•

•

COMMITTEE

STEERING COMMITTEE1. Dr. Satyawan Pudyatmoko, S.Hut., M.Agr.5c.2. Dr. Jamhari, S.P.,M.P.3. Prof. Dr. Ir. Ali Agus, DAA., DEA4. Dr. drh. Joko Prastowo, M.5i.

5. Prof. Dr. Ir. Lilik Sutiarso, M.Eng.6. Prof. Dr.lr. Budi Prasetyo Widyobroto, DEA.,DESS.7. Dr.Budiadi, M.Agr.8. Prof. Dr. Tata Wijayanta, M.Hum .

ORGANIZING COMMITTEE1. Yuny Erwanto, Ph.D.

2. Dr. Hatma Suryatmojo3. Anggoro Cahyo Sukartiko, Ph.D.4. Atus Syahbudin, Ph.D.5. R.Ahmad Romadhoni Surya Putra, Ph.D.6. Dr. Alim Isnansetyo7. Dr. Ngadisih8. Raden Rara Devita Anggraeni, Ph.D.9. Galuh Ratnawulan Sekararum, S.E.1O.Dino Dwi Jayanto, S.Kom.11.AlifYuni Alfiani, S.Kom

12.Sri Wijayanti Suryaningrum, S.Pd.13.Nadhia Nuri Tariana14.Ayu Dipta Kirana15.Aristia Pintakarini

324------------ The 1" UGM International Conference on Tropical Agriculture 2016

Table of ContentsWELCOME FROM ICTA BOARD •....•.......••...........•.......•....•...•......•......•..•................•.. •..·3WELCOMING SPEECH FROM RECTOR··· ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••·······5SCHEDULE •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••··7INVITED SPEAKER ..•....•....•....•.........•...•....•.....................•.... ··..•·..·.. ·.. ····•..•.. · •·..•· ·'0

a. Prof. Dr.rer.nat. Heinz Dieter Isengard 11b. Prof. Mamoru Kanzaki 13c. Dr. Mulyoto Pangestu · · · 15d. Prof. Dr.lr. Mohammad Na'iem M. Agr.5c ·..· · · · ·.. 18e. Prof. Stefaan De Neve · · · 20f. Suadi, S.Pi.,M.Agr.5c., Ph.D · · ·..·..· · 22

LIST PARTICIPANTS ••........•....•..........•....•...................•............ •·..···•··..··..·..•··..··.. ·•····..··24PARALLEL SESSIONS .........•..............•..........•..................•......... ··•·..•·..•·..•··..·..•···..•·····.. 53

ABSTRACTS OF SUSTAINABLE TROPICAL FISHERY ••••••••••••••••••••••••••••••••••••••••••••••••••••66A.11.1SOUTH SEA PEARL SHELL QUALITY INSPECTION USING X-RAY DIGITAL RADIOGRAPHy •.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.69A.11.2CULTIVATION AND CONSERVATION OF INDONESIAN NATIVE FISH (Rasbora lateristriata) THROUGH

FISH FARMER GROUP EMPOWERMENT IN YOGYAKARTA •.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.·70A.l1.3 DEVELOPMENT FISHERIES INDUSTRY; ALTERNATIVE FISH FEED INDUSTRY CASE STUDy •.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.•.71A.11.4NON-SPESIC DEFENCE POTENTIATING ACTIVITY OF ALGINAT FROM SARGASSUM SP.lN

WALKING CATISH (Clarias Sp.) •.•.•.•.•.•.·•.•.•.·•.•.•.·•.•.•.•.•.•.·•.•.•.•.•.•.·•.•.•.•.•.•.•.•.•.··•.•.•.·•.•.•.•.•.•.•.•.•.·•.•.·•.•.·•.•.·•.•.•.•.•.·•.··•.·····•.··············•.···72A.1I1.1AN APPLICATION OF DATA ENVELOPMENT ANALYSIS TO DETERMINE THE EFFICIENCY LEVEL OF

THE FISH AUCTION FACILITY IN COASTAL AREA, JAVA ISLAND···································································•.····•.•.·····•.······•.·73A.1I1.2MOLECULAR IDENTIFICATION OF CHITINOL YTIC BACTERIA ISOLATED FROM SHRIMP PASTE AND

CHARACTERIZATION OF ITS CHITINASE GENES ····•.······•.•.··•.•.•.•.•.·•.·•.•.•.•.·•.•.··•.•.···•.•.·····•.·•.·74A.I.1INTRA-SPECIFIC VARIATION OF THE MITOCHONDRIAL 16S GENE AMONG PERIOPHTHALMUS KALOLO

