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IDENTITAS DAN URAIAN UMUM
1. Judul Penelitian
: PEMBERIAN BAKTERI AKTINOMISETES
DAN Serratia marcescens Strain MBC1 SEBAGAI Plant
Growth-Promoting PADA PERTUMBUAN Lactuca
sativa SECARA HIDROPONIK MENGGUNAKAN
AIR PAYAU
2. Tim peneliti
No Nama Jabatan Bidang keahlian Alokasi waktu
(jam/minggu)
1 Achmad Arifiyanto, S.Si., M.Si. Ketua Mikrobiologi;
Aktinomisetes
15 jam/minggu
2 Hapin Afriyani, S.Si., M.Si. Anggota Kimia; Kimia
organik
15 jam/minggu
3. Objek Penelitian (jenis material yang akan diteliti dan segi penelitian):
Jenis material yang digunakan pada penelitian ini adalah bakteri aktinomisetes
dan Serratia sp. IAA diproduksi dan dikarakterisasi menggunakan
spektrofotometer. Pengaruh pertumbuhan IAA diamati menggunakan kultur
hidroponik media air payau pada tanaman selada.
4. Masa Pelaksanaan
Mulai : bulan: April tahun
2021 Berakhir : bulan: September
tahun 2021
5. Usulan Biaya : Rp35.000.000.-
6. Lokasi Penelitian
Laboratorium Mikrobiologi Jurusan Biologi, Laboratorium Kimia Organik
Jurusan Kimia FMIPA Unila, UPT Lab Terpadu dan Sentra Inovasi Teknologi
(LTSIT) Unila.
7. Instansi lain yang terlibat
8. Kontribusi mendasar pada suatu bidang ilmu
Produksi IAA oleh mikroorganisme merupakan bentuk hormon eksogen
bagi tanaman. Mikroba dapat membantu tanaman bertahan dari kondisi
lingkungan ekstrem termasuk kadar garam. Produksi IAA dan pengaruhnya
terhadap pertumbuhan tanaman selada pada air payau diharapkan dapat
dituangkan dalam bentuk tulisan dalam jurnal internasional bereputasi.
9. Jurnal ilmiah yang menjadi sasaran untuk setiap penerima hibah
a. Biocatalysis and Agricultural Biotechnology (Scopus Q2) by Elsevier Bv.
b. Journal of Microbiological Methods (Scopus Q3) by Elsevier Bv.
c. Biodiversitas, Journal of Biological Diversity (Scopus Q3) by Society
for Indonesian Biodiversity
d. Walailak Journal of Science and Technology (Scopus Q3) by The
College of Grad. Studies of Walailak University
DAFTAR ISI
DAFTAR ISI .............................................................................................................................. 2
RINGKASAN ............................................................................................................................ 1
BAB 1. PENDAHULUAN ........................................................................................................ 2
1.1 Latar belakang dan Permasalahan............................................................................................ 2
1.2 Tujuan Khusus dan Urgensi Penelitian .................................................................................... 2
1.3 Spesifikasi khusus .................................................................................................................... 2
BAB II. TINJAUAN PUSTAKA .............................................................................................. 3
2.1 Rhizobakteria pemacu pertumbuhan tanaman ......................................................................... 3
2.2 Aktinomisetes .......................................................................................................................... 3
2.3 Serratia marcescens ................................................................................................................. 3
2.4 Kultur hidroponik selada ......................................................................................................... 4
BAB 3. METODE PENELITIAN ............................................................................................. 5
3.1 Kultur produksi ........................................................................................................................ 5
3.2 Penapisan dan produksi IAA ................................................................................................... 5
3.3 Ekstraksi dan pemurnian IAA.................................................................................................. 5
3.4 Evaluasi penambahan inokulum bakteri pada kultur selada secara hidroponik ...................... 6
BAB 4. HASIL DAN PEMBAHASAN .................................................................................... 7
4.1 Hasil ......................................................................................................................................... 7
4.2 Pembahasan ............................................................................................................................. 9
DAFTAR PUSTAKA .............................................................................................................. 11
RINGKASAN
Latar belakang: Zat pengatur tumbuh tanaman berperan penting dalam mengontrol
proses biologi dalam jaringan tanaman. Proses pembentukan organ seperti tunas atau
akar merupakan interaksi antara zat pengatur tumbuh eksogen dari lingkungan dengan
zat pengatur tumbuh endogen yang diproduksi oleh jaringan tanaman. Secara alamiah
zat pengatur tumbuh eksogen seringkali dihasilkan oleh mikroba yang berasosiasi
dengan tanaman tersebut, salah satunya auksin. Indole-3- acetic acid (IAA)
merupakan salah satu turunan auksin. Tujuan: Penelitian ini berfokus pada produksi
IAA dan pengaruhnya terhadap pertumbuhan tanaman selada (Lactuca sativa).
Metode: Agensia hayati yang dipakai terdiri atas Streptomyces hygroscopicus GGF4-
i18, Streptomyces sp AB8, Micrococcus luteus, Serratia marcescens MBC1 dan
Streptomyces hygroscopicus subsp. Jinggangensis InaCC A497. IAA diproduksi
menggunakan prekursor 1.0% L-tryptophan. Pengaruh IAA dibuktikan dengan
menginokulasikan bakteri pada tanaman Lactuca sativa (selada) secara hidroponik.
Metode kultur hidroponik yang dipilih yaitu NFT (Nutrient Film Technique). IAA
dikarakterisasi menggunakan HPLC dan kromatografi lapis tipis. TKT: Penggunaan
bakteri non Streptomyces sp terbukti mampu membantu tanaman selada bertahan
terhadap cekaman kadara garam selain membantu menghasilkan hormon IAA pada
cekaman kadar garam menggunakan kultur hidroponik.
Kata kunci: Aktinomisetes, Serratia, IAA, selada, air payau.
BAB 1. PENDAHULUAN
1.1 Latar belakang dan Permasalahan
Selada (Lactuca sativa L.) merupakan jenis sayuran yang digemari masyarakat
Indonesia. Ia juga kaya akan antioksidan, potassium, folat, dan karoten. Ia dapat
membantu pembentukan sel darah putih dan sel darah merah dalam susunan sumsum
tulang, mengurangi resiko kanker, tumor dan penyakit katarak, membantu kerja
pencernaan dan organ-organ di sekitar hati serta menghilangkan gangguan anemia 1.
Gejala fitopatogenik seperti stunting percabangan akar, epinasty daun, hingga
ketidakseimbangan hormon mempengaruhi hasil tanaman di bidang pertanian baik dari
segi kualitas maupun jumlah 2. Perawatan ekstensif diperlukan untuk mengatasi
permasalahan tersebut. Hidroponik pada selada merupakan salah satunya. Metode ini
menggunakan air yang dilarutkan nutrisi di dalamnya sebagai media tumbuh tanaman
untuk menggantikan tanah 3.
Bakteri yang berasosiasi dengan tumbuhan merupakan penghasil senyawa
bioaktif. Mereka menghasilkan fitohormon eksogen yang meningkatkan pertumbuhan dan
metabolisme dalam kondisi stres 4. Sejumlah stres tersebut di antaranya kekeringan,
salinitas, dan kontaminasi tanah oleh minyak bumi 5.
Indole-3- acetic acid (IAA) merupakan fitohormon auksin utama yang lumrah
dieksploitasi. Mikroba penghasil IAA di antaranya Serratia marcescens 6, Micrococcus
yunnanensis 7, Micrococcus luteus 8, dan Streptomyces sp., 9. Mikroba jenis ini dilaporkan
toleran terhadap air payau. Air payau dianggap menjanjikan karena perkembangan
populasi manusia dan perubahan iklim turut mengurangi suplai air tanah 10.
