Polder Tawang Semarang : Study Case of Biotechnological

Application and Waste Water treatment as part of Integrated

Coastal Management
Hermin Pancasakti Kusumaningrum1), Muhammad Zainuri2), Muhammad Helmi2), Hadi
Endrawati2)
1)
Department of Biology, Faculty of Mathematics and Natural Sciences,
2)
Department of Oceanography, Faculty of Fisheries and Marine Sciences,
Diponegoro University.Jl. Prof. Soedarto, SH., Tembalang, Semarang. 50275. Indonesia.
e-mail : gandasakti@yahoo.com
Abstract
Microalgae are microscopic plants that convert solar energy and CO2 into O2
and carbohydrates, then used to synthesize all other biomass constituents.
Contaminant microalgae act as an eutrophication and pollution controlling agent for
the removal of P and N. Polder Tawang Semarang was the ideal example for an
open source outdoor pond of natural microalgae cultures cultivation. It was a subject
and model for biotechnological application and waste water treatment to reduce
pollution and eutrophication marked by blooming of microalgae. Biotechnology
application was used to product biofertilizer from nitrogen-fixing microalgae
contaminant. Cyanobacteria (blue-green algae) have been studied as possible
biofertilizers because of the ability to fix atmospheric N under anaerobic conditions.
The aim of this study was to investigate the potential of contaminant microalgae from
Polder Tawang Semarang as biofertilizers as an initiative study for exploiting their
innate potentials and pollution management. The research result shows that
application of microalgae contaminant from Polder Tawang Semarang in the plant as
biofertilizers has yielded satisfactory results comparing to controlled experiments.

INTRODUCTION

Microalgae including cyanobacteria, was an oxygenic photosynthetic

organisms, largely contribute to the balance between CO2 and O2 in the
atmosphere. They adapted to a wide range of environmental conditions,
including extreme ones, also colonise most aquatic and terrestrial
ecosystems. For the last decades, the occurrence of waterblooms of
planktonic microalgae greatly increased in continental aquatic ecosystems as
a consequence of pollution generated by humans. These proliferations disrupt
ecosystem equilibrium and may be harmful to animals and human due to the
large number of secondary metabolites (hepato- and/or neurotoxins) some
cyanobacteria may produce. In the environment, planktonic cyanobacteria of
the genus Microcystis form waterblooms potentially harmful to animals and
human.

Cyanobacteria, or blue-green algae, include edaphic and filamentous

species also capable in biological nitrogen fixation. Microalgae like

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cyanobacteria (blue-green algae) are capable of both carbon assimilation and
N2 fixation, thereby enhancing productivity in variety of environments. Apart
from fixing atmospheric N2, they secrete a number of biologically active
substances. Tropical conditions such as those in Indonesia provide favourable
environment for the luxuriant growth of these organisms in the natural
ecosystems such as different types of soil, freshwater bodies, oceans, saline
backwaters, estuaries, and also hyper saline saltpans (Benemann, 2002).
Besides their ecological significance, microalgae offer a great potential tool as
an organisms for the biotechnological interest such as marineculture, food,
feed, fuel, fertilizer, medicine and combating pollution (Venkataraman 1981,
Borowitzka, 1988 ). With the advent use of a microalgal biomass, it is of
interest to investigate whether from blooming of contaminant microalgae are
feasible for use as cyanobacterial biofertilizers. Microalgae can synthesize
and operate the nitrogenase complex in oxygenated surroundings because
they derive energy for growth and nitrogen fixation from sunlight. Therefore,
heterocystous cyanobacteria are of interest as biofertilizers (Reynaud and
Metting, 1988). Biofertilizer is defined as inoculant containing active material
of living microorganisms which functions to fix a particular nutrient and
facilitate the availability of soil nutrients to plants. The present work was
carried out to understand the use of cyanobacteria from Polder Tawang
Semarang for biofertilizer.