LESSON 1831 (PISCES: GOBIIDAE) FROM BOGOWONTO ESTUARY, YOGYAKARTA, INDONESiA •.•.··•.•.•.•.•.·•.•.·•.•.•.·•.· 75A.1.2 A SIMPLE OPTIMISED DNA EXTRACTION METHOD FOR MACROALGAE FROM COASTS OF GUNUNGKIDUL,

YOGYAKARTA, USING CTAB-BASED METHOD·················································...............•........•............•.•..•.•..•.•.•.•.•..•.•..•.•...•.•....•.76A.l.3 SASI:TRADITIONAL CONSERVATION TO PROTECT POPULATION OF MANGROVE CRABS

(SCYLLA SERRATA FORSKAL 1775) IN MANGROVE AREA OF LEKSULA VILLAGE OF BURU SELATAN DiSTRICT· •.· 77A.1.4INDONESIAN SUSTAINABLE FISHERIES MANAGEMENT IN GLOBALIZATIONAND REGIONALIZATION OF ASEAN

ECONOMIC COMMUNITY (AEC) 2016·················································· ·····························•.····•.··78

ABSTRACTS OF SUSTAINABLE ANIMAL HEALTH •..............•••...............•.....•............••. 79B.I.1 DIFFERENCES IN THE REARING SYSTEM TO BALI CADLE GASTROINTESTINAL HELMINTHS INFESTATION

IN PRAFI DISTRICT MANOKWARI REGENCY WEST PAPUA PROVINCE ·····························•.·80B.1.2TRYPANOSOMIASIS AND SURRA: VECTOR, DETECTION AND ERADICATION TO ENSURE LIVESTOCK

PRODUCTION IN INDONESiA···· •.····•.······················•.····•.·•.··•.·•.....•......•.........•.........................................................................................81B.I.3 DETECTION THE PRESENCE OF BOVINE VIRAL DIARRHEA VIRUS PERSISTENT INFECTION IN LOW REPRODUCTIVE

PERFORMANCE DAIRY CADLE HERD IN CENTRAL JAVA, INDONESiA································································•.··•.•.•.•.··•.·•.··82B.l1.1FASCIOLOSIS IN CENTRAL JAVA: A CONTINUOUS THREAT ....................•.........•.........................················································83B.II.2DETECTION OF TREMBOLONE ACETATE IN BEEF MUSCLE AND LIVER USING HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY METHOD ..............................................................•.....•.........................................·················································84B.l1.3 ANTIBIOTIC RESISTANCE IN ESCHERICHIA COLI ISOLATED FROM BROILER ASSOCIATED WITH

ENROFLOXACINE AND OXYTETRACYCLINE TREATMENT ........•...•........•..•.•............•....................·················································85B.l1.4SEROLOGICAL AND MOLECULAR BIOLOGICAL DIAGNOSIS FOR LEPTOSPiROSiS··········································•.•.·····•.·•.··•.·86B.l1.5RAPID DETECTION OF Verocytotoxigenic Escherichia coli (VTEC) BACTERIA BY POLYMERASE

CHAIN REACTION (PCR) METHOD AGAINST ISOLATES INOCULATED IN BGLB·····································································87B.II.6 INVESTIGATION OF LEAD CONTAMINATION IN BADUNG RIVER USING FISHES AS BIOINDICA TORS ...•..................... 888.11.7HAEMOPROTOZOA INFECTIONS OF DOMESTIC BIRDS IN HILLY AREAS OF BANGLADESH ·························•.·············•.89

ABSTRACT OF SUSTAINABLE TROPICAL FORESTRy · 90cnu ASSESSMENT OF LANDSLIDE RISK AND DISASTER MANAGEMENT IN WATUGEDE CATCHMENT,

GUNUNGKIDUL DISTRICT .........................................................................•....................•............•.•.•.··•.····•.•.·•.•.·•.•.·•.·•.·•.·•.·•.···················91

-----------------The 1"UGMInternationalConferenceonTropicalAgriculture2016TI

F.V13 ESTABLISHING QUAIL POPULATIONS FOR LESS BIASED EXPERIMENTAL RESULTS 239F.V1.4RESTRlalON ENZYME MAPPING OF CYTOCHROME B GENE ON ANGUS GRADE CATTLE AND OTHER CATTLE

IN THE WORLD ..· ·..· · · · · · · ·· · · 240F.VI,5COMPARATIVE STUDY OF BODY MEASUREMENTS IN DIFFERENT STRAIN OF INDONESIAN LOCAL DUCKS · 241F.VI,6THEEFFECT OF BIRTH TYPE ON PHENOTYPIC PERFORMANCE OF SAPUDI SHEEP IN SAPUDIISLAND, MADURA 242F.VI.7THE MICRORNA-AMPK GENE CANDIDATE IN REGULATE LIPID ACCUMULATION OF BOVINE GRANULOSA CELL

