microalgae. Docosahexaenoic acid (DHA) is a primary structural component of human brain, cerebral cortex, skin, and retina [44]. Eicosapentaenoic acid (EPA), a precursor of DHA, was found to have clinical 4

significance against valproate (VPA)-induced hepatic dysfunction, necrosis, and steatosis [45]. Although both fatty acids can be synthesised by humans from alpha-linolenic acid, the slow rate of synthesis cannot fulfil the demand of the organism. Increased awareness and understanding of the importance of PUFAs in the human diet prompted the search for sustainable sources of these valuable compounds. Autotrophic and heterotrophic microalgae that inhabit the oceans are original producers of these PUFAs in the food chain and have recently attracted the attention of researchers and entrepreneurs alike. New market opportunities have opened for various PUFA containing products. For example, the retail price of DHA supplements are within the range of 10– 15 s/30 capsules containing 0.2 g DHA each, which translates to the retail price of 1700–2500 s/kg of DHA. This could easily offset the high cost of DHA-producing microalgae. Numerous microalgae species in China have been reported to produce high PUFA contents, such as Arthrospira [46], Spirulina platensis [47], Chlorella sp., Nannochloropsis oculata, Nannochloropsis sp., Isochrysis galbana, Phaeodactylum tricornutum and Chaetoceros calcitrans [48]. Liu et al. reported four strains of Aurantiochytrium sp. and Schizochytrium sp. having DHA content between 27% and 44% of total fatty acids [49]. Large-scale production of such microalgae could help to kick start biorefining processes for simultaneous production of DHA and biofuels.

Carotenoids and chlorophylls Microalgae are also producers of important cellular pigments: carotenoids and chlorophylls. These pigments can be successfully applied in food, cosmetics, and pharmaceutical industries.

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Carotenoids are organic pigments including astaxanthin, xanthophylls, zeaxanthin, canthaxanthin, echinenone, and b-carotene. These pigments play an important role for plants and algae, as they absorb excessive light energy and protect chlorophyll from photodamage [50]. In humans, carotenoids serve as antioxidants [51] and precursors of Vitamin A [52]. D. salina, a green unicellular marine halophilic algae grown in sea salt fields, is known for its ability to produce b-carotene up to 14% of dry cell weight under optimised culture conditions [53]. Another pigment, chlorophyll, is the main pigment responsible for capturing light photons by photosynthetic organisms. Most green microalgae like Chlorella, Chlamydomonas, and Ulothrix are therefore abundant in chlorophyll [54]. Chlorophyll derivatives such as sodium copper chlorophyllin are valuable natural colourants used by the textile, food and paper industries [55,56]. Like other natural dyes, chlorophyll is safer and healthier than synthetic alternatives, which is especially important for the food industry. Market price chlorophyllin is about 150 s/kg and therefore is able to offset a considerable amount of the cost of algal biofuel production. Freshwater microalgae, Haematococcus pluvialis, is the major producer of astaxanthin [57]. Under conditions of stress it produces 2–3% (dry weight) of astaxanthin, much higher than yeast Xanthophyllomyces dendrorhous (0.1–0.4% of dry cell weight) or which is present in typical crustacean oils [57]. The price of natural astaxanthin exceeds 5000 s/kg and can easily justify high costs of H. pluvialis cultivation. Other microalgae have also been reported to produce considerable amounts of astaxanthin. For example Coelastrum sp. HA1 isolated from Bohai Bay in China was able to yield 168.9 mg/m2/ d astaxanthin and 18.0 g/m2/d of lipids after 24 hours of cultivation. Additionally reported strains can be harvested by simple sedimentation, achieving 98.2% removal efficiency after settling for 24 hours [58], therefore decreasing energy costs related to harvesting.