MATERIAL AND METHODS

Polder Tawang Semarang is located in the north of Semarang. Polder
Tawang Semarang occupies about 3,205 sq miles [8,300 sq km] in area in
the part of north Coastal region of Central Java. Polder Tawang serve as
outdoor open ponds. Water and microalgal samples were collected aseptically
from Polder Tawang ponds during June – October 2008 on a sterile Duran
bottle 500 ml.
Microalgal flocculants and biomass to make biofertilizers were collected
by manual filtration using bamboo net (mesh size 1000 µm). The liquid
biomass were dried in the temperature room until constant weight. Mixed
microalgal biomass about 500 g were added to the plant. Water samples were
also taken from each site for analyzing physico-chemical and biological

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parameters such as pH, dissolved oxygen, salinity by using standard methods
(APHA, 1975). Microalgae specimens were identified microbiologically the
publications of Tze (1987). 1932. Photomicrography was taken using digital
camera on microscope (Germany). The correlation co-efficient analysis was
made between physico-chemical properties of water and total cyanobacterial
species.

RESULTS AND DISCUSSION

According to microscopic visualization and analysis, as per the diversity
and abundance of microalgae in Polder Tawang Semarang, Microcystis was
dominating in Polder Tawang Semarang (Fig 1). Low diversity of microalgae
was attributed to a massive bloom of Microcystis. Low amount of dissolved
oxygen (0.814 mS/cm) had a significant effect in reducing the other
cyanobacterial population. Microcystis is one of the dominant organisms that
is associated with almost permanent blooms in tropical freshwaters that are
exposed to constant sunshine, warmth, and nutrients like phosphate, silicate,
nitrate, CO2 and lime. Formation of cyanobacterial blooms in freshwater
bodies as illustrated in Fig.2. is essentially due to buoyant nature of these
organisms. Buoyancy of cyanobacteria is imported by the gas vacuoles which
forms dense growth on the water surface in ponds, reservoirs and lakes and
cause serious nuisance because of visual appearance, production of toxins
(Carmichael, 1994) and unpleasant odour produced by substances such as
geosmin (Baudin et al., 2006).

(a) (b) (c)

Fig. 1. Microscopic appearance of contaminat microalgae on Polder Tawang
Semarang (a. 100x, b. 400x, c. 1000x)

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Fig. 2. Blooming of microalgal on Polder Tawang Semarang

Physico-chemical analysis of in Polder Tawang Semarang water

revealed that the pH was 7.0, dissolved oxygen was 0.814 mS/cm, salinity
was 0.03 and temperature was warm 30.1 oC. The correlation co-efficient
analysis of physico-chemical properties of water samples and total
cyanobacterial species revealed the significant positive correlation between
Total Cyanobacterial Species (TCS) and dissolved oxygen (r=0.8; p<0.01).
Climate change and warm temperature have connection with the proliferation
of cyanobacteria blooms. Warming trends lead to stablization of thermal
layers in water, which inhibits vertical circulation and thus lowers dissolved
oxygen in the bottom layer of water.
Studies on the microalgal biodiversity of Polder Tawang Semarang
during July until October 2008 has been made. Although a massive bloom of
Microcystis in Polder Tawang had a significant effect in reducing the other
cyanobacterial population, in any ecosystem, not a single species grows
independently and indefinitely. All the species are interlinked and has cyclic
transformation of nutrients. The physicochemical changes in the environment
may affect particular species and induce the growth and abundance of other
species, which leads to the succession of several species in a course of time.
When microalgae are associated to bacteria on the environment (non axenic),
an interaction happens, which might be good for both; in a way that the
microalga is able to assimilate products of the bacterial activity in the media.
Likewise, the associated microbial flora is implied on the regulation of
physiologic parameters such as pH, temperature and salinity (Moronta et al.,
2006). Some bacteria can enhance bioremediation of wastewater by
microalgae by increasing microalgal proliferation and metabolism, allowing the

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microalgae to clean the water better than when used alone. Azospirillum and
cyanobacteria species may improve mangrove reforestation by increasing the
rate of survival and development of seedlings in an otherwise unfavorable
environment.
The association ability between contaminant microalgae on Polder
Tawang Semarang then was biotechnologically applied to use as biofertilizers
to improve their potential. This application marked successfully result as we
can see on Fig 3. comparing to the control.