LEUTEINIZATION: AN INSILICO STUDy ·· ·· · ·· · · · ··· ·· ·..............................•.................................. 243F.VI,8IMPACT OF BULL'S BREED AT CONCENTRATION OF PROSTAGLANDIN COLLECTED FROM VESICULAR

SEMINALIS BY IN VITRO METHOD ·· ··..··· ·· ··· ·· ·· ··· ·· 244F.VI.9 ANIMAL WELFARE AS STRESS MANAGEMENTTO IMPROVE BEEF CATTLE REPRODUCTION · 245F.V1.10SPERM QUALITY OF BALI CATTLE AFTER GIVEN CRUDE TANIN SUPPLEMENT IN THE SEMEN 246F.VI.11 BLOOD CHEMISTRY PARAMETERS OF ADULT KACANG GOATS 247F.VI.12 THE MOTILITY OF DEG RAM'S SPERMATOZOA BEFORE AND AFTER FREEZING WITH ADDITION

ANTIOXIDANT AND DIFFERENT EXTENDER · ··248F.IV.1 POTENTIAL OF MARMELOSIN BIOACTIVE COMPOUND IN AEGLE MARMELOS TO INHIBIT THE GROWTH OF

BACTERIAL UREASE FOR REDUCE FORMING AMMONIA (NH3) IN EXCRETA 249F.IV.2THE ADDITION EFFECT OF FERMENTED AEGLE MARMELOS FRUIT AND BAMBOO SHOOTS IN

CAmE FECES SLURRY TO THE REDUCTION OF AMMONIA GAS EMISSION 2S0F.IV.3 MEAT CONTAMINATION FROM FARM TO FORK THREATEN HUMAN HEALTH 251F.lV.4 EFFECTSOF ADMINISTRATION OF COMBINATION OF PALM KERNEL MEAL, KATUK LEAF AND BETEL NUT

POWDER ON SENSORY VALUE OF MEAT IN THE GOAT ·252F.lV.s QUANTITATIVE ELEMENTAL ANALYSIS OF VARIOUS POULTRY EGGSHELL CUTICLE USING SCANNING ELECTRON

MiCROSCOPE 2S3F.lV.6 PHYSICAL QUALITY OF EGG OF LAYING HENS FED DIETS CONTAINING PURSLANE (Portulaca oleracea)

RICH IN ALPHA-LINOLENIC ACID ··· ··· ···· ·· · · ··· ···254F.1I1.1HETEROTROPHIC AND AEROBIC AMMONIUM REMOVAL IN WASTEWATER BY Alcaligenes sp. LS2T 2S5F.l11,2MANAGEMENT AND REPRODUCTIVE PERFORMANCES OF ACEH COWS, LOCAL INDONESIAN CATTLE KEPT BY

FARMERS IN A TRADITIONAL SySTEM ··· · · · · · ··· 2S6F.1I1,3ARTIFICIALINSEMINATION ON THE ETAWAH GRADE DOES USING FROZEN SEMENS OF GEMBRONG GOAT 257F.III.4 EDIBLE PORTION OF CARCASS AND OFFALS OF INDONESIAN YEARLING KACANG BUCK FED RUMINALLY

UNDEGRADABLE PROTEIN 258F.lll.s POSTPARTUM ESTRUS VARIATION OF LOCAL COWS AT VILLAGE BREEDING CENTER IN

THE YOGYAKARTA PROVINCE · ·· ·259F.l11.6ANIMAL WELFARE ASSESSMENT SYSTEM FOR SUSTAINABLE CATTLE PRODUCTION IN INDONESIA 260F.lII.7 REPRODUCTIVE PERFORMANCE OF JABRES COW AT KETANGGUNAN DISTRICT, BREBES REGENCY,

CENTRAL JAVA PROVINCE, INDONESIA 261F.V.1 BEEFCATTLE FARMERS ATTITUDE TOWARDS ENVIRONMENTAL AND SOCIOECONOMIC DEVELOPMENT IN

YOGYAKARTA PROVINCE · · · ·· ·· ·· · ·· · ·262F.V.2 MARKET SEGMENTATION OF GOAT MILK CONSUMERS: AN APPROACH USING DEMOGRAPHIC AND

BEHAVIORAL VARIABLES ..·..·· ·· · • ·..•..·· ·· · · ·· 263F.V.3 ANALYSIS OF THE IMPLEMENTATION OF ETTAWA CROSSBRED GOAT MANURE TREATMENT IN THE DISTRICT

