Final Project Report

Rehabilitation of Ex-Mining Quarry Area with

Phytoremediation Plants and Biological Fertilizer

Application

Project Coordinator:

Nisa Rachmania Mubarik1*

Other Researchers:

Hirmas Fuady Putra1

Dini Fitriyanti1

Marini Adani1

Idin Abidin2

1Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Bogor 16680, INDONESIA

2Gunung Ceremai National Park, Kuningan, INDONESIA

September 2016

INDONESIA

1 Contestant profile

Contestant name Dr Nisa Rachmania Mubarik

Contestant occupation Lecturer

University/Organization Bogor Agricultural University

E-mail

Phone

Number of people in your team Four person

2 Project overview

Title Rehabilitation of Ex-Mining Quarry Area

with Phytoremediation Plants and

Biological Fertilizer Application

Contest INDONESIA

Quarry name Hambalang

Prize category Education and Raising Awareness

Habitat and Species Research

Biodiversity Management

Student Project

Beyond Quarry Borders

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ABSTRACT

Several phosphate (P) and sodium (K) solubilizing bacteria, nitrogen fixing bacteria, and indole-3-

acetic acid (IAA) producing bacteria isolates have been isolated from soil in mining limestone Cirebon.

These bacteria were selected, blended, and packaged together into biological fertilizer product. The use

of biological fertilizers on seedling plants could replace synthetic chemical fertilizers on seedling plants.

Phosphate and potassium solubilizing bacteria have the ability to produce a substance that dissolves the

compound P and K bound contained in the soil, so that it becomes available to plants and can replace

synthetic fertilizers (chemical) P and K. Nitrogen-fixing bacteria ( N) can reduce the use of urea fertilizer.

Several bacteria also produce indole-3-acetic acid known as auxin, one of growth promoting hormone.

The basic philosophy underlying this research project is to rehabilitate sandy clay at ex-mining area

based on new technology and low capital expenditure. The experiments conducted were (1) use of

bacterial formulation consist of P and K solubilizing bacteria, N fixing bacteria, IAA producing bacteria as

biofertilizer for seedling plant that planted in ex-mining land in Hambalang, (2) re-vegetation on ex-mining

area by using phytoremediation plants such as leucaena (Leucaena leucocephala), albizia (Albizia

saman), and pterocarpus (Pterocarpus indicus) application with compost and consortium of bacteria on

ripping and without ripping soil treatments, and (3) evaluation of ecological condition on ex-mining area.

Preparation of bacterial formulation was done in Department of Biology, Faculty of Mathematics and

Natural Sciences, Bogor Agricultural University. The field experiments were initially with determined the

location of the research in the reclaimation area near the lake in Hambalang. A 100 m transect line

stretched for ecological observation and vegetation prior to planting. Observation area were divided into

three locations corresponding elevation of the land starting from higher land area (zero point) to lower

land area (100 m from zero point). The re-vegetation experiments has been done on higher land area,

center land area, and lower land area. Each location is divided into six plots with a size of 6 mx 4 m per

plot, consist of planting treatment with leucaena, albizia, and pterocarpus (3 plots) and without planting (3

plots). There were 3 soil treatments i.e. ripping, ripping with soil enrichment with compost and bacterial

consortium), and control without treatments.

The conclusion of the research project that soil treatments with ripping and application with compost

before planting could rehabilitate of ex-mining Hambalang area. The bacterial consortium from similar

quarry condition could use as biofertilizer for seedling plants which planted at the area and making plant

growth better.

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INTRODUCTION

The topsoil is the layer that provides elements of macro and micro essential for the plant. The

existence of microbes in the soil layer plays a role in the provision of nutrients, litter decomposition, and

improve soil structure. The open mining land and excessive use of synthetic fertilizer can lead to

decrease of soil quality and environmental pollution. The previous research project around Cirebon

limestone quarry, we have found 55 isolates of phosphate solubilizing bacteria and 12 isolates of

potassium (K) solubilizing from around of limestone Cirebon quarry (Mubarik et al. 2014). In addition, one

of the K solubilizing bacteria, Burkholderia cepacia KQC.5C.5, was able to release soluble K from soil

after ten days incubation (Anggraeni et al. 2016). Other strain of Burkholderia sp QC3.a.1 was capable of

solubilizing the highest P from Pikovskaya media (Mursyida et al. 2015). Dwiana (2015) reported that