1.2 Tujuan Khusus dan Urgensi Penelitian
Berdasarkan uraian tersebut belum peneliti bermaksud membandingkan
kemampuan produksi IAA oleh aktinomisetes dan Serratia sp., dalam mengendalikan
cekaman salinitas pada selada yang dikultur secara hidroponik.
1.3 Spesifikasi khusus
Menerapkan penambahan mikroba penghasil IAA guna memacu pertumbuhan
selada pada media mengandung air payau secara hidroponik.
BAB II. TINJAUAN PUSTAKA
2.1 Rhizobakteria pemacu pertumbuhan tanaman
Rizomikrobioma memainkan peran kunci dalam akuisisi dan asimilasi hara,
memperbaiki tekstur tanah, mensekresi, dan memodulasi molekul ekstraseluler seperti
hormon, metabolit sekunder, antibiotik, dan pelbagai senyawa sinyal, semuanya mengarah
pada peningkatan pertumbuhan tanaman. Mikroba dan senyawa yang mereka keluarkan
merupakan biostimulan yang berharga dan memainkan peran penting dalam mengatur
respons stres tanaman. Penelitian telah menunjukkan bahwa inokulasi tanaman dengan
rhizobakteria pemacu pertumbuhan tanaman (PGPR) dapat menjadi strategi yang efektif
untuk merangsang pertumbuhan tanaman 11. Pemberian rizomikrobioma penghasil IAA
dilaporkan berpengaruh terhadap jumlah akar lateral 12.
2.2 Aktinomisetes
Aktinomisetes merupakan satu dari sekian banyak rizomikrobioma Gram positif.
Mereka dikenal akan kemampuannya sebagai produsen senyawa bioaktif. Cekaman pH,
salinitas, dan logam berat tidak terlalu berpengaruh karena aktinomisetes banyak
digunakan justru untuk meremidiasi tanah kritis. Keberadaan spora memungkinkan
aktinomisetes bertahan dalam jumlah yang cukup meski lingkungan berada dalam kondisi
lingkungan yang kering 13. Beberapa bakteri anggota ordo aktinomisetales penghasil IAA
di antaranya Streptomyces nobilis WA-3, Streptomyces kunmingenesis WC-3, dan
Streptomyces enissocaesilis TA-3 14. Sementara itu, Micrococcus yunnanensis RWL-2,
dan Micrococcus luteus RWL-3 juga mampu menghasilkan IAA 15.
2.3 Serratia marcescens
Rizomikrobioma Gram negatif penghasil IAA salah satunya ialah Serratia sp.
Serratia adalah bakteri gram negatif famili Enterobacteriaceae. Mereka hidup di air,
tanah, permukaan daun, tubuh serangga, hewan dan manusia 16. Serratia marcescens
MBC1 mampu menghambat pertumbuhan Rigidoporus sp penyebab penyakit jamur akar
putih (JAP) pada tanaman karet 17. Serratia sp dilaporkan mampu menghasilkan IAA
hingga 123.2 mg/mL setelah 144 jam inkubasi 6.
2.4 Kultur hidroponik selada
Setiap 100 g berat basah selada mengandung 1.2 g protein, 0.2 g lemak, 22.0 mg
Ca, 25.0 mg Fe, 162 mg vitamin A, 0.04 mg vitamin B, serta 8.0 mg vitamin C. Metode
hidroponik NFT (Nutrient Film Technique) sering digunakan untuk budidaya selada.
Teknik ini merupakan model budidaya hidroponik dengan meletakkan akar pada lapisan
air yang dangkal menggunakan talang. Air tersebut mengandung nutrisi sesuai kebutuhan
tanaman dan disirkulasikan secara terus-menerus 3. Penelitian mengenai skrining dan
potensi aktinomisetes telah dilakukan sejak tahun 2018 melanjutkan penelitian bidang
penulis dengan biaya mandiri. Road Map (Peta Jalan) dan luaran penelitian yang telah dan
akan dilakukan dapat dilihat pada Gambar 1.
2018-2020
-Eksplorasi
aktinomisetes
-Eksplorasi
bakteri
berpigmen
-Identifikasi strain
menggunakan 16S
rRNA
Output
-Aktivitas enzim
hidrolitik-
-Jenis bakteri
berpigmen terdaftar
Genebank
-Skripsi 3 mahasiswa
-Artikel terbit pada
jurnal Biocatalysis
and Agricultural
Biotechnology
(Scopus Q2)
2020-2022
-Identifikasi
metabolit
antimalaria
-Aplikasi IAA
-Dekolorisasi oleh isolat
aktinomisetes dan
kapang
Output
-Jenis dan struktur
senyawa antimalaria
-Artikel terbit pada jurnal
Biodiversitas Journal of
Biological Diversity
(Scopus Q3)/ hibah PDP
2019-2020
-Artikel diterima pada
jurnal Biocatalysis
and Agricultural
Biotechnology
(Scopus Q2)
2022-2024
-Prebiotik: XOS dan FOS
-Formulasi metabolit
antimalaria
Output
- Jenis dan
struktur
senyawa aktif
pada pigmen
-Tepung prebiotik
-Formula antimalaria
-HAKI Strain
aktinomisetes agen
bioremedias
Gambar 1. Peta jalan penelitian koleksi strain aktinomisetes
BAB 3. METODE PENELITIAN
3.1 Kultur produksi
Streptomyces sp. strain AB8 diremajakan pada media yeast starch agar (YSA).
Serratia marcescens strain MBC1 diperbanyak menggunakan media tryptone soy agar
(TSA). Micrococcus luteus dan Streptomyces hygroscopicus strain GGF4-i18 ditanam
menggunakan media Sodium Agar (NA). Streptomyces hygroscopicus subsp. Jinggangensis
InaCC A497 ditumbuhkan di media ISP4 (International Streptomyces Project). Satu koloni
loop diambil dan diinokulasi ke dalam media cawan agar. Bakteri diinkubasi pada suhu 37o
C selama 3 hari, kecuali aktinomiset yang diinkubasi selama 7 hari.
3.2 Penapisan dan produksi IAA
Media fermentasi yang digunakan adalah media Gause. Komposisinya terdiri dari
pati larut, KNO3, NaCl, MgSO4.7H2O, K 2 HPO4, FeSO4. 7H2O, dan aquades 18. Satu loop
inokulum bakteri ditambahkan ke 100 mL medium Gause, dengan suplementasi L-triptofan
sebagai prekursor. Media Gause tanpa triptofan digunakan sebagai kontrol. Fermentasi
dilakukan dalam kondisi gelap dengan kecepatan 150 rpm pada suhu ruang 19. Pertumbuhan
bakteri diamati setiap hari selama 8 hari yang diukur menggunakan spektrofotometer pada
panjang gelombang 600 nm. Pengamatan dilakukan dua kali (pagi dan sore) dengan
pengukuran triplicate sampling. Metabolit dipisahkan dari kultur menggunakan sentrifugasi
dengan kecepatan 6000 rpm, selama 10 menit.
Sebanyak 1 ml supernatan dimasukkan ke dalam tabung reaksi dan ditambahkan 4
mL pereaksi Salkowski, serta diinkubasi selama 60 menit dalam kondisi gelap. Perubahan
warna diamati dan penyerapannya diukur pada panjang gelombang 530 nm 9. Hari pertama
fermentasi dihitung sebagai hari ke-0.
3.3 Ekstraksi dan pemurnian IAA
Sel bakteri diinokulasi menggunakan media kultur IAA. Kultur diinkubasi dalam medium No.
