Academic Editor: Ganesh D. Saratale
P. subcordiformis is a potentially promising species with commercial,
environmental and technological viability for industrial applications. The great
potential of these microalgae lies in their fast biomass growth, pollution
resistance, and compatibility with different culture media. This study aimed to
determine the efficiency of P. subcordiformis biomass production in a
medium prepared with water from the Bay of Gdańsk. The tested medium
supported high biomass growth rates which reached 317.58
Pushing forward the development and widespread take-up of environmentally-friendly, efficient and cost-effective biomass production technologies represents an ongoing challenge to scientists, as well as a priority issue for operators of energy systems . There have been numerous attempts to demonstrate that typical land energy crops can be used for this purpose [2, 3]. However, the use of such plants requires appropriate agrotechnical procedures, marginal/degraded soils, natural fertilizers, as well as species of energy crops that must be carefully selected, fast-growing and resistant to environmental conditions .
However, this prevailing view has been contested by some opinions and publications. Life cycle analyses (LCA) have shown that mismanagement of traditional energy crop reserves may actually exacerbate greenhouse gas emissions . There is an increasingly common view that intensive use of farmland for growing biofuel crops may lead to decreased global food supply and a significant positive pressure on food prices . Such risks and limitations have also been acknowledged by European Union (EU) legislators. Reports on ILUC (Indirect Land Use Change Impacts), commissioned by the European Commission, call into question the environmental viability of biofuels produced from grain, root/tuber crops or oil crops . As such, the feasibility of using food sources for biofuel production is an increasingly common thread in discussions at the EU level. It is generally assumed that these goals are to be achieved, e.g., by harnessing advanced biofuels made from alternative feedstocks .
Therefore, there is a real need to seek alternative sources of biomass, ones
which would be both commercially and environmentally viable as a source of
energy. Algae possess very high photosynthetic efficiency, can rapidly build
biomass, are resistant to various contaminants and can be sited on land that is
unsuitable for other purposes. Given these considerations, algae may represent a
viable substitute for traditional energy cops. Algae also appear on the European
list of feedstocks which, when used for biofuel production, can be double-counted
for the purposes of determining biofuel energy content [9, 10]. Microalgae biomass
is also a rich source of carbohydrates, proteins, fatty acids, enzymes and
fibers. In addition, algae are rich in vitamins and minerals, including vitamins
A, C, B
A key factor, which in many cases determines the cost-effectiveness of energy production from algal biomass, is the choice of technology used to grow and separate the organisms . Algae can be cultivated using various methods, beginning from strictly monitored methods in technologically advanced designs, to less predictable methods based on open systems . Apart from the technology used to produce the biomass, the type and, most importantly – cost of the growth medium used also play a huge role . Therefore, there have been attempts to identify relatively cheap methods which would harness various types of sewage, leachate, industrial effluent or waste water . Use of natural water as growth medium is a novel area of research that very few papers have dealt with so far . The vast majority of the data available pertain to producing microalgal biomass on media prepared from distilled water and chemical reagents that provide optimal conditions for microalgal growth [23, 24].
One promising species, which may be commercially, environmentally and technologically viable, is P. subcordiformis. Some literature reports show that these microorganisms can be a source of value-added substances such as sugars, proteins or fats, given optimal culture conditions . The great potential of P. subcordiformis algae lies in their fast biomass growth, resistance to various types of pollution, high adaptability, and compatibility with different culture media of various physico-chemical parameters . There is also a fast-expanding body of research on using P. subcordiformis planctonic algae by switching their metabolism towards hydrogen production [27, 28].
This study aimed to determine the efficiency of biomass production on a mixotrophic P. subcordiformis culture. The medium was prepared with Bay of Gdańsk water as the base.
The study was separated into two series with the growth medium as the differentiating factor. Series 1 (S1) utilized deionized water, whereas series 2 (S2) used water sourced from the Bay of Gdańsk. Each of the series was divided into two variants. In variant 1 (V1), the medium was supplemented exclusively with microelements required for optimal growth of P. subcordiformis. In the second variant (V2), the medium was amended with glucose as an external carbon source to expedite the growth of the microalgal population.