Bacillus megaterium QC 7B41 and QC 5C32 isolated from Cirebon quarry could produce indole-3-acetic

acid (IAA or auxin) and N fixed. .Application the B. megaterium showed positive effect on the growth

primary root and lateral root number of seedling plant of leucaena in green house experiment (Yandra

2015). Serratia marcescens QC5C32 has potency as P and K solubilized, indole-3-acetic acid (IAA)

production, and could convert N2 to NH3 by using nitrogenase enzyme activity. The use of S. marcescens

as biofertilizer on seedling plant of leucaena showed a better effect on the dry weight of roots.

The project was aimed to rehabilitate of ex-mining area by planting of phytoremediation plants.

The category of phytoremediation in the project as Phytostimulation or rhizodegradation which use of

rhizospheric associations between plants and symbiotic soil microbes to degrade contaminants from

quarry area. The bacterial consortium was used as biofertilizer for seedling plant which planted on

quarry. The impact of the research results generated technologies for the rehabilitation of ex-mining area

by using compost with consortium of soil bacteria and ecological treatments such as soil ripping and

revegetation.

OBJECTIVES

The research project was conducted to (1) use of bacterial formulation consist of P and K

solubilizing bacteria, N fixing bacteria, IAA producing bacteria as biofertilizer for seedling plant that

planted in ex-mining land in Hambalang, (2) re-vegetation on ex-mining area by using phytoremediation

plants such as leucaena (Leucaena leucocephala), albizia (Albizia saman), and pterocarpus

(Pterocarpus indicus) application with compost and consortium of bacteria on ripping and without ripping

soil treatments, and (3) evaluation of ecological condition on ex-mining area.

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BACKGROUND INFORMATIONS

Location of clay quarry in Hambalang at District Citereup and Gunung Putri, Bogor Region, West

Java Province, Indonesia. The topographic looks like elongated hill and sloping hill with an altitude

between 175-630 meters above sea level and Hambalang Hill are rear of the quarry. Several locations

are still in the reclamation area, namely Parumpung, Pondok Manggu, and Pasir Gadung. There are two

main rivers around the mining area, namely Cileungsi and Ciherang. Both of the river flow to the

springhead namely Citeureup river. Surface water from the the upper area of quarry is collected in the

pond at reclamation activities. Hambalang quarry is located a bit far from the main highway and only a

local road that connects from the village area within 10 km. There are several quarry of other company

near PT Indocement Tunggal Prakarsa TBK (Figure 1). The site area for research project were located

around ex-mining area of sandy clay in Hambalang, Citeureup Bogor. The project has been done since

May until July 2016. The GPS of location and environment data at the project area as shown on Table 1.

Figure 1 Location of Hambalang quarry at Bogor Region, West Java Province, Indonesia (https://www.google.co.jp/maps)

Table 1 Environment data at project area at Hambalang reclamation area on May 2016

No Area Location area with Global Positioning

System (GPS)

Light intensity (lux)

Humidity (%)

Ambient temperature

( 0C)

Wind velocity (Km/h)

1 Higher area (0 m)

S 06°31.737'

E 106°54.750'

1964 39 42.5 9.3

2 Centre area S 06°31.737'

E 106°54.720'

1965 46.5 37.3 8.8

3 Lower area (100 m from 0 m)

S 06°31.737'

E 106°54.696'

1825 46 36.7 7.7

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METHODS

Soil Sampling from Hambalang Quarry and Measurement of Microclimate

Soil was obtained from mining quarry area and around .An amount of 1 Kg soil surface (0-10 cm) from

each soil sampling was taken in polyethylene bag. Whole soil samples were taken with the permission of

PT Indocement Tunggal Prakarsa to analyzed at Bogor Agricultural University.

Microclimate of the project area (temperature and humidity) is measured by sling psychrometer and light

intensity by using lux meter.