1 Gause yang dioptimalkan pada suhu 28° C dengan pengocokan pada 200 rpm selama 6 hari.
Kaldu fermentasi disaring menggunakan kertas saring Whatman No.1 dan filtrat kultur diatur
hingga pH 9 dengan NaOH 1 M untuk menjaga IAA terionisasi dan lebih polar. Filtrat
dipartisi menggunakan 100% etil asetat dan fase organik atas dipulihkan. PH diturunkan
menjadi 3 dengan asam asetat pekat untuk mempertahankan IAA dalam pelarut, dan sampel
diuapkan sampai kering. Senyawa kering dilarutkan dalam 3 mL metanol analitik dan
digunakan untuk analisis KLT dan HPLC. Standar IAA dibuat pada konsentrasi 500 ppm.
Standar IAA dan sampel ekstraksi terlihat pada pelat G silika gel yang didukung aluminium
dan KLT dilakukan dengan fase gerak butanone: etil asetat: etanol: air (3: 5: 1: 1, v / v / v / v)
18. Pelat KLT dikeringkan dan bintik-bintik diamati di bawah sinar UV pada 256 nm.
3.4 Evaluasi penambahan inokulum bakteri pada kultur selada secara
hidroponik
Bakteri diinkubasi pada media Gause No.1 selama 6 hari (sekitar
5.6 × 106 CFU/mL). Inokulum disiapkan sebesar 10 % dari kapasitas tanki media nutrisi
hidroponik. Kultur hidroponik yang dipilih ialah jenis NFT. Alat instalasi hidroponik NFT
yang dibuat sebanyak 5 masing-masing alat digunakan untuk 2 perlakuan kombinasi, 5
kontrol postif, dan 1 kontrol negatif. Dalam 1 alat menggunakan talang sepanjang 2 m dengan
jarak tanam tanaman masingmasing 20 cm sehingga dalam 1 alat instalasi terdapat 10
tanaman. Media tanam yang digunakan yaitu rockwool berukuran 23.5 cm x 9 cm x 3.5 cm
dipotong menjadi 3 cm x 2 cm x 3 cm dan dilubangi. Kemudian benih selada (3-4 cm)
diletakkan pada media tanam. Diagram alir metode penelitian dapat dilihat pada gambar 2.
BAB 4. HASIL DAN PEMBAHASAN
4.1 Hasil
Pada produksi IAA dengan perlakuan penambahan precursor L-triptofan 2mg/mL
diperoleh hasil sebagaimana pada Gambar 2. Laju produksi IAA mengalamai kenaikan pada
hari ke 2 dan hari ke 5. Genus Streptomyces sp lebih unggul daripada Serratia sp dan
Micrococcus sp. Hal ini linier jika dibandingkan dengan data pada Gambar ke 3 yakni
tentang kenaikan kepadatan sel. Di mana Streptomyces sp strain AB8 memiliki kepadatan
tertinggi.
Gambar 2. Produksi IAA oleh bakteri Streptomyces sp AB8, Streptomyces sp InaCC A497,
Streptomyces sp i18, Micrococcus luteus hari ke1 hingga ke 9
Gambar 3. Kepadatan sel bakteri Streptomyces sp AB8, Streptomyces sp InaCC A497, Streptomyces
0.07
0.25
0.15
0.20
0.35
0.07
0.19 0.18 0.190.21
0.110.09 0.09 0.10 0.11
0.07
0.14
0.19
0.280.31
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
1 3 5 7 9
Ab
sorb
ansi
Hari
AB3 INACC PINK I18 ML
0.08
0.23
0.30
0.58
0.150.08
0.21 0.18
0.31
0.170.11
0.26
0.13
0.24 0.26
0.06
0.22 0.23 0.25
0.090.10 0.11 0.10
0.18 0.16
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
1 3 5 7 9
Ab
sorb
asn
i
Hari
AB3 INACC PINK I18 ML
sp i18, Micrococcus luteus pada pengamatan hari ke 1 hingga ke 9
Pertumbuhan tanaman selada secara hidroponik dengan pemberian cekaman air payau terbukti tidak
terhambat. Hal ini patut diduga akibat pengaruh pemberian bakteri dalam media hidroponik. Laju
pertumbuhan terbaik pada kategori daun selada terjadi pada perlakuan menggunakan Serratia sp strain
MBC1 (Gambar 4). Perlakuan control menyusul di peringkat ke dua.
Gambar 4. Pertumbuhan daun selada pada hari ke 16 -28 setelah sebar pada instalasi hidroponik
Kategori pengamatan jumlah helai daun juga menunjukkan pengaruh pemberian bakteri non
Streptomyces sp lebih baik, yakni pada perlakuan penambahan Bakteria Serratia sp dan Micrococcus
sp. (Gambar 5).
Gambar 5. Pertumbuhan jumlah helai daun selada pada hari ke 16 -28 setelah sebar pada instalasi
hidroponik
9.37 10.32 10.74 11.34
12.06 12.2513.22
8.84 9.44 10.63 10.38 10.48 10.95 11.42
9.28 9.52 10.27
8.73 7.40
6.20
1.28
8.86 10.24
11.08 10.90 9.67
6.76
4.735.30 5.72 6.307.35
8.7310.10
11.87
16 18 20 22 24 26 28
Len
gth
gro
wth
(m
m)
Hari
MBC ML i18 AB8 C
4.96
5.81 5.96
4.88 5.08 5.58
6.31
5.23 5.85 5.54
5.00 4.96 4.855.275.23
6.00 5.23
3.92
2.38 2.38
0.42
5.69 6.15
5.81 5.04
3.81
2.35
1.46
2.60 2.81 2.96 3.16
4.143.60 3.86
16 18 20 22 24 26 28
Len
gth
gro
wth
(m
m)
Hari
MBC ML i18 AB8 C
Gambar 6. Pertumbuhan Panjang akar selada pada hari ke 16 -28 setelah sebar pada instalasi
hidroponik
Perlakuan control lebih unggul dalam hal pertumbuhan akar dibandingkan dengan perlakuan yang lain.
Hal ini berrarti perlakuan cekaman kadar garam pada air payau mempengaruhi pertumbuhan kara pada
tanaman selada.
4.2 Pembahasan
Pengaruh cekaman salinitas terhadap pertumbuhan, kenampakan, dan senyawa nutrisi,
terutama senyawa fenolik dan karotenoid, selada romaine (Lactuca sativa L.), tanaman toleran garam
rendah, telah dipelajari. Berat kering, tinggi, dan warna tanaman selada berubah secara nyata dengan
pengairan jangka panjang (15 hari) dengan konsentrasi NaCl yang lebih tinggi (yaitu >100 mM).