Other valuable metabolites Other possible high value co-products from microalgae include polysaccharides, proteins, and other compounds with pharmacological or antioxidant activities. To date, antitumor, antiviral, anticoagulant, and antihyperlipidemia functionalities have been discovered in algae. Polysaccharides of Schizochytrium sp. TIO 1101 showed multiple activities: antioxidant, DPPH radical and hydroxyl radical scavenging [59]. Antioxidant properties were also detected in microwave degraded polysaccharides from other alga Porphyridium cruentum [60]. Various pharmacological activities – anticancer, immunomodularion, and hepatoprotective activities – were found in phycobiliproteins of Porphyridium spp. and S. platensis [53]. Nervonic acid, an important compound in the biosynthesis of nerve cell myelin, was first detected in the Chinese native microalgae strain M. afer HSO-3-1 and may become a valuable source of this pharmaceutical substance [61]. Antimicrobial activity was detected in pressurised liquid extracts obtained from D. salina. Suspected compounds responsible for this activity were beta-cyclocitral, alpha- and beta-ionone, neophytadiene and phytol, all previously identified as antimicrobial agents [13]. The actual commercialisation potential of many of these compounds remains to be determined but there is clear evidence that many of these compounds can achieve high market prices due to their unique properties. There are still many more biotechnologically

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active metabolites in microalgae to be discovered and analysed, as functional activities of many algal metabolites are still unknown. Further studies should be carried out to verify the function of specific compounds and to effectively transfer this research into practice.

Microalgae as cell factories for protein production Production of high value proteins such as antibodies or vaccines in autotrophically cultivated microalgae is a promising method to convert waste CO2 into a high value product [62]. There are several advantages of using microalgae as microbial cell factories over traditional fermentation methods. First and foremost there is a significant potential for the cost reduction. Although currently prohibitively expensive for biofuel production, microalgae cultivation is relatively cheap when compared with the cultivation of many bacterial, yeast and especially mammalian cell cultures. One of the most important features of microalgae is the flexibility of their metabolism towards various sources of carbon and nutrients. This flexibility could be explored to minimise the costs of protein production. Most microalgae can be grown autotrophically, heterotrophically or mixtrophically which opens several possibilities for using them as microbial cell factories. For example, during fully autotrophic cultivation algae can utilise CO2 from industrial sources and grow in a simple inorganic medium. Energy to maintain the cellular processes will be provided by photosynthesis. In the presence of additional reduced carbon source microalgae can switch their metabolism to mixotrophy. Under this metabolism microalgae can utilise two sources of carbon and energy: from photosynthesis and respiration enhancing their growth and resultant target protein productivity. The reduced carbon source could be obtained from waste streams of food industry or any other source that would fit a particular algal strain. Second, microalgaeproduced proteins are not susceptible to viral or prion contamination that could harm humans [62]. This is especially important for therapeutic proteins and a significant concern when using mammalian culture systems [62]. Third, eukaryotic microalgae possess cellular machinery such as chaperones that can process complex mammalian proteins, and ability to introduce post-translational modifications [62]. These features can significantly expand the portfolio of proteins that can be successfully produced in microalgae when compared with other unicellular organisms. Fourth, compared with plant-based systems for protein expression that have similar advantages to those of microalgae, life cycles of microalgae are much shorter. Once appropriate microalgae strain has been selected as a host for protein production, the subsequent steps, that is transformation, screening of transformants and culture scale up can be done much quicker than it is possible for vascular plant systems like tobacco [62]. Last but not least many microalgal strains are considered as GRAS organisms, that is generally regarded as safe, what can simplify the downstream processing of protein products. Analysis of current developments in using algae as microbial cell factories yielded moderate success, and further improvements in protein yield is required to make significant impact as high value products. To date, various, even complex, proteins have been expressed in microalgae [63–65]. Among these, several high value proteins should be emphasised: VEGF (vascular endothelial growth factor), HMGB1 (high mobility group protein B1) [66]