Total plate count of bacteria

In amount of 3.0 g of soil samples is diluted in 27 mL of NaCl 0.85% and it was agitated 120 rpm in

shaking incubator, room temperature (25 oC) for 4 hours. Then, the culture was done with serial dilution

from 10-1

to 10-5

in NaCl 0.85 %. Suspension was spread on Nutrent Agar (NA), Pikovskaya agar with

composition of 1 L medium: 10 g glucose, 5 g Ca3(PO4)2, 0.5 g (NH4)2SO4, 0 .1 g MgSO4.7H2O, 0.5 g

yeast extract, 0.2 g KCl, 0.2 g NaCl, 0.002 g FeSO4.7H2O, 0.002 g MnSO4.7H2O, and 15 % bacto agar)

for phosphate solubilizing bacteria isolation (Nautiyal 1999), and Nitrogen free base medium consist of

media Nitrogen Fixation Bacteria (NFB) consist of 5.0 g/L, K2HPO4 0.5 g/L, MgSO4.7H2O 0.2 g/L, NaCl

0.1 g/L, CaCl2.2H2O 0.02 g/L, indicator of bromthymol blue (BTB) 0.5% in KOH 0.2 M 2 mL and vitamin B

1 mL micronutrient solution 2 mL (CuSO4.5H2O 40 mg/L, ZnSO4.7H2O 0.12 g/L, H2BO3 1.4 g/L,

Na2MoO4.2H2O 1.0 g/L, MnSO4.H2O 1.175 g/L, FeEDTA 1.64% 4 mL, KOH 4.5 g/L. pH 6.8 and added

with 1.8 g/L agar (Okon et al. 1977). The cultures were incubated at room temperature for 48-72 hours.

Consortium of Bacterial Formulation

The carrier material used in this study was talcum with the composition and mixing process of the

material as followed: 200 mL suspension of selected isolate (108 cells/mL), 100 g of talcum, 1 g carboxy

methyl cellulose, and 1 g of CaCO3 (Suryadi et al. 2013). Control treatment was prepared only with sterille

talcum (without suspension bacteria). The viability test of the isolate was routinely done every weeks..

Revegetation of Ex-mining Area

Before revegetation of field experiment on at sandy clay of ex-mining at Hambalang area, data of

ecological condition was measured by by using line transect from upper land area (0 m, zeo spot) to

lower area (100 m) (Putra 2013). The area + 2000 m2 was excavated by using tractor and divided of six

plots (6 m x 4 m per plot) at three regions (upper, centre, and lower) There were 3 soil treatments by

using ripping, ripping with soil enrichment with compost, and control without treatments. Ripping was

done by making 7 line mounds on ripping plots. Compost treatment was used one week before

revegetation treatment on rippling plot and without ripping (non-ripping) (Figure 2).

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A half meter until one meter of plant of pterocarpus (Pterocarpus indicus) and albizia (Albizia

saman) are prepared by researcher from Gunung Ciremai National Park, Kuningan, West Java, Indonesia

and leucaena (Leucaena leucocephala) seedling plants (< 30 cm) are found from Hambalang area. Two

plants of each species are planted randomly on ripping and non-ripping area, and on the treatment with

and without compost application. Bacterial consortium was applied after planted at composting treatments

(Figure 3). Data of plant growth of assisted and spontaneus growing plants are measured and collected

every two weeks from each plant of three region from June until last July 2016.

Statistical Analysis

The data were to analyzed by using analysis of variance with Microsoft Excell 2010 and SAS 16.0.8

(Statistical Analysis System) at P<0.05.

(a) (b) (c)

Figure 2 Ripping (a) and non-ripping (b) plot area treatments. Bacterial application on seddling plant (c)

RESULTS AND DISCUSSION

Collection of Soil Samples

Each 1 kg of soil sample from third regions were analyzed of soil chemical composition (Table 1).

Based on of criteria of soil chemical properties (Hardjowigeno 1995), soil samples from Hambalang ex-

mining are mildly alkaline and have low category for N total, C-N ratio, and CEC. Cation exchange

capacity (CEC) is the total capacity of a soil to hold exchangeable of cations. The data showed that the

soil samples are sandy clay and category as infertile soil (Table 2). The environmental data of

Hambalang ex-mining as shown on Table 1 showed that the ambient temperature is high which caused

by low of covered vegetation so that the intensity of barriers to the sun becomes lower (Putra 2013).