KULONPROGO YOGYAKARTA INDONESIA · ···· · · ··· · ··..·264F.V.4 ROLE OF FEMALE MEMBERS IN SMALLHOLDER FARMERS' FAMILY FOR GOAT FARMING ACTIVITIES IN

THE VILLAGE AT THE UPLAND AREA, YOGYAKARTA INDONESIA 265F.V.s DEVELOPMENT OF MICRO FINANCE INSTITUTIONS MODEL FOR FARMERS GROUP TO SUPPORT BEEF CATTLE

FARMING SUSTAINABILITY: A PILOT STUDY 266F.V.6 THE PERFORMANCE OF MILK PRODUCTION, TOTAL MILK REVENUE AND REPRODUCTION INDICATORS ON

DAIRY SMALLHOLDERS IN DAERAH ISTIMEWA YOGYAKARTA AND EAST JAVA PROVINCE, INDONESIA· ·..· 267F.V.7 PERCEPTION OF BEEF CATTLE FARMERS TOWARD CROP WASTE AS ANIMAL FEED 268F.v.8 USING CAUSAL LOOP DIAGRAM AS A QUALITATIVE INSTRUMENT TO MODEL SMALLHOLDERS: A CASE STUDY OF

GOAT FARMING IN GUMELAR - BANYUMAS ..·· · · ·· · ·· ·• · ·..· · · · ·..·269F.V.9 THE RATE OF TECHNOLOGY ADOPTION ON INTEGRATED PALM OIL AND CATTLE FARMING IN

JAMBI PROViNCE ..· ·..·..· · ·..· ·..·..· · ·.....................................................•............................................. · ·..·273F.V.10 SUMMARy ·..· · · ·..· ·..·..·..·..··..·..· · ·· ·..·..··..·..··..·274

LIST OF POSTER SESSIONS .••.••.••..•..••.•••••••..•.•.•..••••••.•••••••••••••••••••••.••••••••••••••••• ••·•••••····276ABSTRACTS OF POSTER SESSiONS· •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••···••••·•·••284

1. IDENTIFICATION OF POTENTIAL FISHERY COMMODITY FOR SUPPORTING SMES OF PROCESSED FISHERYPRODUCT OF WEST KALIMANTAN, INDONESIA (A CASE STUDY IN SAMBAS REGENCy) · · · 285

2. INTESTINAL NEUROTROPISM OF NEWCASTLE DISEASE VIRUS FIELD ISOLATES IN LAYER CHICKEN · ·2863. HOST PLANTS OF WEAVER ANT (OECOPHYLLA SMARAGDINA) IN GREEN AREA OF

UNIVERSITAS GADJAH MADA CAMPUS· · · · · · · · ·..· · ·..· · · · · ·..·2874. ETlHNOBOTANICAL STUDY OF SACRED FOREST OF BULUPITU, KEBUMEN, CENTRAL JAVA 288

The'· UGM International Conference on Tropical Agriculture 2016

1st International Conference on Tropical Agriculture (ICTA) 2016

Characteristics of Alcaligenes sp. LS2T Heterotrophic and Aerobic Ammonium Removal for Potential Livestock’s

Wastewater Treatment

A.R. Azkarahmana, Y. Erwantoa, W. Hadisaputroa, L.M. Yusiatia, and N.A. Fitriyantoa*

aFaculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna No.3 Kampus UGM Bulaksumur, Yogyakarta, Indonesia

Abstract

Alcaligenes spp. was found to have the ability to grow and remove ammonium in the medium through the nitrification-denitrification process. This ability was also known as an ammonium treatment in organic wastewater including livestock’s

wastewater. In order to further understand its potential to remove ammonium in livestock’s wastewater; microbial growth, ammonium, nitrite, and nitrate production of Alcaligenes sp. LS2T were investigated in phosphate medium at different carbon/nitrogen ratios (C/N ratios) along with constant airflow. Ammonium sulfate was used as sole nitrogen source with acetate and citrate as an organic carbon source in a separate medium. Results showed that Alcaligenes sp. LS2T could utilize ammonium as sole nitrogen source which associated with acetate and citrate as carbon source at different C/N ratios, resulting in ammonium removal, production of nitrite and nitrate at various concentrations in the medium. Highest ammonium removal was found in the acetate C/N 28 medium where 94.44% of initial ammonium was removed. Keywords: Alcaligenes sp. LS2T; ammonium removal; heterotrophic nitrification