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and M-SAA (mammary-associated serum amyloid) [67]. Similar studies have been conducted in China where allophycocyanin [68] and TRIAL (tumour necrosis factor-related apoptosis-inducing ligand) [69] have been successfully expressed in Chlamydomonas reinhardtii. Many studies have also focused on expression of antigens for recombinant or edible vaccines [70–73]. Large-scale production of these compounds could easily offset the high cost of algal biomass production. Expression of recombinant proteins in algal chloroplast have been successfully performed for many proteins, and levels of 0.5% of total soluble protein are often obtained [67,74] but nuclear expression of foreign proteins results in considerably lower yields [75]. Chloroplast expression has its advantages over nuclear one such as: generally higher expression levels due to lack of gene silencing mechanisms, transgene containment, and possibility of integrating entire cassettes in a prokaryotic-like manner [62]. These advantages come at the expense of more limited post-translational modifications than those of proteins expressed from the nucleus. Despite numerous advantages protein production in microalgae suffers from smaller yields than those obtained with alternative organisms such as bacteria, yeast, mammalian cells and vascular plants. There are several reasons why current yields of expressed proteins are lower than these of alternative hosts. The list includes, but is not limited to: gene silencing mechanisms (particularly strong for nuclear constructs), lack of strong inducible promoters that would work over broad range of hosts, codon usage incompatibility, and significant effects of introns on nuclear expression levels. Over the years several strategies have been developed to get around these problems [62], however in order to fully utilise the potential of microalgae as microbial cell factories for protein production fundamental advances in understanding of the molecular mechanisms of protein production in microalgae are required. Once the comprehensive understanding of protein synthesis regulation is achieved, then the improvement of target protein yields can have a significant impact on the development of algal microbial cell factories.

Algal biorefineries – towards increased economic and environmental sustainability Cultivation of microalgae for the sole production of biofuel is not economically feasible even at high oil prices due to large energy costs associated with algae cultivation, harvesting, and processing [76]. These costs are also higher than those for terrestrial biomass [77]. High-energy consumption also results in poor energy return on investment (EROI) from current microalgae to biofuels processes [78,79]. Proposed increased competitiveness of microalgal biofuels at even higher prices than fossil fuels is not likely to materialise. The energy input used for algae processing comes directly or indirectly from fossil fuels, therefore rise of these prices will directly translate to the increase of the price of algal biofuel. Until breakthrough technologies in algae cultivation, dewatering, and harvesting are developed and implemented integration of biofuel production with high value co-products in an integrated system can provide a transition solution that will ensure that public and private interest (and investment) in microalgal biotechnology is maintained. Simultaneous production of multiple compounds from microalgae is the solution that will allow further development of microalgal technologies that will hopefully result 6

New Biotechnology  Volume 00, Number 00  February 2015

in an economically and environmentally feasible process of converting microalgae to biofuels. High value products such as PUFAs, antioxidants, colourants, proteins, and other compounds can be used to offset the high cost of producing biofuels and to generate multiple products. These products can be oriented at different markets depending on the chosen microalgal strain. For the successful implementation of this scheme the selection of microalgal strains for biofuel production should take into consideration multiple products from the initial stages of the screening process (Fig. 1). When searching for a promising microalgal strain from culture collections or environmental isolates three aspects need to be taken into consideration. The first stages of screening organisms for biofuel production should focus on various high value compounds such as PUFAs, carotenoids or ability to express heterologous proteins. The secondary screening should focus on the potential of a given strain for biofuel production, that is the ability to produce carbohydrates or lipids that could become feedstocks for biofuel production. Tertiary screening should look at selecting strains with optimal resource use efficiency, such as inexpensive growth medium, possibility of using recycled nutrients, and CO2 from flue gasses, among others. All products (at least one high value product and one biofuel product) should be produced in a biorefinery system to maximise financial and environmental profits resulting from integration. The scheme of a proposed biorefinery to produce at least one high value product and one biofuel is presented in Fig. 2. First, key assumptions regarding proposed processes need to be made. These include site analysis and selecting the most suitable combination of strains and their products for a particular location. Several site specific factors need to be considered: water supply and characteristics, land topography, climate conditions, access to nutrients, and carbon sources [80]. Second, an algae cultivation system needs to be selected. Algae cultivation systems can be broadly divided into two categories: open and closed. Open systems offer lower capital and operating costs at the expense of control of the cultivation conditions. A detailed explanation of differences between these two modes has been extensively reviewed on numerous occasions [81–83] and is beyond the scope of this article. Algae cultivation requires numerous inputs such as water, carbon, nutrients, light, and energy that should be optimised for each cultivation process and recycled if possible to minimise the environmental impact of the process. Harvested biomass should be separated into two fractions: the high value product and residual feedstock for biofuel production, such as carbohydrates or lipids. The high value product should be purified to meet appropriate quality standards, and residual biomass could then be used for biodiesel or bioethanol (or other alcohol) production depending on the character of residual biomass. Post-conversion residues, glycerol from biodiesel production, and residual algal and yeast biomass from bioethanol production should then be explored for recycling their nutrients (nitrogen, phosphorus and trace elements) and carbon (e.g. CO2 from yeast fermentation) back to algae cultivation. Final residues could be explored as animal feed if they still present sufficient nutritional value or converted into energy by anaerobic digestion or combustion to generate at least some of the energy for algae cultivation, harvesting, and processing.