(a) (b)
Fig. 3. Application of Polder Tawang contaminant microalgae as biofertilizers
(a) control without adding microalgae (b) after addition of microalgae

Application of biotechnology to Polder Tawang microalgae contaminant

was using microalgae as free-living cyanobacteria community that under
some conditions, are beneficial for plants. They will promote plant growth in
two different ways: (1) They directly affect the metabolism of the plants by
providing substances that are usually in short supply, as listed in Table 1.
These cyanobacteria are capable of fixing atmospheric nitrogen, of
solubilizing phosphorus and iron, and of producing plant hormones, such as
auxins, gibberelins, cytokinins, and ethylene. Additionally, they improve a
plant's tolerance to stresses, such as drought, high salinity, metal toxicity, and
pesticide load. One or more of these mechanisms may contribute to the
increases obtained in plant growth and development that are higher than
normal for plants grown under standard cultivation conditions.

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 Table 1. Nutritive component of microalgae
No Component amount
Chlorella sp. Dunaliella sp. Polder Tawang
microalgae
1 Protein 31.57 31.44 46.2
2 Lipid 8.49 7.86 3.8
3 Carbohydrate and others 15.36 11.05 31.7 (crude)
4 Ash 37.18 36.86 33.2
5 Water 7.40 7.42 7.8
6 Laurat Acid 1.625 1.614 46.2
7 Miristat Acid 4.227 3.958
8 Palmitat acid 35.287 31.584
9 Palmitaleat acid 35.287 31.584
10 Stearat acid 18.004 17.855
11 Oleat acid 6.489 6.247
12 Linoleat acid 13.885 12.986
13 Linooleat acid 0.658 0.559
14 Fikosa Penta Enoit acid 2.876 2.776
15 Dokosa Hexa Enoit acid 1.596 1.468

Some cyanobacteria also play indirectly promote plant growth by

preventing the deleterious effects of phytopathogenic microorganisms
(bacteria, fungi, and viruses). They produce substances that harm or inhibit
other microbes, but not plants, by limiting the availability of iron to pathogens
or by altering the metabolism of the host plant to increase itsesistance to
pathogen infection. Biocontrol cynobacteria may also fix nitrogen or produce
phytohormones. These advantages make microalgal contaminant was
potential and suitable as biofertilizers, therefore reducing environmental
pollution and can help managing waste water treatment . In the next future,
fundamental and applied research with cyanobacterial biofertilizer
technologies will focused on flooded rice cultivation for which there is some
evidence that agronomically significant quantities of crop available N are
provided.

CONCLUSION AND PROSPECTS

The present study concluded inspite of the fact that cyanobacteria
Microcystis are ubiquitous in Polder Tawang Semarang , their population
dynamics are often influenced by the available nutrients and the physico-
chemical conditions of the ecosystem. Biotechnological application of
microalgae contaminant from Polder Tawang Semarang in the plant as

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biofertilizers or plant growth-promoting agent has yielded satisfactory results
comparing to controlled experiments. Cyanobacteria promoting plant use
multiple mechanisms to promote plant growth, or mechanisms such as
nitrogen-fixation. Hopefully, the results will be promising under applications
on agricultural conditions. The public will be more sympathetic to the concept
of cyanobacteria inoculants. In the near future, cyanobacteria growth-
promoting agent will be part of solutions to agricultural and environmental
problems.
.
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_______________, H.P. E. Kusdiyantini , T. Yuwono, dan J. Soedarsono,

2006b. Determination of a Green Algae Species From Jepara,
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Production. Jour. Coastal Dev. Oktober (10) 1: 33-46

______________, H.P. dan J. Soedarsono, 2006c. Molecular Determination

of Dunaliella sp. to detecting 1-Deoxy-D-Xylulose-5-phosphate
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Solo. 26-27 Agustus 2006

Kusumaningrum , H.P. J. Soedarsono, dan E. Kusdiyantini. 2006d. Molecular

Characterization of A Green Algae Isolate by 16S rRNA in
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Chlorella sorokiniana to pH, salinity and temperature in axenic and non
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on temperate irrigated soils in Washington State USA. Biological
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(1979). Generic assignments, strain histories and properties of pure
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Sze, P. 1993. A Biology of The Algae. 2nd Ed. Wm.C. Brown. p:5–80.