Introduction

Development in livestock’s industry was followed by the intensive farming system, causing accumulation of ammonium in livestock’s wastes area. Excess of ammonium was known to cause several environmental problems, one of it is eutrophication. Therefore, ammonium removal in wastewater treatment became essential to overcome this problem thus the sustainable farming system could be achieved. Conventional ammonium removal in wastewater treatment plants consist of nitrification by autotrophic bacteria under aerobic conditions1, and difficult to apply because autotrophic bacteria are vulnerable to the high concentration of ammonium and organic matters with C/N ratio of 0-22. Thus, wastewater pretreatment must be done to reduce the C/N ratio3 or diluting the wastewater4,5. Other researches have been done to observe potential of various heterotrophic microbial which could live in the higher C/N ratio medium and able to remove ammonium on livestock’s wastes

like on dairy wastewater6, piggery wastewater7, and poultry manure8. Alcaligenes spp. was known as a heterotrophic bacteria that could be used for removing ammonium in

wastewater9 through simultaneous heterotrophic nitrification and aerobic denitrification1,10,11. Alcaligenes sp. LS2T is one bacteria that could remove ammonium in the medium12, allegedly through heterotrophic nitrification and aerobic denitrification process. In this research, we further observe the potential of Alcaligenes

sp. LS2T for livestock’s wastewater treatment. Investigation on its characteristics to remove ammonium at different C/N ratios in the phosphate medium was done, nitrite and nitrate production which was also the result of the nitrification-denitrification process was also determined.

Materials and Methods

Microorganisms and culture conditions

Alcaligenes sp. LS2T was collected from the soil around layer farm in Yogyakarta, Indonesia, then cultured and stocked in agar medium in the Laboratory of Animal Skins, By-products, and Animal Wastes Technology, Faculty of Animal Science, Universitas Gadjah Mada. The phosphate medium was prepared by dissolving the following in 1 L of distilled water: (NH4)2SO4 0.472 g, MgSO4·7H2O 0.05 g, K2HPO4 0.2 g, NaCl 0.12 g, MnSO4·4H2O 0.01 g, and FeSO4 0.01 g. Sodium acetate and trisodium citrate were used as organic carbon

source separately in the medium, abbreviated as acetate medium and citrate medium, respectively. The growth of Alcaligenes sp. LS2T in this experiments were performed in shaken Erlenmeyer flasks containing 500 ml medium and 1% preculture of Alcaligenes sp. LS2T on a rotary shaker at 120 rpm with constant airflow.

Shaking culture experiment

In order to elucidate the influence of carbon to nitrogen molar ratio (C/N) on the nitrifying capacity, the amount of N-ammonium was fixed at approximately 100 mg·L-1 while the amount of carbon was adjusted to the appropriate C/N ratio which was 7, 14, 21 and 28. The ammonium removal ability was investigated in the acetate medium and citrate medium separately. Samples were taken periodically to examine changes in growth, concentrations of ammonium, nitrite, and nitrate.

Analytical methods

The growth of Alcaligenes sp. LS2T was monitored by measuring the optical density at 600 nm (OD600 nm) of the culture medium using a spectrophotometer (Shimadzu, Japan)11. Culture samples were centrifuged at 10.000 rpm for ammonium, nitrite and nitrate analysis. Ammonium concentration was analyzed by Nessler’s reagent

photometry method13. Nitrite concentration was determined by N-(1-naphtalene)-diaminoethane photometry method13. Nitrate concentration was measured by using EPA14 modified colorimetric, brucine method. All tests were conducted in triplicate.

Results

Growth characteristics

The growth of Alcaligenes sp. LS2T at different C/N ratios in the acetate and citrate medium in shaking culture showed at Fig. 1. Growth curves were established by measurement of the OD value at 600 nm.

Fig. 1. The Growth of Alcaligenes sp. LS2T at different C/N ratios in the (A) acetate medium; (B) citrate medium. Closed square: C/N7;

open square: C/N 14; closed circle: C/N 21; open circle: C/N 28.

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

0 9 18 27 36 45 54 63 72

Op

tica

l de

nsi

ty (

OD

60

0)

time (h)

(A)

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

0 9 18 27 36 45 54 63 72

Op

tica

l de

nsi

ty (

OD

600)

time (h)

(B)

In the acetate medium, the log phase of the bacterium was all similar started at 3 h. However, the growth peak in the C/N 7 medium was relatively lower than any other medium, and the stationary phase starts later than any other acetate medium, indicating that carbon source was deficient for cell synthesis. However, the shorter stationary phase was found in the C/N 14 medium which lasts for 9 hours while the longest stationary phase was found in the C/N 28 medium which lasts for 24 hours. In the citrate medium under same conditions, the log phase of the bacteria was all similar started at 3 h. The growth peak in the citrate C/N 7 medium was also relatively lower, and the stationary phase was also started later than any other citrate medium. The growth phase trend in citrate medium was relatively same than in the acetate medium, with longest stationary phase was found in the citrate C/N 21 and 28 medium which lasts for 21 hours and the shortest was found in the citrate C/N 14 medium which lasts for only 9 hours.