Namun, tidak ada perbedaan signifikan yang diamati dalam pertumbuhan dan penampilan antara
kontrol, semua perawatan jangka pendek (2 hari; 50, 100, 500, dan 1000 mM), dan irigasi jangka
panjang dengan konsentrasi garam rendah. Selain itu, pada selada romaine yang diberi irigasi jangka
panjang dengan 5 mM NaCl, kandungan karotenoid total meningkat tanpa perubahan warna, dan
kandungan karotenoid utama dalam selada romaine, lutein dan beta-karoten, masing-masing meningkat
37 dan 80%. Tidak ada perbedaan yang diamati pada kandungan lutein dan beta-karoten pada selada
yang diberi perlakuan jangka pendek. Kandungan fenolik selada romaine menurun dengan irigasi garam
jangka pendek, sedangkan tidak ada perbedaan yang signifikan antara perlakuan terkena irigasi jangka
panjang. Penelitian ini menunjukkan bahwa irigasi jangka panjang dengan konsentrasi garam yang
relatif rendah, daripada irigasi jangka pendek dengan konsentrasi garam yang tinggi, dapat
meningkatkan kandungan karotenoid dalam selada romaine tanpa menyebabkan tradeoff dalam hasil
4.81
5.76 5.82 5.31
6.28
7.72
8.56
4.84 5.54 5.40 5.18 5.40 5.30
6.32
5.43 5.75 5.76 4.95
4.18 4.18
0.58
5.93 6.23
5.38 4.62
4.17
2.85
2.00
5.43
6.476.95
7.347.71
8.308.87
-
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
16 18 20 22 24 26 28
Len
gth
gro
wth
(m
m)
Hari
MBC ML i18 AB8 C
atau kualitas visual 20
Sementara itu pengaruh kadar garam pada akar dijelaskan oleh Arif et al 2019 21 menurutnya,
akar tanaman menunjukkan plastisitas morfologi dan memainkan peran penting dalam toleransi
terhadap berbagai tekanan edafik. Penelitian ini bertujuan untuk mengetahui respon morfogenik akibat
salinitas pada sifat akar dan rambut akar dua varietas rapeseed, BARI Sarisha-8 dan Binasarisha-5, pada
tahap reproduksi dan mengetahui pengaruhnya terhadap pertumbuhan reproduksinya. Percobaan
dilakukan pada budidaya hidroponik. Dua perlakuan, 0 mM NaCl sebagai kontrol dan 100 mM NaCl,
diberikan 55 hari setelah perkecambahan. Tanaman yang terpapar 100 mM NaCl selama tujuh hari
menunjukkan kerusakan yang lebih besar pada daun, bunga, dan siliquae dibandingkan dengan kontrol.
Panjang rambut akar pada akar lateral orde pertama dan ketiga, kerapatan rambut akar pada akar lateral
orde pertama, dan panjang akar lateral orde ketiga secara signifikan lebih besar sebesar 91%, 22%,
29%, dan 48% , masing-masing, dalam kondisi yang dirawat dibandingkan dengan kontrol. Peningkatan
luas permukaan akar diperkirakan sebesar 20% di bawah kondisi stres garam menunjukkan bahwa
respon spontan tanaman untuk menyerap lebih banyak air dan nutrisi memungkinkan tanaman untuk
mengatasi kondisi stres. Hasil penelitian ini menunjukkan bahwa program pemuliaan stres di masa
depan harus mempertimbangkan plastisitas sifat akar secara intensif.
Salinitas yang tinggi mengurangi produktivitas dan kualitas tanaman. Rhizobakteri tanah
pemacu pertumbuhan tanaman (PGPR) meningkatkan pertumbuhan tanaman dan toleransi cekaman
abiotik melalui mediasi berbagai mekanisme fisiologis dan molekuler. Salinitas yang tinggi secara
signifikan mengurangi pertumbuhan tanaman dan produksi biomassa, penyerapan nutrisi, kandungan
air relatif daun, kandungan pigmen, atribut pertukaran gas daun, dan kandungan flavonoid dan fenolik
total dalam jagung. Namun, kandungan osmolit (misalnya, protein larut, prolin, dan asam amino bebas),
penanda stres oksidatif, dan tingkat antioksidan enzimatik dan non-enzimatik meningkat pada jagung
di bawah salinitas tinggi. Di sisi lain, inokulasi Serratia liquefaciens KM4 secara signifikan mengurangi
penanda stres oksidatif, tetapi meningkatkan pertumbuhan jagung dan produksi biomassa bersama
dengan pertukaran gas daun yang lebih baik, osmoregulasi, sistem pertahanan antioksidan, dan
penyerapan nutrisi di bawah tekanan garam. Selain itu, ditemukan bahwa semua peningkatan ini disertai
dengan peningkatan regulasi gen terkait stres (APX, CAT, SOD, RBCS, RBCL, H+-PPase, HKT1, dan
NHX1), dan penurunan regulasi gen kunci dalam biosintesis ABA (NCED). Secara keseluruhan,
hasilnya menunjukkan peran menguntungkan dari Serratia liquefaciens KM4 dalam meningkatkan
pertumbuhan tanaman dan toleransi cekaman garam pada jagung dengan mengatur homeostasis ion,
potensi redoks, pertukaran gas daun, dan ekspresi gen terkait stres. Bakteri selain Genus Serratia sp
yang dilaporkan toleran terhadap cekaman garam adalah Genus Micrococcus sp 22. Meski banyak
dilaporkan species Streptomyces sp mampu tolerant terhadap kadar garam 23, pada penelitian ini strain
i18 dan AB8 terbukti tidak tolerant terhadap kadar garam. Hal ini ditandai terjadinya kematian pada
tanaman selada pada perlakuan penambahan kedua isolate tersebut
Secara umum performa pertumbuhan tanaman selada berpengaruh dengan penambahan
cekaman garam. Jenis bakteri juga menunjukkan pengaruh yang signifikan dalam membantu
memoderasi cekaman garam.
Gambar 7. Pengamatan pengaruh pemberian cekaman garam pada pertumbuhan selada secara
hidroponik.
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BIODIVERSITAS ISSN: 1412-033X Volume 22, Number 7, July 2021 E-ISSN: 2085-4722 Pages: 2817-2823 DOI: 10.13057/biodiv/d220731
Short Communication:
In vitro antimicrobial and antimalarial screening of a crude extract of
Streptomyces sp. AB8 isolated from Lapindo Mud Volcano Area,
Sidoarjo, Indonesia
ACHMAD ARIFIYANTO1,, ENDAH SETYANINGRUM1, NISMAH NUKMAL1, TITIK NUR AENY2 1Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Lampung, Jl. Prof. Soemantri Brojonegoro No 1, Gedong Meneng,
Rajabasa, Bandarlampung 35144, Lampung, Indonesia, Tel.: +62-721 704625 Fax. +62-721 704625, email: [email protected] 2Department of Plant Protection, Faculty of Agriculture, Universitas Lampung, Jl. Prof. Soemantri Brojonegoro No 1, Gedong Meneng, Rajabasa,
Bandarlampung 35144, Lampung, Indonesia
Abstract. Arifiyanto A, Setyaningrum E, Nukmal N, Aeny TN. 2021. Short Communication: In vitro antimicrobial and antimalarial screening of a crude extract of Streptomyces sp. AB8 isolated from Lapindo Mud Volcano Area, Sidoarjo, Indonesia. Biodiversitas 22:
2817-2823. Streptomyces is a potential bacterial genus that has been investigated extensively as a source of natural microbial compounds. Its potential metabolites have been widely developed for pharmaceutical, pathogen control, and other applications in agriculture. This study aimed to determine the ability of the Streptomyces sp AB8 crude extract in inhibiting Plasmodium and pathogenic microbes. Streptomyces was cultured on Gause synthetic media for 10 days. The fermented broth culture media has dissolved in a 1:1 mixture of ethyl acetate and methanol. Biochemical characterization of this isolate has carried out using the standard methods. In-vitro antimalarial activity assay was performed using a chloroquine-sensitive Plasmodium falciparum strain 3D7. Fresh type O-positive human erythrocytes were suspended at 4 percent hematocrit in a complete medium to maintain culture. The inhibitory concentration (IC50) was determined using probit analysis. The results showed the extract of Streptomyces sp. AB8 contains phenolic
and alkaloids. Streptomyces sp. AB8 extract can inhibit Dickeya zeae N-Unila 5, Dickeya zeae N-Unila 10, Aspergillus sp IK3, and Escherichia coli growth. The results also showed that the ICs0 value of extract against P. falciparum 3D7 was 17.56 ug/mL. Further research was needed to determine the types of purified bioactive compounds and their bioactivity.