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Maret 2003.

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Metting, B. (1981). The systematics and ecology of soil algae. Botanical
Review, 47, 196-312.

Metting, B. (1986). Population dynamics of Chlamydomonas and its influence

on soil aggregate stabilization in the field. Applied & Environmental
Microbiology 51, 1161-1 164.

_______, B. (1987). Dynamics of wet and dry aggregate stability from a three-
year microalgal soil conditioning experiment in the field. Soil Science,
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In Algae and Human Affairs (C.A. Lembi & R.A. Waaland, eds.), pp.
335-370 Cambridge University Press; Cambridge.

Results and Discussion

9
 ..
Cyanobacteria (blue-green algae) have been studied as possible
biofertilizers for rice cultivation
because of the abilify dfheterocystous species to fix atmospheric N
under aerobic conditions.

Recent studies have shown that eukaryotic single-celled microalgae under

development as biological soil conditioners will colonize moist temperate soils
when large inocula can be applied (Metting, 1986 & 1987).
Metting, B. (1981). The systematics and ecology of soil algae. Botanical
Review, 47, 196-312.
Metting, B. (1986). Population dynamics of Chlamydomonas saja0 and its
influence on soil
aggregate stabilization in the field. Applied & Environmental Microbiology
51, 1161-1 164.
Metting, B. (1987). Dynamics of wet and dry aggregate stability from a three-
year microalgal soil
conditioning experiment in the field. Soil Science, 143, 139-143.
Metting, B., Rayburn, W.R. & Reynaud, P.A. (1988). Algae and agriculture. In
Algae and Human
Affairs (C.A. Lembi & R.A. Waaland, eds.), pp. 335-370 Cambridge University
Press; Cambridge.

In outdoor cultures algae are exposed to highly variable, often extreme,

environments, in particular for light. Settling by spontaneous bioflocculation is
one potential low-cost process. once the algal biomass has been produced
with high yield and harvested with great efficiency, that is at low overall cost.
Although open ponds can be of low cost, these, and the supporting systems (e.g.CO2
injection) have yet to be demonstrated at the large scales required.
High levels of nitrogen source in the environment is also eliminating
heterocystous forms, since nitrogen free media is commonly used for the
isolation and purification of heterocystous cyanobacteria. The significant
positive correlation between the cyanobacterial diversity and micronutrients
(zinc and nitrite) was observed and also reported by Govindasamy & Azaraiah
(1999). In the present study the significant positive correlation was observed
between the Total Cyanobacterial Species (TCS) and dissolved oxygen
(r=0.9803; p<0.01), TCS and bicarbonate (r= 0.9928; p<0.01) and TCS and
carbonate (r=0.941; p<0.05).

The following pictures are of partner project sites where phosphate levels
have been high enough in recent years to nourish a bloom of cyanobacteria.

10
Most of the blooms have been of anabaena or microcystis species. Both are
photosynthetic and nitrogen-fixing, and tend to form mats on the water’s
surface. Microcystis is pictured below. mats of cyanobacteria on the surface
of the water. mats of cyanobacteria on the surface of the water. n Southeast
Asia, the benefits of cyanobacteria are also being studied. Floating ferns that
house colonies of Anabaena species in their fronds have been introduced into
paddies; the cyanobacteria serves as an inexpensive nitrogen-fixing fertilizer
for rice cultivation. In several countries, students are also studying the anti-
cancer substances in various cyanobacteria species and writing grant
proposals for community harvesting of cyanobacteria for medicinal research
purposes. This research will be extended to include other partners through
iEARN. Students are working on a revised protocol for phosphate monitoring,
and reviewing research on the effect of nitrates on phosphate levels. They
are also studying the connections between climate change and the
proliferation of cyanobacteria blooms. Early research indicates that warming
trends lead to stablization of thermal layers in water, which inhibits vertical
circulation and thus lowers dissolved oxygen in the bottom layer of water.
This causes phosphates to be liberated from sediments. Phosphates are
key nutrients for plants and animals, including humans. They are also
pollutants which reach the environment through use of phosphate detergents,
fertilizers and pesticides. In Seattle, the Billings Middle School study of
phosphates began in 2003 when nearby Green Lake was closed to the public
due to a bloom of cyanobacteria. Cyanobacteria are nourished by phosphate
pollution, and under certain conditions produce neurotoxins which may cause
illness or even death. The bloom occluded and warmed the lake, thus killing
fish and other aquatic fauna. The smell was offensive, causing some area
residents to experience nausea, headaches, and/or difficulty breathing.
Contaminated water also caused skin irritations. The dominant forms of
cyanobacteria were Anabaena species, pictured below. Rebecca Timson.
Role of Schools in Sustainable Development.
http://www.unescobkk.org/fileadmin/user_upload/apeid/Conference/papers/timson_6B.doc.