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Table 2 Chemical composition of soil sample from ex mining Hambalang area

Soil Region

pH 1:5 H

2O

C Org (%)

N Total (%)

P (ppm)

K (cmol /Kg)

CEC (cmol/Kg)

Texture (%)

Sand Silt Clay

Higher area

7.99 0.46 0.09 10.5 0/19 7.92 34.16

52.13 13.71

Centre area

8.01 0.69 0.12 12.4 0.24 9.51 30.99 54.48 14.53

Lower area 8.13 0.69 0.12 9.8 0.22 12.28 36.42 45.78 17.80

CEC: Cation exchange capacity

Abundance of Soil Bacteria

The abundance of soil bacteria was measured before land treatment and after plant treatments. There

are increasing of soil bacteria after one month after revegetation as much as log 1.17 (Table 3).

Enhancement of total bacteria after revegetation can be caused by plant produce exudates or

metabolites that used as nutrient for bacteria and soil microbes. Rhizobacteria which grow around roots

plant also produce chemical signal to invite the bacteria of the same species to create a consortium near

roots plant (Madigan et al.. 2008). The bacteria could categorize as phosphate solubilizing bacteria,

nitrogen fixing bacteria, plant growth promoter that benefit to plants (Table 4). Treatment with biofertilizer

on plant seedling could increase the population of bacteria on soil. Previous measurement of total

bacteria which contained in compost is log 7.80.

Consortium of bacterial population which used as biofertilizer consist of Bacillus sp. QC7B41 and

Bacillus QC5C32. Both bacteria are categorized as phosphate solubilizing bacteria, nitrogen fixing

bacteria, and plant growth promoter (IAA producing bacteria). The bacterial consortium of both bacteria

was stable on talcum as carrier substance up to 6 weeks incubation with log number of bacteria 6.94

(Mulyani 2016).

Table 3 The abundance of soil bacteria before land treatment and after revegetation

No Region Total of bacteria (log) Increasing of log number of bacteria

Before land treatment After revegetation

1 Higher area 3.94 6.70 2.76

2 Centre area 4.85 7.11 2.26

3 Lower area 5.43 6.6 1.17

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Table 4 Total plate count of phosphate solubilizing and N fixing bacteria after plant treatments

No Region Total of bacteria (log)

Phosphate solubilized N fixed

1 Higher area 5.77 6.47

2 Centre area 5.30 6.27

3 Lower area 4.77 5.53

Evaluation of Ecological Condition on Ex-Mining Area

Data of field treatments was analyzed using Excell and SAS programs at P<0.05.. Both results

showed that P > 0.05 or no significant between treatments. This is presumably because the data is taken

in a short time (two months) and the plants used are not the same on the age and plant height. However,

there was a tendency that the treatment with ripping and adding with compost and bacteria showed

higher than control (non-ripping or without ripping) for plant assisted (Figure 3). The use of bacteria and

compost on condition ripping treatment has no effect on plants that grow spontaneously in the ex-mining

area at Hambalang.

Figure 3 Comparison of plant productivity between vegetation in various soil conditions

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Comparison of plant productivity among species showed the tendency that the treatment with

ripping and adding with compost and bacteria showed higher than ripping treatment and control (non-

ripping) for pterocarpus and leucaena (Figure 4). Enhancement of leucaena plant cover showed that it

was higher than other plants. It was suggested that all leucaena which used were seedling plants (<30

cm) while albizia and pterocarpus were used varying in size between 0.5m until 1 m at the beginning of

the planting. Data of plant height increment and trunk diameter increment showed the same tendency that

leucaena are showed higher than other plants (Appendix 1 and Appendix 2).

Figure 4 Comparison of productivity among species of plants in various soil conditions

CONCLUSIONS

Soil treatments with ripping and application with compost before planting could rehabilitate of ex-

mining Hambalang area. The bacterial consortium from similar quarry condition could use as biofertilizer

for seedling plants which planted at the area and making plant growth better.