Heterotrophic nitrification

In order to investigate the correlation between microbial growth, ammonium removal, and release of products during heterotrophic nitrification, Alcaligenes sp. LS2T was inoculated into acetate and citrate medium under different C/N ratios. Cultures were sampled every 12 hours for analysis. Fig. 2 shows the changes by nitrification (ammonium, nitrite, and nitrate) during the 96-h growth period.

In the acetate medium, highest ammonium removal after 96-h was found in C/N 28 medium, about 94.44% of initial ammonium removed; followed by C/N 21, C/N 14 and C/N 7 which removed 93.49%, 93.12% and 93.04% of initial ammonium respectively. Furthermore, highest nitrite production was found in C/N 14 medium (0.79 mg·L-1), followed by C/N 28 (0.69 mg·L-1), C/N 21 (0.68 mg·L-1) and C/N 7 (0.63 mg·L-1). However, highest nitrate production was found in C/N 28 medium (35.53 mg·L-1), while in the C/N 7 medium was 22.59 mg·L-1; 23.31 in C/N 14 and 26.71 mg·L-1 in the C/N 21 medium.

Fig. 2. Nitrification products of Alcaligenes sp. LS2T in acetate medium at (A) C/N 7; (B) C/N 14; (c) C/N 21; (d) C/N 28. Closed square: NH4

+-N; closed circle: NO2--N; closed triangle: NO3

--N.

0.64

0.66

0.68

0.7

0.72

0.74

0.76

0.78

0.8

0 12 24 36 48 60 72 84 96

0

10

20

30

40

50

60

70

80

90

NO

2-co

nce

ntr

atio

n (

mg.

L-1)

time (h)

NH

4+an

d N

O3-

con

cen

trat

ion

(m

g·L-1

)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 12 24 36 48 60 72 84 96

0

10

20

30

40

50

60

70

80

90

NO

2-co

nce

ntr

atio

n (

mg.

L-1)

time (h)

NH

4+ a

nd

NO

3-co

nce

ntr

atio

n (

mg·

L-1)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 12 24 36 48 60 72 84 96

0

10

20

30

40

50

60

70

80

90

NO

2-co

nce

ntr

atio

n (

mg.

L-1)

time (h)

NH

4+an

d N

O3-

con

cen

trat

ion

(m

g·L-1

)

0.62

0.64

0.66

0.68

0.7

0.72

0.74

0.76

0.78

0.8

0.82

0.84

0 12 24 36 48 60 72 84 96

0

10

20

30

40

50

60

70

80

90

NO

2-co

nce

ntr

atio

n (

mg.

L-1)

time (h)

NH

4+an

d N

O3-

con

cen

trat

ion

(m

g·L-1

)

(A) (B)

(D) (C)

In the citrate medium after 96-h growth period, highest ammonium removal was found in the C/N 21 medium, with 89.98% of initial ammonium removed; followed by C/N 28 with 88.45%, C/N 14 with 86.36% and C/N 7 with 83.49% of initial ammonium removed. Highest nitrite production was found in the C/N 7 and 14 medium which both produced 0.27 mg·L-1, while in the C/N 21 and 28 medium both produced 0.24 mg·L-1

nitrite. The C/N 7 medium was found to produce highest nitrate production, with 23.89 mg·L-1 of nitrate produced after 96-h, followed in the C/N 21; C/N 14 and C/N 28 medium which produced 22.45 mg·L-1; 22.08 mg·L-1 and 20.13 mg·L-1 of nitrate respectively. Heterotrophic nitrification process in citrate medium under different C/N ratios shown in Fig. 3 below.

Fig. 3. Nitrification products of Alcaligenes sp. LS2T in citrate medium at (a) C/N 7; (b) C/N 14; (c) C/N 21; (d) C/N 28. Closed square: NH4

+-N; closed circle: NO2--N; closed triangle: NO3

--N.

Discussion

The previous study done by Gutama12 showed that Alcaligenes sp. LS2T has the ability utilize ammonium as a nitrogen source for metabolism. Various studies showed that some Alcaligenes spp. has the ability to perform heterotrophic nitrification under aerobic condition1,10,11. In this study, we investigate the growth profiles of Alcaligenes sp. LS2T at different C/N ratios in the acetate and citrate medium. The results indicate that log phase by Alcaligenes sp. LS2T was found in 3-h in both acetate and citrate medium at all C/N ratios researched. In both acetate and citrate medium with C/N 7 stationary phase was found later than in any other C/N ratio medium. Furthermore, the growth was relatively lower this could be caused the lack of carbon source for metabolism, resulting in less ammonium removal in the medium. Highest ammonium removal in acetate medium was found in C/N 28, and for citrate, the medium was found in 21, both was known to achieve earlier and longer stationary phase than in any other C/N in the respective medium. The correlation between earlier and longer stationary phase with higher ammonium removal is because, in heterotrophic nitrification, the assimilation ratio is as high as heterotrophic nitrification ratio15,16, the assimilated nitrogen is associated with cell growth, and the nitrogen removal rate is affected by cell doubling time17.Therefore, microorganisms with earlier and longer stationary phase can take ammonium quickly into the cell, resulting in more effective ammonium removal18.