Keywords: Alkaloids, antimalarial, antimicrobial, phenolic, Streptomyces
INTRODUCTION
Malaria is a disease caused by the Plasmodium parasite,
is one of the deadliest diseases in tropical countries (Hay et
al. 2004). It was transmitted by the bite of a female
Anopheles sp. infected with Plasmodium. Malaria in
humans was caused by five different Plasmodium species, i.e., Plasmodium falciparum, Plasmodium vivax,
Plasmodium malariae, Plasmodium ovale, and
Plasmodium knowlesi (Singh et al. 2013).
Unfortunately, the Plasmodium sp. parasite was
resistant to antimalarial drugs such as chloroquine,
amodiaquine, mefloquine, and artemisinin, according to
many studies (Cui et al. 2015). Malaria research was
actively being conducted to combat parasite resistance. The
analysis of metabolites from different plants has been
performed (Alkandahri et al. 2019; Fatmawaty et al. 2017;
Okokon et al. 2017; Orabuezea et al. 2020; Zeleke et al. 2017), as well as metabolites from microorganisms to
combat multi-drug resistant Plasmodium. Metabolites
derived from microbes were considered to be more
desirable due to their shorter life cycles.
Streptomyces, a Gram-positive bacteria, is a member of
the actinobacteria phylum which is known to have
antimicrobial, anticancer (El-Naggar and El-Ewasy 2017),
anti-inflammatory (Gomathi and Gothandam 2019), and
antioxidant (Li et al. 2018). Several Streptomyces, such as
Streptomyces hygroscopicus subsp. hygroscopicus (Fitri et
al. 2019; Nugraha et al. 2020), Streptomyces SUK10 (Zin
et al. 2017), and Streptomyces spectabilis BCC 4785 (Isaka
et al. 2002) have been studied for their antimalarial activity. Streptomyces sp. AB8 was obtained from the
Lapindo mud volcano (Arifiyanto et al. 2020), has not been
investigated for its antimalarial activity. This study aimed
to conduct an initial screening of the antimicrobial and
antimalarial activity of Streptomyces sp. AB8.
MATERIALS AND METHODS
Culture and fermentation
Streptomyces sp. strain AB8 was rejuvenated on yeast
starch agar (YSA) medium, Yeast Starch Agar medium
consisted of 2.0 g yeast extract (OxoidTM), 10.0 g soluble
starch (Sigma-Aldrich), 15.0 g of agar powder (Sigma-Aldrich), and 1000 mL distilled water adjusted to a pH 7.3.
Bacteria were cultured at room temperature for 1–5 days.
The bacterial stock was cultured on a slanting agar
medium. According to Arifiyanto et al. (2021) and Ezeonu
Manuscript received: 5 April 2021. Revision accepted: 22 June 2021.
BIODIVERSITAS 22 (7): 2817-2823, July 2021
2818
and Ejikeme (2016), Bacteria were characterized by several
biochemical tests.
Gause's medium consists of soluble starch, KNO3,
NaCl, MgSO4.7H2O, K2HPO4, FeSO4.7H2O, and distilled
water was used as the fermentation medium (Lin et al.
2012). One loop of bacterial inoculum was added to 100
mL of Gause's medium. The fermentation was carried out
at a speed of 150 rpm in a shaking incubator at room
temperature. Bacterial growth was observed for 10 days
and measured using a spectrophotometer at 600 nm wavelength. A free-cell supernatant was separated from
cell biomass using centrifugation at a speed of 6000 rpm,
for 10 minutes. The supernatant was filtered through filter
paper and extracted using a 1:1 methanol-ethyl acetate. A
rotary evaporator was used to evaporate the solvent at 40-
50oC for 40 minutes to obtain concentrate extract and was
subsequently lyophilized into powder (Setyaningrum et al.
2021).
Antimicrobial activity
Fourteen day-olds of Aspergillus IK3 in a test tube
were added with sterile distilled water parasite, then one percent molasses and 0.5 mL Tween 80 were added to the
test tube, and homogenized using a vortex mixer. Conidia's
suspension separated, then fungal spore cell density was
adjusted to 108 cells/mL using a hemocytometer. It was
observed under a microscope with 400x magnification
(Agustina et al. 2019; Rosa et al. 2020).
Some swabs of bacterial suspension ages 18-24 hours
were taken and then put into a test tube containing 5 mL of
sterile 0.9% NaCl so that the turbidity was comparable to
that of 0.5 McFarland suspension (Darmawan et al. 2017;
Putri et al. 2021). E. coli and Dickeya zeae were grown on Mueller
Hinton Agar (MHA) at a 108 CFU/mL density in a Petri
dish. One loop of Streptomyces sp. AB8 aged 24-hour was
inoculated on media and challenged in the center of MHA
media that has been inoculated with bacteria. An antifungal
test was conducted on Potato Dextrose Agar media against
Aspergillus IK3. Clear zones formation was observed after
24 hours of incubation at room temperature (Sumardi et al.
2020).
In vitro antimalarial assay
The in vitro antimalarial activity test was carried out
using chloroquine-sensitive Plasmodium falciparum strain 3D7. As much as 1 mg of the extract was dissolved in 100
μL of DMSO (concentration 10.000 μg/mL) as a stock
solution. The stock solution was diluted serially.
Synchronous parasites (ring stage) with about 1%
parasitemia were used in this test. Fresh type O-positive
human erythrocytes were suspended at 4 percent
hematocrit in a complete medium to maintain culture
(Baniecki et al. 2007). Using a high-throughput liquid
handler, parasites were supplemented into a 96-well plate.
Two microliters of the test solution with various
concentrations were added to the well. The final
concentrations of the extract were 100, 10, 1, 0.1, and 0.01
μg/ mL. The gas mixture (O2 5%, CO2 5%, N2 90%) was
applied to the test plate that has been inserted into the
chamber. The chamber containing the test plate was
incubated for 48 hours at 37°C. The culture was then
harvested and a thin blood layer was made with 20%
Giemsa staining. Data were analyzed by counting the number of infected
erythrocytes per 1000 normal erythrocytes to determine the
growth percentage of Plasmodium and inhibitory activity
of the extract against Plasmodium. A blood smear was
observed under a microscope. The growth percentage was
calculated using the following formula;
(1)
Where: D0 is the initial hour of growth (%)
The inhibition ability is expressed as a percentage using
the formula below:
(2)
Where:
Xu = % growth on the test solution
Xk = % growth on the negative control
The two formulas above were used to determine the
concentration of extract to inhibit the growth of parasites by 50%. The lethal concentration 50 (LC50) was estimated
using probit analysis based on data of the inhibition
percentage.
A B
Figure 1. A. Colonies of Streptomyces AB8 on Yeast Starch Agar; and B. The Gram-stained colony observed under microscope with 1000x magnification
SETYANINGRUM et al. – Antibacterial and antimalarial of Streptomyces sp AB8
2819
RESULTS AND DISCUSSION
Antimicrobial activity of Streptomyces extract
Streptomyces sp. AB8 bacteria were isolated from
rhizosphere soil in the Lapindo mud volcano field,
Sidoarjo. Previous research showed that Streptomyces sp.
