1

Polder Tawang Semarang : Study Case of BiotechnologicalApplication and Waste Water treatment as part of Integrated

Coastal ManagementHermin 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

AbstractMicroalgae 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 forthe removal of P and N. Polder Tawang Semarang was the ideal example for anopen source outdoor pond of natural microalgae cultures cultivation. It was a subjectand model for biotechnological application and waste water treatment to reducepollution and eutrophication marked by blooming of microalgae. Biotechnologyapplication was used to product biofertilizer from nitrogen-fixing microalgaecontaminant. Cyanobacteria (blue-green algae) have been studied as possiblebiofertilizers because of the ability to fix atmospheric N under anaerobic conditions.The aim of this study was to investigate the potential of contaminant microalgae fromPolder Tawang Semarang as biofertilizers as an initiative study for exploiting theirinnate potentials and pollution management. The research result shows thatapplication of microalgae contaminant from Polder Tawang Semarang in the plant asbiofertilizers 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

2

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 METHODSPolder 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

3

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 TawangSemarang (a. 100x, b. 400x, c. 1000x)

4

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

5

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.

6

Table 1. Nutritive component of microalgae

amountNo ComponentChlorella sp. Dunaliella sp. Polder Tawang

microalgae1 Protein 31.57 31.44 46.22 Lipid 8.49 7.86 3.83 Carbohydrate and others 15.36 11.05 31.7 (crude)4 Ash 37.18 36.86 33.25 Water 7.40 7.42 7.86 Laurat Acid 1.625 1.6147 Miristat Acid 4.227 3.9588 Palmitat acid 35.287 31.5849 Palmitaleat acid 35.287 31.58410 Stearat acid 18.004 17.85511 Oleat acid 6.489 6.24712 Linoleat acid 13.885 12.98613 Linooleat acid 0.658 0.55914 Fikosa Penta Enoit acid 2.876 2.77615 Dokosa Hexa Enoit acid 1.596 1.468

46.2

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 PROSPECTSThe 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

7

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.

.

REFERENCES

APHA, -1975- Standard methods for theexamination of water and wastewater(14thedn.), American Public Health Association,Washington, D.C.

Aquacop, 1983. Algal Food Cultures At The Centre Oceanologique duPacifique. In McVey, J.P and J.R. Moore. CRC Handbook of Mariculture: Vol. I. Crustacean Aquaculture. CRC Press.

Baudin, I., O. Cagnard, J.J. Grandguillaume, dan Z. Do-Quang. 2006. Algaeand Associated toxins and metabolites : methodology for riskassessment and risk management. Water Practice and Tech. Vol I.No.4. : 1- 12

Borowitzka, M.A. 1988. Vitamins and fine chemicals from Microalgae. In:Microalgal Biotechnology (Eds.) Borowitzka, M.A.and Borowitzka, L.J.,Cambridge University Press. pp 153 - 196.

Carmichael, W.W. 1994. The toxins of cyanobacteria. Science America 270:78 - 86.

Kannaiyan, S. 1985. Algal biofertilizers for low land rice. Tamil NaduAgricultural University, Coimbatore. pp. 14.

Rippka, R., J. Deruelles, J.B Waterbury, M. Herdman & R.Y.Venkataraman,G.S. 1981. Blue-green algae for rice production. FAO Soil Bulletins 16:33 - 42.

Venkataraman, L.V. 1983. A Monograph on Spirulina platensis -Biotechnology and application. Department of Science and Technology,New Delhi.

Benemann, J. 2002, A Technology Roadmap for Microalgae Biofixation.Report to DOE NETL/IEA

Bidwell, J.P. dan Spotte S. 1983. Artificial Sea Water Formulas and Methods.Jones & Bartlett. p:324-325.

8

Kusumaningrum, H.P. J. Soedarsono., T. Yuwono, dan E. Kusdiyantini. 2004.The Effect of Various Salinity Level to the Growth and Characterizationof Dunaliella sp Isolated from Jepara Waters, in Laboratory Scale.ILMU KELAUTAN, Indonesia Journal of Marine Sciences. 9(3). ISSN0853 – 7291. September . (9):136 –140

_______________, H.P. dan J. Soedarsono, 2006a. Molecular Determinationof A Green Algae Isolate to detecting 1-Deoxy-D-Xylulose-5-phosphateSynthase (DXS) Gene in Improvement of Carotenoid Production. ILMUKELAUTAN, Indonesia Journal of Marine Sciences. 9(3). ISSN 0853 –7291. Juni(11) 1: 79-86

_______________, H.P. E. Kusdiyantini , T. Yuwono, dan J. Soedarsono,2006b. Determination of a Green Algae Species From Jepara,Indonesian Coastal Region Based on Microbiological, Ecophysiologicaland Molecular Characterization for Improvement of CarotenoidProduction. Jour. Coastal Dev. Oktober (10) 1: 33-46

______________, H.P. dan J. Soedarsono, 2006c. Molecular Determinationof Dunaliella sp. to detecting 1-Deoxy-D-Xylulose-5-phosphateSynthase (DXS) Gene in Improvement of Carotenoid Production.Proceeding Seminar Nasional Perhimpunan Mikrobiologi Indonesia.Solo. 26-27 Agustus 2006

Kusumaningrum , H.P. J. Soedarsono, dan E. Kusdiyantini. 2006d. MolecularCharacterization of A Green Algae Isolate by 16S rRNA inImprovement of Carotenoid Production. Proceeding Seminar NasionalSPMIPA. 9 September 2006

Moronta, R., R. Mora and E. Morales. 2006. Response of the microalgaChlorella sorokiniana to pH, salinity and temperature in axenic and nonaxenic conditions. Rev. Fac. Agron. (LUZ). 23: 27-41

Reynaud and Blaine Metting. 1988. Colonization potential of Cyanobacteriaon temperate irrigated soils in Washington State USA. BiologicalAgriculture and Holticulture. Vol.0144 : 8765-8788.

Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M. and Stanier, R.Y.(1979). Generic assignments, strain histories and properties of purecultures of cyanobacteria. Journal of General Microbiology, 111, 1-61.

Sze, P. 1993. A Biology of The Algae. 2nd Ed. Wm.C. Brown. p:5–80.

Zainuri, M, Endang Kusdiyantini, Widjanarko, Joedoro Soedarsono andTriwibowo Yuwono. 2003a. Preliminary study of yeast Phaffiarhodozyma as pigment Source to the carotenoid contents of tigershrimp (Penaeus monodon Fabricius) . Ilmu Kelautan. (1):47-52. EdisiMaret 2003.

9

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

Metting, B. (1986). Population dynamics of Chlamydomonas and its influenceon soil aggregate stabilization in the field. Applied & EnvironmentalMicrobiology 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,143, 139-143.

_______, B., Rayburn, W.R. and 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.

Results and Discussion

10

..

Cyanobacteria (blue-green algae) have been studied as possiblebiofertilizers for rice cultivationbecause of the abilify dfheterocystous species to fix atmospheric Nunder aerobic conditions.

Recent studies have shown that eukaryotic single-celled microalgae underdevelopment as biological soil conditioners will colonize moist temperate soilswhen large inocula can be applied (Metting, 1986 & 1987).Metting, B. (1981). The systematics and ecology of soil algae. BotanicalReview, 47, 196-312.Metting, B. (1986). Population dynamics of Chlamydomonas saja0 and itsinfluence on soilaggregate stabilization in the field. Applied & Environmental Microbiology51, 1161-1 164.Metting, B. (1987). Dynamics of wet and dry aggregate stability from a three-year microalgal soilconditioning experiment in the field. Soil Science, 143, 139-143.Metting, B., Rayburn, W.R. & Reynaud, P.A. (1988). Algae and agriculture. InAlgae and HumanAffairs (C.A. Lembi & R.A. Waaland, eds.), pp. 335-370 Cambridge UniversityPress; 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.

11

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

12

and terrestrial ecosystems. For the last decades, the occurrence ofwaterblooms of planktonic cyanobacteria greatly increased in continentalaquatic ecosystems as a consequence of pollution generated by humans.These proliferations disrupt ecosystem equilibrium and may be harmful toanimals 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 waterbloomspotentially harmful to animals and Man.

In order to elucidate the developmental cycle of these microorganisms andtheir acclimation capabilities to variations in environmental parameters, wehave chosen the hepatotoxic strain Microcystis aeruginosa PCC 7806 as amodel system.The algae are a polyphyletic, artificial assemblage of O2-evolving,photosynthetic organisms (and secondarily nonphotosyntheticevolutionary descendants) that includes seaweeds (macroalgae) and ahighly diverse group of microorganisms known as microalgae.Phycology, the study of algae, developed historically as a disciplinefocused on the morphological, physiological and ecological similaritiesof the subject organisms, including the prokaryotic bluegreen algae(cyanobacteria) and prochlorophytes. Eukaryotic algal groups representat least five distinct evolutionary lineages, some of which includeprotists traditionally recognized as fungi and protozoa. Ubiquitous inmarine, freshwater and terrestrial habitats and possessing broadbiochemical diversity, the number of algal species has been estimatedat between one and ten million, most of which are microalgae. Theimplied biochemical diversity is the basis for many biotechnological andindustrial applications.F B Metting Jr.J. of Industrial Microb and Biotech, Springerberlin/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 ofthe periphyton community. Because they are photosynthetic organisms, theyare discussed here with the algae. The "bluegreens" are importantenvironmentally because they can fix nitrogen and store phosphorus, so theycan thrive when nutrient and other environmental conditions do not favor theother algae. They can form noxious blooms in ponds, lakes, and reservoirs.These blooms can cause oxygen depletion (resulting in fish kills), and tasteand odor problems in drinking water, and can sometimes be toxic to livestock.In streams, the bluegreen algae are usually less troublesome, and are foundin waters ranging from pristine to severely polluted.

Examples of bluegreen algae: Anabaena, Aphanizomenon, Microcystis,Oscillatoria, Phormidium.

13

World over, a few passionate people are trying to figure out ways to derive oilfrom various species of algae in order to make biodiesel. Algae present a veryinteresting proposition because they have few of the drawbacks otherfeedstock have. (a) Algae for instance have oil yields that are over a hundredtimes higher than that for soy, implying that for the same amount of oil, theyrequire only a fraction of the area required to grow soy! (b) Owing to theirability to grow practically anywhere, algae as feedstock also do not contributeto large-scale deforestation. (c) Since they are not part of the human foodchain at present, algae oil also present no conflicts with our existing foodsupplies.