At four different C/N ratios in both media, nitrite, and nitrate were formed by Alcaligenes sp. LS2T during heterotrophic ammonium removal process. The experimental results showed that the formation of nitrate is

0

0.1

0.2

0.3

0.4

0.5

0 12 24 36 48 60 72 84 96

0

20

40

60

80

100

120

NO

2-co

nce

ntr

atio

n (

mg.

L-1)

time (h)

NH

4+an

d N

O3-

con

cen

trat

ion

(m

g·L-1

)

0

0.1

0.2

0.3

0.4

0.5

0 12 24 36 48 60 72 84 96

0

20

40

60

80

100

NO

2-co

nce

ntr

atio

n (

mg.

L-1)

time (h)

NH

4+an

d N

O3-

con

cen

trat

ion

(m

g·L-1

)

0

0.1

0.2

0.3

0.4

0.5

0 12 24 36 48 60 72 84 96

0

20

40

60

80

100

NO

2-co

nce

ntr

atio

n (

mg.

L-1)

time (h)

NH

4+an

d N

O3-

con

cen

trat

ion

(m

g·L-1

)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 12 24 36 48 60 72 84 96

0

20

40

60

80

100

NO

2-co

nce

ntr

atio

n (

mg.

L-1)

time (h)

NH

4+an

d N

O3-

con

cen

trat

ion

(m

g·L-1

)

(C)

(A) (B)

(D)

higher than nitrite. Nitrite and nitrate concentration in this research demonstrate a whole time nitrification process until 96-h, these results correspond to present study that indicates heterotrophic nitrite/nitrate production is linked with growth, supports that heterotrophic nitrification can take place during entire growing phase19,20. Nitrate as dominant product in the process of heterotrophic nitrification by Alcaligenes sp. LS2T is similar with A. faecalis strain NR10, Achromobacter sp.GAD3 and Comamonas sp. GAD421. While other strains like A.

faecalis C1611 and Bacillus methylotrophius strain L722 had nitrite as the dominant product over nitrate in the process of heterotrophic nitrification.

Carbon dependency has been shown in this research. A possible reason is that ammonium is oxidized by a sequence involved in some way with the metabolism of a certain or specific carbon source that could provide the acceptor molecules for the synthesis of intermediate organic nitrogen compounds23. Results of this research also showed that even if the C/N ratio is as high as 28, which is too high for autotrophic nitrifying bacteria, Alcaligenes sp. LS2T still exhibits nitrification ability. This results showed Alcaligenes sp. LS2T potential in livestock’s wastewater treatment like in piggery wastewater which C/N ratio was around 5-207 or even chicken manure which C/N ratio was around 824.

Conclusion

Alcaligenes sp. LS2T was capable of removing ammonium in the medium through nitrification process, utilizing ammonium in the medium for cell synthesize and producing nitrite and nitrate with organic carbon sources. The highest ammonium removal was found in the acetate medium at C/N 28; this indicates that Alcaligenes sp. LS2T could perform heterotrophic nitrification under relatively high C/N ratio. Results of this research suggest deeper research on the ammonium removal ability of Alcaligenes sp. LS2T which is in the production of hydroxylamine and nitrogen gas and its application on various livestock’s wastewater, so that clearer nitrogen pathway in this process could be conducted.

Acknowledgement

This research was supported by Program Penelitian Unggulan UGM 2016 DPKM Management contract number 690/UN1-P.III/LT/DIT-LIT/2016. The authors also thank Laboratory of Animal Skins, By-products, and Animal Wastes Technology, Faculty of Animal Science, Universitas Gadjah Mada for providing research needs.

References

1. Joo, HS, Mitsuyo H, Makoto S. Characteristics of Ammonium Removal by Heterotrophic Nitrification-Aerobic Denitrification by Alcaligenes faecalis No. 4. J. Biosci Bioeng 2005;100:184-191.

2. Itokawa, H, Hanaki K, Matsuo T. Nitrous oxide production in high-loading biological nitrogen removal process under low COD/N ratio condition. Water Res 2001;35:657–664.