AB8 could inhibit the growth of pathogenic bacteria
(Staphylococcus aureus ATCC 6538P and Escherichia coli
ATCC 25922) (Arifiyanto et al. 2020). Dickeya zeae as a
causative agent of stem and root rot in agricultural and
plantation commodities (Aeny et al. 2020) was also inhibited by Streptomyces sp. AB8 (Table 1). The results
suggested that crop damage caused by D. zeae could be
controlled by the extract of Streptomyces sp. AB8 to
overcome economic losses. These bacteria could also
inhibit the growth of Aspergillus IK3, a cockroach
pathogen, and Escherichia coli, which was isolated from
wastewater around the campus of the University of
Lampung.
Streptomyces is a Gram-positive bacteria that belong to
the order Actinobacteriales with branched mycelium and
chain-shaped. These bacteria could grow in a variety of environments, such as rhizosphere, marine debris,
weathered leaf litter, plant endophytes, and insect
symbionts (Mathew et al. 2020). The initial screening
revealed that Streptomyces sp. AB8 could utilize glucose,
fructose, cellulose, and lactose, galactose, sucrose, chitin,
mannan, and amylum (Table 2) as its carbon source. As a
decomposer group of microbes, Streptomyces has the
ability to degrade complex molecules such as cellulose and
mannan (Sasongko et al. 2015). Chitin is the main
component of fungal cell walls. Streptomyces ability to
decompose chitin potentially used to control mold. (Okay et al. 2013).
Biochemical characterization of Streptomyces sp. AB8
The development of new drug candidates has been
driven by the resistance of pathogenic microbes to
antibiotics and the risk to human health they pose.
Microbial metabolites are increasingly being used to treat
harmful bacteria. Streptomyces, a Gram-positive bacteria,
have been shown to restrict the growth of various harmful
bacteria. This bacterial community was thought to be in the
middle of the bacteria-mold spectrum producing superior
secondary metabolites such as triterpenoids, flavonoids,
and alkaloids, and antibiotics. Gram-negative bacteria and pathogenic fungi were a challenge for humans health (Al-
Ansari et al. 2019). Some of them were responsible for
infection in humans. Pathogenic bacteria such as
Enterobacter aerogenes and Proteus mirabilis were
confirmed to be inhibited by Streptomyces radiopugnans,
Streptomyces atacamensis, Streptomyces fenghuangensis,
Streptomyces verrucosisporus, and Streptomyces mangrove
isolates (Al-Ansari et al. 2019). In humans, Aspergillus
genera cause diseases such as localized infections, deadly
illnesses, allergic responses, and inhaled conidia (Mousavi
et al. 2016). Production of lyase, chitinase, protease, and
cellulase enzymes by Streptomyces is suspected of
supporting antifungal activity. These enzymes can damage
the cell wall of the mold, which was rich in components
that were easily degraded by these enzymes. Therefore,
Streptomyces spp could be used as antifungals (de Lima
Procópio et al. 2012).
There are several pathways of antimicrobial
mechanisms of Streptomyces compounds. In general,
antimicrobial mechanisms are the interactions of
pathogenic microorganisms' biochemical, genetic, and cellular structures. (Chevrette et al. 2019). Metabolites of
Streptomyces spp may cause the inhibition of DNA
division. Disorders of RNA synthesis affect the ability of
pathogens to produce proteins that play an important role in
the cellular structure of membranes, functional enzymes,
and the cell walls of pathogenic microbes. They were also
inhibited pathogenic metabolism and structural breakdown
(de Lima Procópio et al. 2012).
Alkaloids and phenols were detected in synthetic Gause
broth after the fermentation process (Table 2). Alkaloids
are present in a variety of plants and animals, including microbes. Alkaloids extracted from biological compounds
had long been used to treat a range of symptoms (Zhou et
al. 2013), such as cancer, painkillers, high blood pressure,
nervous system damage, Parkinson's disease, and
antimalarials were among them. Meanwhile, indole
alkaloids isolated from Streptomyces sp. CT37 has been
demonstrated to inhibit Candida albicans ATCC 10231 at
low concentrations (Fang et al. 2020).
Table 1. The diameter of the growth inhibition by Streptomyces sp. AB8
Microbes Colony diameter
(x̄ mm)
Clear zone
(x̄ mm)
Escherichia coli 0.56 0.40 Dickeya zeae N-Unila 5 1.60 0.70
Dickeya zeae N-Unila 10 1.50 0.50 Aspergillus sp IK3 0.20 0.10
Table 2. Biochemical characters of Streptomyces sp. AB8
Test Result Test Result
Glucose + Phenol + Fructose + Alkaloids + Cellulose + Flavonoids - Lactose + Saponin - Galactose + Triterpenoids - Sucrose + Anthraquinone glycosides -
Chitinase + Tannin - Mannanase + Catalase + Lipase - Indole + Amylase + Protease +
Note: + : positive reaction; - : negative reaction
BIODIVERSITAS 22 (7): 2817-2823, July 2021
2820
-2 0 2
2
4
6
8
Gro
wth
(%
)
Log Doses (ug/mL)
Growth (%)
DoseResp Fit of Growth
-2 0 2
0
30
60
Inhib
itio
n (
%)
Log Doses (ug/mL)
Inhibition (%)
DoseResp Fit of Inhibition
A B
-2 -1 0 1 2
0
10
20
30
40
50
60
70
80
Gro
wth
(%
)
Log dose (0.01, 0.1, 1, 10, and 100) ug/mL
Growth (%)
Inhibition (%)
D E
Figure 2. The effect of Streptomyces extracts on growth (A), growth inhibition (B), the ratio of growth to inhibition (C), the IC50 value of extract against Plasmodium parasites (D)
Alkaloids, which have a wide structural variation, are
distinguished by the presence of a specific nitrogen atom.
The majority of alkaloids have only one nitrogen atom, but others had up to five. This nitrogen was discovered in the
form of a primary amine (RNH2), secondary amine
(R2NH), or tertiary amine (R3NH) (R3NH) (Cushnie et al.
2014). Many alkaloids contain oxygen in addition to
carbon, hydrogen, and nitrogen (Cushnie et al. 2014).
Figure 3 showed that there was H bonded NH at an
absorbance range of 3070-3350 cm-1. This was presumed
as a sign of the presence of alkaloids. The acetate and
benzoic groups occurred at 1073.23 cm-1 and above,
followed by double bond -CH=CH- (cis) at 650-750 cm-1.
Phenols also present in the liquid media after the fermentation process (Table 2). It was confirmed by the
emergence of spectroscopy bands around 990 until 1060
cm-1. The secondary cyclic alcohols are found at these
spectra. Phenols donate hydrogen to react with reactive
nitrogen and oxygen species as an antioxidant (Pereira et
al. 2009). Phenols also have a variety of biochemical and
pharmacological properties such as antiviral, anticancer,
antimalarial, and anti-inflammatory activities.
Antimalarial activity Streptomyces extract at a concentration of 100 µg/mL
was able to inhibit parasite growth up to 70.92%, with an
average parasite growth rate of 2.15%. Parasitic growth
was suppressed at a rate of 4.31 % with an inhibitory
percentage of 41.84 percent at a concentration of 10
μg/mL. Extract concentration of 1 μg/mL resulted in
parasite growth of 5.96% with an inhibition percentage of
19.23%. While a concentration of 0.1 μg/mL resulted in
inhibition of 6.55% with parasite growth of 6.93 and a
concentration of 0.01 μg/mL resulted in inhibition of 1%
with growth of 7.33%. Overall, the treatment resulted in lower growth compared to the control. The increasing
extract concentration was linear with inhibition, and vice
versa, parasite growth increased with decreasing concentration
(Figure 2). The inhibitory concentration to suppress a half
parasites population (IC50) was achieved at 17.56 μg/mL.