www.elsevier.com/locate/nbt Please cite this article in press as: Li, J. et al., Biological potential of microalgae in China for biorefinery-based production of biofuels and high value compounds, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.02.001

NBT 756 1–9 New Biotechnology  Volume 00, Number 00  February 2015

Environmental isolates from preselected sites

Secondary screening

• PUFAs • Carotenoids and other pigments • Pharmaceucal funconalies (ancancer, anmicrobial etc.) • Proteins of high commercial value

• high content of carbohydrates (starch) • high content of lipids (triglycerides)

Profiling and selecon for high value compounds

Profiling and selecon as biofuel feedstock

Terary screening • Inexpensive growth medium components • Growth on recycled nutrients • Carbon ulisaon efficiency • Culture stability

Profiling and selecon for opmal resource ulisaon

FIGURE 1

Scheme of microalgae screening process for biorefinery producing high value compounds and biofuels.

Strain, site, products selecon

Residual products

Carbon

Light

Culvaon

Conversion of residues

Nutrients

Water

Harvesng

Biomass processing (dewatering, drying, fraconaon)

Energy

Residues

Carbohydrate or lipid-rich biomass

Biofuel product

High value product

FIGURE 2

Scheme of microalgal biorefinery producing one high value compound and biofuel. Following annotations are used: solid lines – subsequent steps; double lines – final products; double arrows – inputs; dotted lines – opportunities for recycling resources.

Conclusions Microalgae abundance and diversity in China show promise of identifying suitable strains for the development of algal biorefinery. Implementation of ‘high value product first’ principle should make the integrated process economically feasible even at current costs of algae cultivation. The capacity of microalgae in producing various high value by-products with wide application in medicine, food and cosmetic industries can significantly improve the

prospects of algal biofuel production. Development of methods that combine high yield production of desired metabolites with their purification and conversion of resulting biomass to biofuels is important for the development of algal biotechnologies. Finding the appropriate combination of strains and methods that will allow simultaneous extraction of high value products from biomass and its pre-treatment for biofuel production may be challenging but will be more than offset by overall improvements of

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Research Paper

Primary screening

Microalgae resources

Culture collecons

RESEARCH PAPER

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economy of the algae to biofuels technology. These can provide a catalyst for the development of large-scale biofuel technologies that will not compete for land or freshwater resources.

Acknowledgements Q3 The authors would like to thank National Natural Science

Foundation of China for Young International Scientists

New Biotechnology  Volume 00, Number 00  February 2015

Grant no. 31450110424 and Shenzhen Municipal Government for Special Innovation Fund for Shenzhen Overseas High-level Personnel KQCX201405211502553 to MD. The authors greatly appreciate the help of an editor Priscilla L. Young for carefully editing our manuscript. All authors contributed to the preparation of the manuscript.

Q4 References Research Paper

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Research Paper

New Biotechnology  Volume 00, Number 00  February 2015