3. Khin, T, Annachhatre AP. Novel microbial nitrogen removal processes. Biotechnol Adv 2004;22:519–532. 4. Rostron, WM, Stuckey DC, Young AA. Nitrification of high strength ammonia wastewaters: comparative study of immobilization media.

Water Res 2001;35:1169–1178. 5. Jung, JY, Chung YC, Shin HS, Son DH. Enhanced ammonia nitrogen removal using consistent biological regeneration and ammonium

exchange of zeolite in modified SBR process. Water Res2004;38:347–354. 6. Neerackal, GM, Pius MN, Hung-Soo J, Xiang W, Craig SF, Joseph HH, Marc WB. Potential application of Alcaligenes faecalis strain

No.4 in mitigating ammonia emissions from dairy wastewater. Biores Technol 2016;206:36-42. 7. Joo, HS, Mitsuyo H, Makoto S. Piggery wastewater treatment using Alcaligenes faecalis strain no.4 with heterotrophic nitrification and

aerobic denitrification. Water Research 2006;40:3029-3036. 8. Posmanik, R, Amit G, Ali N. Effect of high ammonia loads emitted from poultry-manure digestion on nitrification activity and nitrifier-

community structure in a compost biofilter. Ecol Eng 2014;62:140-147. 9. Pai, S, Chong N, Chen C. Potential application of aerobic denitrifying bacteria as bioagents in waste water treatment. Biores Technol

1999;68:179-185. 10. Zhao, B, Qaing A, Yi LH, Jin SG. N2O and N2 production during heterotrophic nitrification by Alcaligenes faecalis strain NR. Biores

Technol 2012;116:379-385. 11. Liu Y, Yao W, Yi L, Hua A, Yongkang L. Nitrogen removal characteristics of heterotrophic nitrification-aerobic denitrification by

Alcaligenes faecalis C16. Chinese J Chem Eng 2015: 827 - 834. 12. Gutama, R. 2015. Karakteristik mikrobia Alcaligenes sp.LS2T dan kemampuannya dalam mereduksi senyawa amonia. Skripsi. Sajarna

Peternakan Fakultas Peternakan. Yogyakarta: UGM. 13. APHA.Standard methods for the examination of water and wastewater, 19th ed. American Public Health Association 1995. 14. EPA. Method 352.1: Nitrogen, Nitrate (Colorimetric, Brucine) by spectrophotometer. United States Environmental Protection Agency

1971. 15. Jetten, MSM, Logemann S, Muyzer G, Robertson LA, de Vries S, van Loosdrecht MCM, Kuenen JG. Novel principles in the microbial

conversion of nitrogen compounds. Antonie van Leeuwenhoek 1997;71:75–93. 16. Kuenen, JG, Robertson LA. Combined nitrification and dentrification processes. FEMS Microbiol Rev 1994;15:109–117. 17. Gupta, AB, Gupta SK. Simultaneous carbon and nitrogen removal from high strength domestic wastewater in an aerobic RBC biofilm.

Wat. Res 2001;35:1714-1722.

18. Joo, HS, Mitsuyo H, Makoto S. Improvement in ammonium removal efficiency in wastewater treatment by mixed culture of Alcaligenes

faecalis No. 4 and L1. J Biosci Bioeng 2007;103:166-173. 19. Papen, H, von Berg R. Amost probable numbermethod (MPN) for the estimation of cell numbers of heterotrophic nitrifying bacteria in

soil. Plant Soil 1998;199:23–130. 20. Yang, XP, Wang SM, Zhang DW, Zhou LX. Isolation and nitrogen removal characteristics of an aerobic heterotrophic nitrifying–

denitrifying bacterium, Bacillus subtilis A1, Bioresour Technol 2011;102: 854–862. 21. Chen, Q, Ni JR. Heterotrophic nitrification–aerobic denitrification by novel isolated bacteria. J Ind Microbiol Biotechnol 2011;38:

1305–1310. 22. Zhang, QL, Liu Y, Ai GM, Miao LL, Zheng HY, Liu ZP. The characteristics of a novel heterotrophic nitrification–aerobic

denitrification bacterium, Bacillus methylotrophicus strain L7. Bioresour Technol 2012;108: 35–44. 23. Witzel, KP, Overbeck HJ. Heterotrophic nitrification by Arthrobacter sp. (strain 9006) as influenced by different cultural conditions,

growth state and acetate metabolism. Arch Microbiol 1979;122:137–143. 24. Weerayutsil, P, Umaporn K, Kulyakorn, K. Optimum ratio of chicken manure and napier grass in single stage anaerobic co-digestion.

Energy Procedia 2016;100:22-25.