SETYANINGRUM et al. – Antibacterial and antimalarial of Streptomyces sp AB8
2821
Figure 3. FT-IR spectra of crude extract produced by Streptomyces sp AB8
Several past studies have reported on the mechanism of alkaloids in preventing the growth of plasmodial and
microbiological infections. Alkaloids inhibit nucleic acid
synthesis by suppressing the enzyme dihydrofolate
reductase in cell-free (Raimondi et al. 2019). It can prevent
cell division by inhibiting the Z-ring formation in bacteria
(Keffer et al. 2013). Another study by Godlewska-
Żyłkiewicz et al. (2020) showed that alkaloids are
sufficient to initiate bacterial homeostasis and disrupt the
stability of the outer membrane and cytoplasmic (Cushnie
et al. 2014). Alkaloids were also able to restrain fimbria-
dependent biofilm formation in E. coli (Notarte et al.
2019). Streptomyces cellulosae strain TES17 produced
phenolic compounds with strong antioxidant properties and
effective in damaging lung cancer cell lines (Rani et al.
2018). The antibacterial and antimalarial mechanism of
phenolic compounds possibly due to the ability to inhibit
respiratory electron transport systems, reduced outer
membrane containing lipopolysaccharides, disrupts the
structure of the cell wall, and the synthesis of cell wall
becomes inefficient, affect the permeability, and disrupt the
cell division protein FtsZ-ring (Aldulaimi et al. 2019).
The findings on Streptomyces extract's antimalarial activity were used as a starting point. Further study to
determine its efficacy using an ex vivo or in vitro approach
is needed (Sinha et al. 2017). Peter’s Test or 4-day
suppression test, histopathological observation, and
cytotoxicity for non-cell-targeted are required (Kifle et al.
2020). A metabolite is categorized as antiplasmodial if this
metabolite can suppress parasite growth by more than 30%
compared to negative controls after completing a set of in
vivo tests (Castro et al. 1996). In conclusion, the qualitative
test approach was supported by infrared spectroscopic data
indicating the presence of phenolic compounds and alkaloids from the bacterial metabolite Streptomyces AB8.
The crude extract of Streptomyces sp AB8 can inhibit
Plasmodium, with an IC50 value of 17.56 μg/mL. The
extract of Streptomyces sp. AB8 could inhibit the growth of
Dickeya zeae N-Unila 5, Dickeya zeae N-Unila 10,
Aspergillus sp IK3, and Escherichia coli. These results on
antimalarial and antimicrobial are preliminary results.
ACKNOWLEDGEMENTS
We would like to thank everyone who contributes to
this research. This work was partly supported by grants
from Universitas Lampung, Indonesia (1661/UN26.21/PN/
2021). The authors have declared that there was no conflict of interests.
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Achmad Arifiyanto, S.Si., M.Si.
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Streptomyces hygroscopicus subsp. hygroscopicus strain I18:
Incubation Time and Tryptophan Concentration Effects on
Indole-3-Acetic Acid (IAA) Hormone Production
Mia Fitriani1, Achmad Arifiyanto1*, Sumardi1, Martha Lulus Lande1, Christina
Nugroho Ekowati1, Titik Nur Aeny2, Hapin Afriyani3
1Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Lampung, 2Department of Plant
Protection, Faculty of Mathematics and Natural Sciences, Universitas Lampung, 3Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Lampung,
* Corresponding author: [email protected]
Abstract. Plant hormones are chemical messengers that regulate a wide range of physiological activities in plants. Microbes need
a definite time to synthesize a compound. The microbial life cycle consists of several phases, often requiring a shorter incubation
time than in complex organisms such as plants, animals, and humans. This study aimed to determine the optimum incubation time
and the best tryptophan levels of Streptomyces hygroscopicus subsp. hygroscopicus strain i18 in Indole-3-acetic acid (IAA)
hormone. IAA has synthesized on liquid ISP4 medium using L-tryptophan supplementation as a precursor. Cell density was
measured every day for 4-11 days using the spectrophotometric approach to track the incubation time. The doses of L-Tryptophan
were 0, 1, 2, 3, 4, 5, and 6 mg/mL, respectively. The levels of IAA produced by bacteria were measured and compared with standard
IAA. Streptomyces hygroscopicus subsp. hygroscopicus strain i18 was obtained the optimum incubation time at 9th days with an
IAA level of 12.30 μg/mL. Supplementing with 5 mg/mL L-tryptophan resulted in the highest IAA level of 17.90 μg/mL. This
study would be worthwhile in assisting agricultural product quality improvement through field application for further research.
Keywords: amino acid, IAA, incubation period, plant growth hormone, and Streptomyces
INTRODUCTION
Plant hormones have required as chemical messengers that impact a plant's ability to respond to the environment and
speed up its growth 1,2. Rhizosphere bacteria can create plant hormones, also known as phytohormones. Because they
dwell in plant roots, these bacteria include plant growth promoter rhizobacteria (PGPR). They live in colonies and aid
in the growth of plants as well as the prevention of illness. Auxin-producing rhizobacteria include Actinomycetes,
Azospirillum, Rhizobium, Pseudomonas, Bacillus, Micrococcus, Kocuria, and Enterobacter 3.
Streptomyces was a soil bacteria member of Actinomycetes whose capable of producing plant hormones, including
Indole-3-acetic acid (IAA) 4. This hormone belongs to the auxin family and served as a signaling molecule in plant
development, root elongation, and enzyme activation. It improved nutrient intake by developing longer roots and
increased lateral root hairs. It also has a significant impact on plant development 5.
Several factors, such as incubation duration and Tryptophan content, can alter the amount of IAA generated by
Actinomycetes 6. In maize and peas, supplementing precursors in the form of Tryptophan to the environment enabled
seed germination and root elongation 7. Streptomyces sp has potentially produced IAA that helped plant growth.
Streptomyces sp had generated biosurfactants and antimicrobial performances from the rhizosphere of the Sidoarjo
mud area 8.
Streptomyces sp. strain i18 had identified as Streptomyces hygroscopicus subsp. hygroscopicus and was isolated from
pineapple plantation soil 9. Metabolite extracted from this strain contained saponins, triterpenoids, and anthraquinone.
It showed the potential to inhibit the growth of Plasmodium parasites 10. Unfortunately, information regarding the
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ability of this strain to produce IAA was not yet available. This study intended to determine i18 bacteria' ability to
produce IAA at the optimal time and the effect of the dose of tryptophan amino acid supplementation.
MATERIALS AND METHODS
Bacterial culture
Bacteria were cultured on International Streptomyces Project medium 4 (Inorganic salts-starch) and incubated at room
temperature for 4-7 days. The composition of the medium consisted of 10 g Soluble Starch, 1 g MgSO4 x 7H2O, 1 g
NaCl, 2 g (NH4)2SO4, 2 g CaCO3, 1 L distilled water, 1 mL Trace Salts solution (0.1 g FeSO4 x 7H2O, 0.1 g MnCl2 x
4H2O, 0.1 g ZnSO4 x 7H2O, 100 mL distilled water), and 20 g agar. A loop of inoculum was taken and put in a liquid
inorganic salts-starch medium, incubating for 7th days at room temperature for a 10 mL starter 11.
Incubation time
In 90 mL liquid inorganic salts-starch media, Tryptophan 2 mg/mL was added together with a 10 mL starter and
shaken at 10,000 rpm in the dark. The production medium was taken 2 ml and centrifuged at 3000 rpm for 30 minutes.