The study used halophilous microalgae of the species P. subcordiformis,
sourced from the UTEX collection (The University of Texas at Austin). The
P. subcordiformis inoculum was transferred to the agar medium in a 20
The profile of the microalgal biomass is presented in Table 2. Initial
microalgal biomass with dry organic matter levels (VS) and chlorophyll levels
during the exact experiment were kept at 200 mg
|Mineral solids (MS)||% TS||73.97 |
|Volatile solids (VS)||% TS||26.03 |
|Total nitrogen (TN)||mg/g VS||5.32 |
|Total organic carbon (TOC)||mg/g VS||50.74 |
|Total carbon (TC)||mg/g VS||54.34 |
|Protein||mg/g VS||33.25 |
|Lipids||% TS||18.0 |
|C:N ratio||–||10.21 |
The study used growth media with different profiles depending on the experimental series. In series 1 (control), the medium was primarily composed of deionized water supplemented with trace element-rich sea salt (Aqua Medic Reef Salt) to ensure that salinity levels were close to 30 ppt.
Series 2, on the other hand, utilized water collected from May to September from
the Bay of Gdańsk. Before being used to grow P. subcrodiformis, it
was first filtered through Eurochem medium-grade Ø 12.5 qualitative filter
paper, then sterilized in a Tuttnauer 2840 EL-D autoclave at 121
|Parameter||Unit||Bay of Gdańsk||Series 1 (S1)||Series 2 (S2)|
|Variant 1 (V1)||Variant 2 (V2)||Variant 1 (V1)||Variant 2 (V2)|
|Ammonia nitrogen||mg NH
|Total nitrogen||mg TN/dm
|Total phosphorus||mg TP/dm
|Chlorine (I)||mg Cl
|Chlorine (II)||mg Cl
|Iron (II)||mg Fe
|Iron (III)||mg Fe
The exact experiment was conducted in a New Brunswick BioFlo 115 bioreactor with
an active volume of 2.0 dm
The taxonomic analysis of the algal biomass was done using an MF 346 biological microscope with an Optech 3MP camera. The study included a qualitative analysis of the microalgal biomass used as inoculum in the reactors. Chlorophyll was determined spectrophotometrically after extraction with 90% acetone. The TS, VS, and MS were measured gravimetrically. Total protein content was estimated by multiplying TN by a protein conversion factor of 6.25. Lipids were determined using the Soxhlet method with a Buchi extraction apparatus. The pH determination procedure was as follows: 10 g of the homogenized air-dried sample was weighed out to a 100 mL beaker; 50 mL of distilled water was added; the mixture was then stirred; and the pH of the resultant sample was finally measured with a calibrated apparatus. Total carbon (TC), total organic carbon (TOC), and total nitrogen (TN) were determined using a Shimadzu TOC analyzer.
The water collected from the Bay of Gdańsk for growing P. subcordiformis was tested for salinity, dissolved oxygen, and pH using a Hach Lange HQ 440D multi-parameter meter and an Aqua Medic Marine Control Digital salinity meter. Total nitrogen, ammonia nitrogen, total phosphorus, orthophosphates, sulphates, chlorine compounds, iron compounds, and COD were determined using Hach Lange cuvette tests and an UV/VIS DR 5000 spectrophotometer. The same methodology was applied to monitor the levels ofessential nutrients in the culture, i.e., TN and TP. Light intensity was measured with a HANNA HI 97500 luxometer. Glucose concentration in the growth medium was monitored using a YSI 2700 Select analyzer.
Each experimental variant was conducted in triplicate. The statistical analysis
was conducted and determination coefficient R
The biomass concentration in S1V1 was 3203.3
Trends in VS concentration in the growth medium.
Trends in chlorophyll a levels.
Trends in TN concentration in the growth medium.
Trends in TP concentration in the growth medium.
TN take-up per specific gain of P. subcordiformis VS.
TP take-up per specific gain of P. subcordiformis VS.