Cyanobacteria, oxygenic photosynthetic procaryotes, largely contribute to the

balance between CO2 and O2 in the atmosphere. Adapted to a wide range of
environmental conditions, including extreme ones, they colonise most aquatic

11
and terrestrial ecosystems. For the last decades, the occurrence of
waterblooms of planktonic cyanobacteria greatly increased in continental
aquatic ecosystems as a consequence of pollution generated by humans.
These proliferations disrupt ecosystem equilibrium and may be harmful to
animals and Man due to the large number of secondary metabolites (hepato-
and/or neurotoxins) some cyanobacteria may produce. n the environment,
planktonic cyanobacteria of the genus Microcystis form waterblooms
potentially harmful to animals and Man.
In order to elucidate the developmental cycle of these microorganisms and
their acclimation capabilities to variations in environmental parameters, we
have chosen the hepatotoxic strain Microcystis aeruginosa PCC 7806 as a
model system.
The algae are a polyphyletic, artificial assemblage of O2-evolving,
photosynthetic organisms (and secondarily nonphotosynthetic
evolutionary descendants) that includes seaweeds (macroalgae) and a
highly diverse group of microorganisms known as microalgae.
Phycology, the study of algae, developed historically as a discipline
focused on the morphological, physiological and ecological similarities
of the subject organisms, including the prokaryotic bluegreen algae
(cyanobacteria) and prochlorophytes. Eukaryotic algal groups represent
at least five distinct evolutionary lineages, some of which include
protists traditionally recognized as fungi and protozoa. Ubiquitous in
marine, freshwater and terrestrial habitats and possessing broad
biochemical diversity, the number of algal species has been estimated
at between one and ten million, most of which are microalgae. The
implied biochemical diversity is the basis for many biotechnological and
industrial applications.
F B Metting Jr.J. of Industrial Microb and Biotech, Springer
berlin/Heidelberg, Vol 17 Numbers 5-6/Nov 1996, 477-489
Cyanophyta: Bluegreen algae. Because of their bacteria-like morphology,
they are sometimes classified as bacteria, but they are an important part of
the periphyton community. Because they are photosynthetic organisms, they
are discussed here with the algae. The "bluegreens" are important
environmentally because they can fix nitrogen and store phosphorus, so they
can thrive when nutrient and other environmental conditions do not favor the
other algae. They can form noxious blooms in ponds, lakes, and reservoirs.
These blooms can cause oxygen depletion (resulting in fish kills), and taste
and odor problems in drinking water, and can sometimes be toxic to livestock.
In streams, the bluegreen algae are usually less troublesome, and are found
in waters ranging from pristine to severely polluted.
Examples of bluegreen algae: Anabaena, Aphanizomenon, Microcystis,
Oscillatoria, Phormidium.

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World over, a few passionate people are trying to figure out ways to derive oil
from various species of algae in order to make biodiesel. Algae present a very
interesting proposition because they have few of the drawbacks other
feedstock have. (a) Algae for instance have oil yields that are over a hundred
times higher than that for soy, implying that for the same amount of oil, they
require only a fraction of the area required to grow soy! (b) Owing to their
ability to grow practically anywhere, algae as feedstock also do not contribute
to large-scale deforestation. (c) Since they are not part of the human food
chain at present, algae oil also present no conflicts with our existing food
supplies.

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