The separated supernatant was taken 1 ml and reacted with 2 ml Salkowski solution, incubated for 60 minutes in the
dark, and observed for a pink to red color change. It was measured on days 4-11 with a spectrophotometer with a
wavelength of 530 nm. The data obtained were analyzed using ANOVA and BNT follow-up test 12.
Tryptophan supplementation
The starter was taken in 10 mL and placed in 90 mL of liquid inorganic salts-starch media with 1, 2, 3, 4, 5, and 6
mg/mL of Tryptophan, respectively. They had incubated on a shaker incubator for the optimum incubation time
previously achieved. Culture media containing bacteria without Tryptophan was assigned as a control. The production
medium was taken 2 ml and centrifuged at 3000 rpm for 30 minutes. One milliliter of the separated supernatant was
mixed with two milliliters of Salkowski solution and incubated in the dark for 60 minutes. The data were analyzed
using the non-parametric Kruskal-Wallis test after measurements using a spectrophotometer with a wavelength of 530
nm 13.
RESULTS AND DISCUSSIONS
The presence of IAA can be detected by reacting with the supernatant of the Salkowski reagent. The interaction
between IAA and Fe causes free electrons from the ligand to fill the "d" orbital in Fe3+ with IAA, resulting in the
formation of a complex bond [Fe2 (OH) 2 (IA)4]. The color change indicates the high content of IAA produced is
proportional to the density of the red color. It had seen in Figure 1.
FIGURE 1. Red color change on IAA production with the addition of Tryptophan 0-6 mg/mL
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During the incubation time of days 4-11, Strain i18 was adequate to produce the IAA hormone. The optimum
incubation time for strain i18 occurred on day 9 with a value of 12.30 μg/mL. The culture began to enter the optimal
production phase of IAA after day 7, Table 1.
TABLE 1. Production of IAA on days 4-11 by Streptomyces hygroscopicus subsp. hygroscopicus strain i18
Tryptophan administration on IAA production had a significant effect, indicated by the Asymp value. In a non-
parametric test using the Kruskal Walis method, the Significant value is 0.008. The Tryptophan concentration of 5
mg/mL resulted in IAA levels of 17.90 g/mL, which was the highest among other Tryptophan dosing treatments (Table
2).
TABLE 2. Tryptophan supplementation effect at IAA content produced by Streptomyces hygroscopicus subsp.
hygroscopicus strain i18
Day IAA concentration (μg/mL)
4 3.90 ± 0.01
5 6.90 ± 0.09
6 8.00 ± 0.02
7 8.20 ± 0.09
8 8.60 ± 0.04
9 12.30 ± 0.10
10 11.00 ± 0.06
11 8.00 ± 0.10
Tryptophan concentration (mg/mL) IAA concentration (μg/mL)
0 2.90 ± 0.01
1 8.60 ± 0.01
2 11.20 ± 0.01
3 14.80 ± 0.01
4 16.30 ± 0.06
5 17.90 ± 0.06
6 16.80 ± 0.03
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The ability of rhizobacterial isolates to produce IAA was considered a beneficial method for identifying helpful
microbes, and they have a significant impact on plant growth 14. IAA was an inorganic-volatile compound produced
by Streptomyces spp 15. The production of secondary metabolites was the ability of each organism. Secondary
metabolites biosynthesis began at the end of the logarithmic or exponential phase until the end of the stationary phase,
after cell division and multiplication stop 16.
3 4 5 6 7 8 9 10 11 12
0
2
4
6
8
10
12IA
A c
on
ce
ntr
atio
n (
ug
/mL
)
Incubation time (day)
IAA concentration (ug/mL)
FIGURE 2. The relation of incubation time to IAA concentration.
0 1 2 3 4 5 6
0
2
4
6
8
10
12
14
16
18
20
IAA
co
nce
ntr
atio
n (
ug
/mL
)
Tryptophan concentration (mg/mL)
IAA concentration (ug/mL)
FIGURE 3. The relation of tryptophan doses to IAA concentration.
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IAA hormone production was increased by nutrient sources available during the incubation phase, while IAA
production had decreased by nutrients that began to deplete during the incubation period 17. Types of nutrients that
influenced IAA production were carbon, nitrogen, and precursor type. The oxidase and peroxidase enzymes were
released, which lowered the amount of IAA produced 18. Thus, due to the availability of sufficient nutrients, there is
a pattern of increases from the beginning of the 4th day to the peak on the 9th day (Figure 2).
TABLE 3. Summary of bacterial species, ideal incubation time, total incubation time, liquid medium, IAA levels
produced by dosages, and L-tryptophan doses in a variety of comparative sources
No Strain
The peak
period of
output (day;
hour)
Duration of
incubation
(days)
Liquid
Media
Optimal
IAA content
(μg/mL)
L -tryptophan
optimal doses
(mg/mL)
Sources
1 Streptomyces
fradiae NKZ-259
6; 144 14 Gause’s
No.1
20.46 2 .00 4
2 Streptomyces
hygroscopicus subsp.
hygroscopicus strain i18
9; 216 11 ISP 4 17.90 5.00 This
result
3 Streptomyces sp. MS1 5; 120 7 Yeast
malt
extract
(YM)
125.48 2 .00 19
4 Streptomyces sp. BR27 5; 120 7 104.13 1 .00
5 Streptomyces sp
VSMGT1014
5: 120 8 ISP 2 15.96 2.00 20
6 Streptomyces sp. PT2 5; 120 10 Yeast
extract-
tryptone
3.63 5.00 6
At the beginning of the logarithmic stage, the yield of IAA produced was lower, presumably because the content of
enzymes utilized to convert Tryptophan to IAA remained low (Figure 3). The yield of IAA produced was lower at the
beginning of the logarithmic stage due to a lack of enzyme concentration and bacterial cells. In contrast, at the end of
the logarithmic phase, IAA production entered following bacteria increasing growth. Monooxygenase, IAM
hydrolase, indole pyruvate decarboxylase and IAAld dehydrogenase were utilized during Tryptophan bioconversion
into IAA. They were produced quite a lot and were active in line with the growth rate 21.
Unfortunately, the cell density data at the time of the IAA measurement has not been measured in this study. These
data help reveal that bacterial cell growth is closely related to the production of IAA as a metabolite. Table 3 informs
us that the type of production media affects the amount of IAA produced. Likewise, the dose of tryptophan as a
precursor in each strain of Streptomyces spp gave different results in producing IAA.
Tryptophan has been identified as a major precursor of IAA and plays an important role in modulating the level of
IAA biosynthesis in bacteria. Tryptophan in the rhizosphere was obtained from the degradation of root cells, microbial
cells, and root exudates. IAA produced by bacteria stimulates the growth of a plant. On the other hand, IAA controlled
microbial antibiotic synthesized 22. This growth triggers the exudation of root organic compounds which are used for
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the growth of these bacteria 23.1 Tryptophan is used as a source of carbon and energy, which will break down into
several compounds involved in metabolic pathways. This provides a mutually beneficial role between the bacteria and
the plant itself 24.
CONCLUSION
On the 9th day of testing, with an IAA concentration of 12.30 μg/mL, Streptomyces hygroscopicus subsp.
hygroscopicus strain i18 produced the most IAA. The addition of 5 mg/mL of Tryptophan produced an IAA of 17.90
μg/mL as the optimum amount of Tryptophan. It was required to count the number of bacterial cells to assess the
relationship between IAA production and cell growth, and as well as to quantify the amount of IAA tested with various
strains for field applications.
ACKNOWLEDGMENT
We'd like to thank everyone who contributes to this research. This work was partly supported by grants from
Universitas Lampung, Indonesia (1661/UN26.21/PN/2021).
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