Efficiency of TN removal by P. subcordiformis.
|Experimental series/variant||Nutrient take-up per specific gain of P. subcordiformis VS|
|Experimental series/variant||Efficiency of nutrient removal by P. subcordiformis|
|Experimental series/variant||VS and chlorophyll in growth medium at the end of the growth period|
|* – values in italics are significant at p |
The r value in S2V1 was 317.58
Efficiency of TP removal by P. subcordiformis.
Changes in glucose levels in the growth medium.
Use of natural water as growth medium is a novel area of research that very few papers have dealt with so far . Most publications focus on producing P. subcordiformis biomass on media prepared from distilled water and chemical reagents that provide optimal conditions for microalgal growth .
Water from Bay of Gdańsk was chosen in order to determine whether natural
water is a viable medium for high-volume production of P. subcordiformis
biomass. The final concentration was 3493.3
The study found that the waters from the coastal zone of the Baltic Sea were too
nutrient-deficient to support rapid growth of P. subcordiformis biomass. Therefore, the medium had to be supplemented with nitrogen and
phosphorus from external sources. Ran et al.  reported similar findings
after growing P. subcordiformis in a medium prepared with water from the
southern part of Bohai Bay (China). This led to the conclusion that a
satisfactory rate of algal growth can only be achieved with micronutrient
supplementation. The final biomass levels in the study ranged from 1.85 to
There are also literature reports confirming that Platymonas sp.
microalgae can be grown in a blend of municipal sewage and industrial (textile)
effluent . The chlorophyll levels were found to be 2.8
Most studies use closed culture systems due to the need to monitor and culture
multiple process parameters that directly affect microalgal growth in hydrogen
production systems . Most literature reports focus on PBRs with active
volumes of 250-5000 cm
In the present study, we also utilized a bioreactor capable of monitoring the
process parameters important for micoalgal cultivation, and grew a culture of
P. subcordiformis. The final biomass concentration exceeded 3200
Xie et al.  also found decreased chlorophyll content in P.
subcordiformis cultures stimulated by added glucose. The chlorophyll content in
the photoautotrophic culture (without external carbon source) was, on average,
twice as high than in the mixotrophic culture with added glucose . A similar
trend was observed by Faraloni et al.  in a Chlamydomonas
reinhardtii culture grown in a medium made from wastewater rich in organic acids
and carbohydrates. Chlorophyll content was 25 mg/dm
Fluctuations in the level of photosynthetic pigments can occur in response to changes in microbial metabolism in photoautotrophic and mixotrophic conditions. In the autotrophic culture, microalgal growth is fuelled directly by photosynthesis (which in turn is driven by light energy). In mixotrophic environments, an additional source of energy is provided in the form of an external carbon substrate (easily degradable organic compounds). However, this additional energy source inhibits photosynthesis, which stimulates the formation of pigments in microalgae such as P. subcordiformis .
Growing P. subcordiformis in water sourced from the Bay of Gdańsk led to high biomass growth rates, comparable with those observed when using growth media based on deionized water and chemical reagents. It was found that water sourced from the coastal zone of the Baltic Sea was too nutrient-deficient to support rapid growth of P. subcordiformis biomass, therefore, the medium had to be supplemented with nitrogen and phosphorus from external sources. Amendment of the growth medium with glucose significantly limited chlorophyll production in microalgal cells, thus resulting in lower chlorophyll levels than in the variants with no external carbon source. The glucose-amended variants had significantly higher nitrogen and phosphorus levels in the medium at the end of the growth period. The use of mixotrophic culture allowed to obtain significantly higher efficiency of P. subcordiformis culture grow characterized by the final concentration of dry organic matter. The use of natural sea waters has been proven to be justified from a technological and economic point of view.
MDu and MDę designed the research study. MDu and AN performed the research. MZ provided help and advice. MDu, MDę and MZ analyzed the data. MDę and JK wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.
The manuscript was supported by Project financially supported by Minister of Education and Science in the range of the program entitled “Regional Initiative of Excellence” for the years 2019–2022, project no. 010/RID/2018/19, amount of funding: 12,000,000 PLN, and the work WZ/WB-IIŚ/2/2019, funded by the Minister of Education and Science.
The authors declare no conflict of interest.