THE ADVANTAGES OF THE RESEARCH FOR THE COMPANY

One of the advantages of the research for the company is soil treatment with compost and potential

bacterial consortium could replace using of top soil for rehabilitation of ex-mining area which need high

cost then the method. It is assumed that for one day working, it needs two workers to do ripping on

land for 96 m2. Payment for workers Rp 100,000/person/day. If ripping will be done for one hectare

required cost Rp 20,833,333 with approximately Rp 20,850,000. The compost price of Rp 1500 / kg and

compost is required 7 tons for one hectare, the cost required Rp 10,500,000. Cost for bacterial

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formulation Rp 500,000 per Kg. It is required 5 Kg/ha with cost Rp 2,500,000. The total of cost of Rp

33.850,000 approximately 2507.4 USD (1 USD = Rp13,500).

ACKNOWLEDGMENT

We thank and appreciate to Quarry Life Award Indonesia at PT Indocement Tunggal Prakarsa Tbk.

Palimanan for start up financial support of this project, thank to Head of Department of Biology, Faculty

of Mathematics and Natural Sciences, Bogor Agricultural University and Head of Gunung Ciremai

National Park for research supporting. Special thank for students i.e. Ahmad Suryadi, Kamila, Rina, Aris,

Abid, and Dijah who assisted in field experiments.

REFERENCES

Dwiana A. 2015. Isolation and characterization of Indole-3 acetic acid producing bacteria from limestone quarry [undergraduate thesis] In Bahasa Indonesia. Bogor (ID): Bogor Agricultural University.

Angraini E. 2015. The study of potassium solubilizing bacteria from limestone mining area Palimanan,

Cirebonquarry [thesis]. In Bahasa Indonesia. Bogor (ID): Bogor Agricultural University Hardjowigeno S. 1995. Soil Science. 2

nd Edition. In Bahsa Indonesia.. Jakarta (ID): Akademika Presindo.

Madigan MT, Martinko JM, Dunlap PV, Clark DP. 2008. Brock Biology of Microorganism. San Francisco

(US): Pearson Benjamin Cummings. Mubarik NR, Wibowo RH, Angraini E, Mursyida E, Wahdi E. 2014. Exploration of bacterial diversity at

Cirebon quarry [final report]. Bogor (ID): www.quarrylifeaward.ae/ system/files/winners. Mulyani. 2016 Cells viability and identification of bacteria that isolated from post-mining limestone at

Cirebon. [undergraduate thesis] In Bahasa Indonesia. Bogor (ID): Bogor Agricultural University. Mursyida E, Mubarik NR, Tjahjoleksono A. 2015. Selection and identification of phosphate-potassium

solubilizing bacteria from the area around the limestone mining in Cirebon quarry. Research Journal of Microbiology 10 (6): 270-279. DOI: 10.3923/jm.2015.270.279.

Nautiyal CS. 1999. An efficient microbiological growth medium for screening phosphate solubilizing

microorganisms. FEMS Microbiology Letter 170:265-270. Okon Y, Albrecht SL, Burris RH. 1977. Methods for growing Spirilum lipoferum and for counting it in pure

culture and in assosiation with plants. Applied of Environmental of Microbiology 33:85-88. Putra HF. 2013. Evaluation of ecological function and revegetation level of xx-mining of Lead at Air

Mungkul, Kabupaten Belitung [thesis]. In Bahasa Indonesia. Bandung (ID): Institute Technology of Bandung.

.Suryadi D, Susilowati DN, Riana E, Mubarik NR. 2013. Management of rice blast disease (Pyricularia

oryzae) using formulated bacterial consortium. Emirates Journal of Food and Agriulture. 25: 349-357.

Yandra RF. 2015. The examination of potential bacteria that producing Indole-3-acetic acid (IAA) from

limestone land at planting leucaena (Leucaena leucocephala). In Bahasa Indonesia. Bogor (ID): Bogor Agricultural University.

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Appendix 1 Comparison of plant height increment between species of plants in various soils conditions

Appendix 2 Comparison of the trunk diameter increment between species of plants in various soils condition

1.667

1.2

4.15

1.61.417

2.483

1.2831.5

1.917

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Tru

nk

dia

met

er in

crem

ent (

mm

)

Treatment

Pterocarpus Albizia Leucaena

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Appendix 3 Monitoring of reclamation model (July 2016): Measuring of plant cover, biomass, rood trees diameter

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Appendix 4 Paper of previous research and used as reference

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